Journal articles on the topic 'Male germline regulatory module'

ABSTRACTIn sexually reproducing metazoans, spermatogenesis is the process by which uncommitted germ cells give rise to haploid sperm. Work in model systems has revealed mechanisms controlling commitment to the sperm fate, but how this fate is subsequently executed remains less clear. While studying the well-established role of the conserved nuclear hormone receptor transcription factor, NHR-23/NR1F1, in regulating C. elegans molting, we discovered that NHR-23/NR1F1 is also constitutively expressed in developing primary spermatocytes and is a critical regulator of spermatogenesis. In this novel role, NHR-23/NR1F1 functions downstream of the canonical sex-determination pathway. Degron-mediated depletion of NHR-23/NR1F1 within hermaphrodite or male germlines causes sterility due to an absence of functional sperm, as depleted animals produce arrested primary spermatocytes rather than haploid sperm. These spermatocytes arrest in prometaphase I and fail to either progress to anaphase or attempt spermatid-residual body partitioning. They make sperm-specific membranous organelles but fail to assemble their major sperm protein into fibrous bodies. NHR-23/NR1F1 appears to function independently of the known SPE-44 gene regulatory network, revealing the existence of an NHR-23/NR1F1-mediated module that regulates the spermatogenesis program.

Abstract. Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs). These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys) are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs) are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28) during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of human fertility preservation strategies.

PHD finger protein 7 ( Phf7 ) is a male germline specific gene in Drosophila melanogaster that can trigger the male germline sexual fate and regulate spermatogenesis, and its human homologue can rescue fecundity defects in male flies lacking this gene. These findings prompted us to investigate conservation of reproductive strategies through studying the evolutionary origin of this gene. We find that Phf7 is present only in select species including mammals and some insects, whereas the closely related G2/M-phase specific E3 ubiquitin protein ligase ( G2e3 ) is in the genome of most metazoans. Interestingly, phylogenetic analyses showed that vertebrate and insect Phf7 genes did not evolve from a common Phf7 ancestor but rather through independent duplication events from an ancestral G2e3 . This is an example of parallel evolution in which a male germline factor evolved at least twice from a pre-existing template to develop new regulatory mechanisms of spermatogenesis.

Male breast cancer (MBC) is a rare and understudied disease compared with female BC. About 15% of MBCs are associated with germline mutation in BC susceptibility genes, mainly BRCA1/2 and PALB2. Hereditary MBCs are likely to represent a subgroup of tumors with a peculiar phenotype. Here, we performed a whole transcriptome analysis of MBCs characterized for germline mutations in the most relevant BC susceptibility genes in order to identify molecular subtypes with clinical relevance. A series of 63 MBCs, including 16 BRCA2, 6 BRCA1, 2 PALB2, 1 RAD50, and 1 RAD51D germline-mutated cases, was analyzed by RNA-sequencing. Differential expression and hierarchical clustering analyses were performed. Module signatures associated with central biological processes involved in breast cancer pathogenesis were also examined. Different transcriptome profiles for genes mainly involved in the cell cycle, DNA damage, and DNA repair pathways emerged between MBCs with and without germline mutations. Unsupervised clustering analysis revealed two distinct subgroups, one of which was characterized by a higher expression of immune response genes, high scores of gene-expression signatures suggestive of aggressive behavior, and worse overall survival. Our results suggest that transcriptome matched with germline profiling may be a valuable approach for the identification and characterization of MBC subtypes with possible relevance in the clinical setting.

Abstract Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Previously it has been shown that fetal alcohol exposure increases the stress response partly due to lowering stress regulatory proopiomelanocortin (Pomc) gene expression in the hypothalamus via epigenetic mechanisms for multiple generations in mixed-breed rats. In this study we assess the induction of heritable epigenetic changes of Pomc-related variants by fetal alcohol exposure in isogenic Fischer 344 rats. Using transgenerational breeding models and fetal alcohol exposure procedures, we determined changes in hypothalamic Pomc gene expression and its methylation levels, plasma corticosterone hormone response to restraint stress, and anxiety-like behaviors using elevated plus maze tests in fetal alcohol-exposed offspring for multiple generations in isogenic Fischer rats. Fetal alcohol-exposed male and female rat offspring showed significant deficits in POMC neuronal functions with increased Pomc gene methylation and reduced expression. These changes in POMC neuronal functions were associated with increased plasma corticosterone response to restraint stress and increased anxiety-like behavior. These effects of fetal alcohol exposure persisted in the F1, F2, and F3 progeny of the male germline but not of the female germline. These data suggest that fetal alcohol exposure induces heritable changes in Pomc-related variants involving stress hyperresponsiveness and anxiety-like behaviors which perpetuate into subsequent generations through the male germline via epigenetic modifications.

The gametophyte represents the sexual phase in the alternation of generations in plants; the other, nonsexual phase is the sporophyte. Here, we review the evolutionary origins of the male gametophyte among land plants and, in particular, its ontogenesis in flowering plants. The highly reduced male gametophyte of angiosperm plants is a two- or three-celled pollen grain. Its task is the production of two male gametes and their transport to the female gametophyte, the embryo sac, where double fertilization takes place. We describe two phases of pollen ontogenesis—a developmental phase leading to the differentiation of the male germline and the formation of a mature pollen grain and a functional phase representing the pollen tube growth, beginning with the landing of the pollen grain on the stigma and ending with double fertilization. We highlight recent advances in the complex regulatory mechanisms involved, including posttranscriptional regulation and transcript storage, intracellular metabolic signaling, pollen cell wall structure and synthesis, protein secretion, and phased cell–cell communication within the reproductive tissues.

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, we compare the sex determination mechanism that operates in the germline with that in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL is maintained by a positive autoregulatory feedback loop in which Sxl protein insures its continued synthesis by binding to Sxl pre-mRNA and thereby imposing the productive (female) splicing mode. The gene splicing-necessary factor (snf), which encodes a component of U1 and U2 snRNPs, participates in this RNA splicing control. Here we show that an increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. We also show that female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu which control female-specific processes in the ovary.

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

Sexual reproduction depends on the production of haploid gametes, and their fusion to form diploid zygotes. Here, we discuss sperm production and function in a molecular and functional evolutionary context, drawing predominantly from studies in model organisms (mice, Drosophila , Caenorhabditis elegans ). We consider the mechanisms involved in establishing and maintaining a germline stem cell population in testes, as well as the factors that regulate their contribution to the pool of differentiating cells. These processes involve considerable interaction between the germline and the soma, and we focus on regulatory signalling events in a variety of organisms. The male germline has a unique transcriptional profile, including expression of many testis-specific genes. The evolutionary pressures associated with gene duplication and acquisition of testis function are discussed in the context of genome organization and transcriptional regulation. Post-meiotic differentiation of spermatids involves very dramatic changes in cell shape and acquisition of highly specialized features. We discuss the variety of sperm motility mechanisms and how various reproductive strategies are associated with the diversity of sperm forms found in animals.

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6- O sulfate groups on HS. Loss of 6- O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.

The differentiation of sperm from morphologically unremarkable cells into highly specialised free-living, motile cells requires the co-ordinated action of a very large number of gene products. The expression of these products must be regulated in a developmental context to ensure normal cellular differentiation. Many genes essential for spermatogenesis are not used elsewhere in the animal, or are expressed elsewhere, but using a different transcription regulation module. Spermatogenesis is thus a good system for elucidating the principles of tissue-specific gene expression, as well as being interesting in its own right. Here, I discuss the regulation of gene expression during spermatogenesis inDrosophila, focussing on the processes underlying the expression of testis-specific genes in the male germline.

Abstract Nonautonomous inductive signals from the soma and autonomous signals due to a 2X karyotype determine the sex of Drosophila melanogaster germ cells. These two signals have partially overlapping influences on downstream sex determination genes. The upstream OVO-B transcription factor is required for the viability of 2X germ cells, regardless of sexual identity, and for female germline sexual identity. The influence of inductive and autonomous signals on ovo expression has been controversial. We show that ovo-B is strongly expressed in the 2X germ cells in either a male or a female soma. This indicates that a 2X karyotype controls ovo-B expression in the absence of inductive signals from the female soma. However, we also show that female inductive signals positively regulate ovo-B transcription in the 1X germ cells that do not require ovo-B function. Genetic analysis clearly indicates that inductive signals from the soma are not required for ovo-B function in 2X germ cells. Thus, while somatic inductive signals and chromosome karyotype have overlapping regulatory influences, a 2X karyotype is a critical germline autonomous determinant of ovo-B function in the germline.

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction inArabidopsis thaliana. DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte,DMEexpression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte,DMEexpression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements ofDMEusing reporter genes, showing that a small region, which surprisingly lies within theDMEgene, controls its expression in male and female companion cells.DMEexpression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the nulldme-2mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities ofDMEand combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.

Pollen grains represent the highly reduced haploid male gametophyte generation in angiosperms. They play an essential role in plant fertility by generating and delivering twin sperm cells to the embryo sac to undergo double fertilization. The functional specialization of the male gametophyte and double fertilization are considered to be key innovations in the evolutionary success of angiosperms. The haploid nature of the male gametophyte and its highly tractable ontogeny makes it an attractive system to study many fundamental biological processes, such as cell fate determination, cell-cycle progression and gene regulation. The present mini-review encompasses key advances in our understanding of the molecular mechanisms controlling male gametophyte patterning in angiosperms. A brief overview of male gametophyte development is presented, followed by a discussion of the genes required at landmark events of male gametogenesis. The value of the male gametophyte as an experimental system to study the interplay between cell fate determination and cell-cycle progression is also discussed and exemplified with an emerging model outlining the regulatory networks that distinguish the fate of the male germline from its sister vegetative cell. We conclude with a perspective of the impact emerging data will have on future research strategies and how they will develop further our understanding of male gametogenesis and plant development.

The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.

Abstract One in 54 children in the United States is diagnosed with autism spectrum disorder. De novo germline and somatic mutations cannot account for all cases of autism spectrum disorder, suggesting that epigenetic alterations triggered by environmental exposures may be responsible for a subset of autism spectrum disorder cases. Human and animal studies have shown that exposure of the developing brain to general anesthetic agents can trigger neurodegeneration and neurobehavioral abnormalities, but the effects of general anesthetics on the germline have not been explored in detail. We exposed pregnant mice to sevoflurane during the time of embryonic development when the germ cells undergo epigenetic reprogramming and found that more than 38% of the directly exposed F1 animals exhibit impairments in anxiety and social interactions. Strikingly, 44–47% of the F2 and F3 animals, which were not directly exposed to sevoflurane, show the same behavioral problems. We performed ATAC-seq and identified more than 1200 differentially accessible sites in the sperm of F1 animals, 69 of which are also present in the sperm of F2 animals. These sites are located in regulatory regions of genes strongly associated with autism spectrum disorder, including Arid1b, Ntrk2, and Stmn2. These findings suggest that epimutations caused by exposing germ cells to sevoflurane can lead to autism spectrum disorder in the offspring, and this effect can be transmitted through the male germline inter- and transgenerationally.

Abstract Abstract 3488 Germline heterozygous mutations in GATA2 have been reported to cause familial myelodysplasia–acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infection (monoMAC syndrome), dendritic cell, myeloid and NK cell lymphopenia (DCML), and Emberger syndrome (lymphedema and MDS). GATA2 is a zinc finger transcription factor that plays a crucial role in regulating growth of hematopoietic progenitors. In some pedigrees, patients or family members have manifestations of bone marrow failure. We hypothesized that patients with aplastic anemia (AA) may harbor mutations in GATA2. The coding regions and regulatory regions of GATA2 were sequenced in 99 patients with confirmed AA. Sequences from 100 normal individuals as well as published human genomes from unaffected individuals (dbSNP build 135 and 1000 Genomes Project) were used as controls. Genetic variants were confirmed in hematopoietic and somatic tissues. We identified 4 heterozygous mutations in regulatory regions of GATA2 in 5 patients. In two patients, a mutation at nucleotide 59T>G in exon 1 of isoform 2 was identified; both had severe AA in early adulthood refractory to immunosuppressive therapy. We noted this 59T>G mutation in two unrelated individuals with severe disseminated mycobacterial disease. We identified a mutation at nucleotide 20G>A in exon 2 of isoform 1, in a 3 year-old male with hepatitis-associated severe AA whose disease was refractory to multiple rounds of immunosuppressive therapy. Another mutation was present in 38G>A in exon 2 of isoform 1 in a 32 year old male with moderate AA and paroxysmal nocturnal hemoglobinuria (PNH). We also identified the exon 2 38G>A mutation in a patient with disseminated mycobacterial disease where reduced transcription of the mutant 38G>A allele was noted on RT-PCR. Finally, an intron 5, c.512+573 G>A variant was identified in an 18 year old male with severe AA who progressed after immunosuppressive therapy to MDS/AML. This variant, which causes a disruption of the FLI1 binding site, has also been found to be pathogenic in monoMAC syndrome. In summary, a subset of patients with AA were found to have mutations in GATA2 suggesting a role for the gene in the pathogenesis of bone marrow failure. It also may identify patients at higher risk of infectious complications, those who may have less advantageous responses to immune suppression, and command earlier bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.

ABSTRACTMaintenance of germ cell sexual identity is essential for reproduction. Entry into the spermatogenesis or oogenesis pathway requires that the appropriate gene network is activated and the antagonist network is silenced. For example, in Drosophila female germ cells, forced expression of the testis-specific PHD finger protein 7 (PHF7) disrupts oogenesis, leading to either an agametic or germ cell tumor phenotype. Here, we show that PHF7-expressing ovarian germ cells inappropriately express hundreds of genes, many of which are male germline genes. We find that the majority of genes under PHF7 control in female germ cells are not under PHF7 control in male germ cells, suggesting that PHF7 is acting in a tissue-specific manner. Remarkably, transcriptional reprogramming includes a positive autoregulatory feedback mechanism in which ectopic PHF7 overcomes its own transcriptional repression through promoter switching. Furthermore, we find that tumorigenic capacity is dependent on the dosage of phf7. This study reveals that ectopic PHF7 in female germ cells leads to a loss of sexual identity and the promotion of a regulatory circuit that is beneficial for tumor initiation and progression.

Human germ cell development is regulated in a spatio-temporal manner by complex regulatory networks. Here, we summarize results obtained in germ cell tumors and respective cell lines and try to pinpoint similarities to normal germ cell development. This comparison allows speculating about the critical and error-prone mechanisms, which when disturbed, lead to the development of germ cell tumors. Short after specification, primordial germ cells express markers of pluripotency, which, in humans, persists up to the stage of fetal/infantile spermatogonia. Aside from the rare spermatocytic tumors, virtually all seminomas and embryonal carcinomas express markers of pluripotency and show signs of pluripotency or totipotency. Therefore, it appears that proper handling of the pluripotency program appears to be the most critical step in germ cell development in terms of tumor biology. Furthermore, data from mice reveal that germline cells display an epigenetic signature, which is highly similar to pluripotent cells. This signature (poised histone code, DNA hypomethylation) is required for the rapid induction of toti- and pluripotency upon fertilization. We propose that adult spermatogonial cells, when exposed to endocrine disruptors or epigenetic active substances, are prone to reinitiate the pluripotency program, giving rise to a germ cell tumor. The fact that pluripotent cells can be derived from adult murine and human testicular cells further corroborates this idea.

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.

The classic roles of mitochondria in energy production, metabolism, and apoptosis have been well defined. However, a growing body of evidence suggests that mitochondria are also active players in regulating stem cell fate decision and lineage commitment via signaling transduction, protein modification, and epigenetic modulations. This is particularly interesting for spermatogenesis, during which germ cells demonstrate changing metabolic requirements across various stages of development. It is increasingly recognized that proper male fertility depends on exquisitely controlled plasticity of mitochondrial features, activities, and functional states. The unique role of mitochondria in germ cell ncRNA processing further adds another layer of complexity to mitochondrial regulation during spermatogenesis. In this review, we will discuss potential regulatory mechanisms of how mitochondria swiftly reshape their features, activities, and functions to support critical germ cell fate transitions during spermatogenesis. In addition, we will also review recent findings of how mitochondrial regulators coordinate with germline proteins to participate in germ cell-specific activities.

Sexual differentiation of inflorescences and flowers is important for reproduction and affects crop plant productivity. We report here on a molecular study of the process of sexual differentiation in the immature inflorescence of oil palm (Elaeis guineensis). This species is monoecious and exhibits gender diphasy, producing male and female inflorescences separately on the same plant in alternation. Three main approaches were used: small RNA-seq to characterise and study the expression of miRNA genes; RNA-seq to monitor mRNA accumulation patterns; hormone quantification to assess the role of cytokinins and auxins in inflorescence differentiation. Our study allowed the characterisation of 30 previously unreported palm MIRNA genes. In differential gene and miRNA expression studies, we identified a number of key developmental genes and miRNA-mRNA target modules previously described in relation to their developmental regulatory role in the cereal panicle, notably the miR156/529/535-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene regulatory module. Gene enrichment analysis highlighted the importance of hormone-related genes, and this observation was corroborated by the detection of much higher levels of cytokinins in the female inflorescence. Our data illustrate the importance of branching regulation within the developmental window studied, during which the female inflorescence, unlike its male counterpart, produces flower clusters on new successive axes by sympodial growth.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

Abstract P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry+, Δ2-3)99B. For H(hsp/CP)2 and P(ry+, Δ2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry+, Δ2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype.

Processing of pre-mRNA into mRNA is an important regulatory mechanism in eukaryotes that is mediated by the spliceosome, a huge and dynamic ribonucleoprotein complex. Splicing defects are implicated in a spectrum of human disease, but the underlying mechanistic links remain largely unresolved. Using a genome-wide association approach, we have recently identified single nucleotide polymorphisms in humans that associate with nonobstructive azoospermia (NOA), a common cause of male infertility. Here, using genetic manipulation of corresponding candidate loci in Drosophila, we show that the spliceosome component SNRPA1/U2A is essential for male fertility. Loss of U2A in germ cells of the Drosophila testis does not affect germline stem cells, but does result in the accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes and mature sperm. Lack of U2A causes insufficient splicing of mRNAs required for the transition of germ cells from proliferation to differentiation. We show that germ cell-specific disruption of other components of the major spliceosome manifests with the same phenotype, demonstrating that mRNA processing is required for the differentiation of spermatogonia. This requirement is conserved, and expression of human SNRPA1 fully restores spermatogenesis in U2A mutant flies. We further report that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogonial differentiation in Drosophila. Collectively, our findings uncover a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differentiation in the male testis, suggesting that spliceosome defects affecting the differentiation of human spermatogonia contribute to NOA.

Abstract Introduction: Neuroblastoma (NBL) is the most common type of extracranial pediatric solid tumor. NBL exhibits a wide spectrum of phenotypes, from low-risk cases that spontaneously regress to high-risk cases with 50% mortality, and it is responsible for 12% to 15% of deaths due to childhood cancer. While genome-wide association studies have identified a handful of risk loci, the germline genetic origins of sporadic NBL remain largely unknown. Notably, NBL is one of several childhood cancers that tend to co-occur with specific birth defects, which could point to novel genetic origins. For example, the risk of neuroblastoma is increased 3- to 7-fold among children with certain congenital heart defects (CHD). It is hypothesized that this co-occurrence arises from pleiotropic defects in the neural crest cells that are progenitors of both NBL and some heart structures. Based on these lines of evidence, we hypothesize that novel germline risk loci for NBL can be identified through pleiotropic analyses of CHD and NBL cohorts. Therefore, in this study we seek to identify germline risk loci for neuroblastoma by jointly analyzing CHD and NBL data sets with a primary focus on de novo variants. Methods: Using whole genome sequencing data from the Gabriella Miller Kids First Data Resource Center, we will identify NBL and CHD associated loci by testing for high rates of de novo variants (DNV) in each cohort separately and in the two cohorts combined. The testing strategy compares the rate of DNV to the expected locus-specific mutation rate. In our primary analyses, we will focus on genes known to be associated with CHD in order to reduce the multiple testing penalty for that family of loci. A broader analysis will explore coding and regulatory loci genome wide to identify loci associated jointly with NBL and CHD. Results: We acquired whole genome sequencing data of parent-offspring trios for NBL (n=609) and CHD (n=711) from the Gabriella Miller Kids First Data Resource Center. The probands in the NBL cohort was 54% male and 81% Non-Hispanic White, with a median age at diagnosis of 16 months. The probands in the CHD cohort was 59% male and 60% Non-Hispanic White. The most common CHD phenotypes were ventricular septal defect (46%), tetralogy of Fallot (32%), hypoplastic left heart syndrome (28%), atrial septal defect (25%), and right aortic arch (25%). Analyses to identify DNV are ongoing. Conclusion: The identification of germline risk loci associated with NBL will help to inform the developmental origins of this important childhood cancer and may inform novel risk stratification protocols and therapies. This study also aims to help explain the NBL-CHD co-occurrence, and that could shed light on the development of other neural crest cell derived cancers. Citation Format: Alexander Renwick, Yao Yu, Chad Huff, Philip J. Lupo. De novo variants associated with neuroblastoma and congenital heart defects: evidence of pleiotropic effect from 1311 WGS trios [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2506.

ABSTRACT In the nematode C. elegans, there are two sexes, the self-fertilizing hermaphrodite (XX) and the male (XO). The hermaphrodite is essentially a female that makes sperm for a brief period before oogenesis. Sex determination in C. elegans is controlled by a pathway of autosomal regulatory genes, the state of which is determined by the X:A ratio. One of these genes, tra-2, is required for hermaphrodite development, but not for male development, because null mutations in tra-2 masculinize XX animals but have no effect on XO males. Dominant, gain-of-function tra-2 mutations have now been isolated that completely feminize the germline of XX animals so that they make only oocytes and no sperm and, thus, are female. Most of the tra-2(dom) mutations do not correspondingly feminize XO animals, so they do not appear to interfere with control by her-1, a gene thought to negatively regulate tra-2 in XO animals. Thus, these mutations appear to cause gain of tra-2 function in the XX animal only. Dosage studies indicate that 5 of 7 tra-2(dom) alleles are hypomorphic, so they do not simply elevate XX tra-2 activity overall. These properties suggest that in the wild type, tra-2 activity is under two types of control: (1) in males, it is inactivated by her-1 to allow male development to occur, and (2) in hermaphrodites, tra-2 is active but transiently inactivated by another, unknown, regulator to allow hermaphrodite spermatogenesis; this mode of regulation is hindered by the tra-2(dom) mutations, thereby resulting in XX females.

Pollen grain is a unique haploid organism characterized by two key physiological processes: activation of metabolism upon exiting dormancy and polar tube growth. In gymnosperms and flowering plants, these processes occur in different time frames and exhibit important features; identification of similarities and differences is still in the active phase. In angiosperms, the growth of male gametophyte is directed and controlled by its microenvironment, while in gymnosperms it is relatively autonomous. Recent reviews have detailed aspects of interaction between angiosperm female tissues and pollen such as interactions between peptides and their receptors; however, accumulated evidence suggests low-molecular communication, in particular, through ion exchange and ROS production, equally important for polar growth as well as for pollen germination. Recently, it became clear that ROS and ionic currents form a single regulatory module, since ROS production and the activity of ion transport systems are closely interrelated and form a feedback loop.

Coronary artery disease (CAD) is a major cause of end-stage cardiac disease. Although profound efforts have been made to illuminate the pathogenesis, the molecular mechanisms of CAD remain to be analyzed. To identify the candidate genes in the advancement of CAD, microarray dataset GSE23766 was downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and pathway and gene ontology (GO) enrichment analyses were performed. The protein-protein interaction network was constructed and the module analysis was performed using the Biological General Repository for Interaction Datasets (BioGRID) and Cytoscape. Additionally, target genes-miRNA regulatory network and target genes-TF regulatory network were constructed and analyzed. There were 894 DEGs between male human CAD samples and female human CAD samples, including 456 up regulated genes and 438 down regulated genes. Pathway enrichment analyses revealed that DEGs (up and down regulated) were mostly enriched in the superpathway of steroid hormone biosynthesis, ABC transporters, oxidative ethanol degradation III and Complement and coagulation cascades. Similarly, geneontology enrichment analyses revealed that DEGs (up and down regulated) were mostly enriched in the forebrain neuron differentiation, filopodium membrane, platelet degranulation and blood microparticle. In the PPI network and modules (up and down regulated), MYC, NPM1, TRPC7, UBC, FN1, HEMK1, IFT74 and VHL were hub genes. In the target genes-miRNA regulatory network and target genes—TF regulatory network (up and down regulated), TAOK1, KHSRP, HSD17B11 and PAH were target genes. In conclusion, the pathway and GO ontology enriched by DEGs may reveal the molecular mechanism of CAD. Its hub and target genes, MYC, NPM1, TRPC7, UBC, FN1, HEMK1, IFT74, VHL, TAOK1, KHSRP, HSD17B11 and PAH were expected to be new targets for CAD. Our finding provided clues for exploring molecular mechanism and developing new prognostics, diagnostic and therapeutic strategies for CAD.

Regulatory phosphorylation of the Cdc2p kinase by Wee1p-type kinases prevents eukaryotic cells from entering mitosis or meiosis at an inappropriate time. The canonical Wee1p kinase is a soluble protein that functions in the eukaryotic nucleus. All metazoa also have a membrane-associated Wee1p-like kinase named Myt1, and we describe the first genetic characterization of this less well-studied kinase. The Caenorhabditis elegans Myt1 ortholog is encoded by the wee-1.3 gene, and six dominant missense mutants prevent primary spermatocytes from entering M phase but do not affect either oocyte meiosis or any mitotic division. These six dominantwee-1.3(gf) mutations are located in a four amino acid region near the C terminus and they cause self-sterility of hermaphrodites. Second-site intragenic suppressor mutations in wee-1.3(gf) restore self-fertility to these dominant sterile hermaphrodites, permitting genetic dissection of this kinase. Ten intragenic wee-1.3 suppressor mutations were recovered and they form an allelic series that includes semi-dominant,hypomorphic and null mutations. These mutants reveal that WEE-1.3 protein is required for embryonic development, germline proliferation and initiation of meiosis during spermatogenesis. This suggests that a novel, sperm-specific pathway negatively regulates WEE-1.3 to allow the G2/M transition of male meiosis I, and that dominant wee-1.3 mutants prevent this negative regulation.

H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Here we used genetic studies to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But our finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, we discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDC’s role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, our findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming.

Abstract Abstract 149 Background. There are few established factors that predict risk of follicular lymphoma (FL) transformation, and only two studies have evaluated the role of germline genetic variation and risk of transformation. One study reported an association of a BCL6 SNP (397C>G) with risk of transformation, and a second study reported an association with rs6457327, which is in the HLA region of 6p21. This study evaluated the risk of FL transformation in the modern treatment era with germline SNPs that were previously reported to be associated with FL transformation (BCL6SNPs and rs6457327), along with SNPs (N=45) previously associated with either risk of FL or FL prognosis. Methods. Newly diagnosed FL patients were prospectively enrolled from 2002–2009 into the Molecular Epidemiology Resource of the University of Iowa/Mayo Clinic Lymphoma SPORE. Peripheral blood for genotyping was collected at enrollment, and clinical and treatment data were abstracted from medical records. All patients were actively followed for disease progression, retreatment, transformation and death. For this analysis, only FL patients with grade I-IIIa were included. Patients with composite DLBCL, FL grade IIIb, or other evidence of transformation at diagnosis were excluded. Transformation was based on either 1) biopsy confirmation of FL grade IIIb, DLBCL, or higher grade B-cell lymphoma; or 2) clinical indicators of transformation based on the published literature, including sudden rise in LDH, rapid discordant localized nodal growth, new involvement of unusual extranodal sites, new B-symptoms, or hypercalcemia. Genotyping was conducted using a custom Illumina iSelect panel. Cox regression was used to estimate Hazard Ratios (HRs) and 95% confidence intervals (CI) for individual SNPs with time to transformation, with death modeled as a competing risk. Each SNP was modeled with the most prevalent homozygous genotype used as the reference group, and each SNP was modeled as having a log-additive (per minor allele) effect in the regression model. An ordinal test was used to assess the trend, and p<0.05 was considered statistically significant. Results. There were 631 newly diagnosed grade I-IIIa FL patients, of whom 532 had DNA and were successfully genotyped. The median age at diagnosis was 60 years (range 23–93) and 53% were male. FLIPI distribution was 42% 0–1, 34% 2 and 24% 3–5. Initial therapies included observation (32%), rituximab (R) monotherapy (13%), alkylator-based chemotherapy +/− R (20%), and anthracycline-based chemotherapy +/− R (19%). At a median of 59 months of follow-up (range, 11–110), 58 patients died, 254 had an event (death, progression or retreatment) and 54 patients (10%) transformed. Transformation was biopsy proven in 43 of the 54 patients (80%). The SNP rs6457327 on chromosome 6p that was previously reported to be associated with transformation was positively associated with risk of transformation in our data (HR=1.40; 95%CI 0.97–2.04; p=0.07), although this association was weaker but still consistent with the prior report. Further, we evaluated additional typed SNPs from the HLA region of 6p, and found additional associations (p<0.05) with HLA-DRA (3 SNPs), HLA-DQA2 (2 SNPs), HLA-DOA (2 SNPs) and HLA-DPB1/HLA-DPA2 (2 SNPs), of which rs10484569 from HLA-DPB1/HLA-DPA2 was the most strongly associated with risk of transformation (HR=3.01; 95%CI 1.48–6.09; p=0.002). None of the BCL6 or other previously reported risk SNPs were associated with transformation. Of the SNPs previously associated with FL prognosis, only the FCGR2A rs1801274 (HR=1.41; 95%CI 0.94–2.10; p=0.09) and CD46 (CD46 molecule, complement regulatory protein) rs2466571 (HR=1.72; 95%CI 1.16–2.55; p=0.007) were associated with risk of transformation. For CD46, one additional SNP (rs2796269) was also associated with transformation (HR=0.61; 95%CI 0.40–0.95; p=0.03). Further adjustment for FLIPI score and treatment (observation versus all other) did not alter these associations. Conclusions. The previously reported association of rs6457327 with risk of transformation in the modern treatment era was replicated. In addition, several HLA loci and two FL prognosis SNPs were linked to transformation risk. The remaining published FL risk or prognosis SNPs were not associated with transformation. Our results support a role for germline genetic variation in the HLA region, FCRGR2A and CD46 in transformation of FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Extreme temperature conditions seriously impair male reproductive development in plants; however, the molecular mechanisms underlying the response of anthers to extreme temperatures remain poorly described. The transcription factor phytochrome-interacting factor4 (PIF4) acts as a hub that integrates multiple signaling pathways to regulate thermosensory growth and architectural adaptation in plants. Here, we report that SlPIF4 in tomato (Solanum lycopersicum) plays a pivotal role in regulating cold tolerance in anthers. CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9-generated SlPIF4 knockout mutants showed enhanced cold tolerance in pollen due to reduced temperature sensitivity of the tapetum, while overexpressing SlPIF4 conferred pollen abortion by delaying tapetal programmed cell death (PCD). SlPIF4 directly interacts with SlDYT1, a direct upstream regulator of SlTDF1, both of which (SlDYT1 and SlTDF1) play important roles in regulating tapetum development and tapetal PCD. Moderately low temperature (MLT) promotes the transcriptional activation of SlTDF1 by the SlPIF4–SlDYT1 complex, resulting in pollen abortion, while knocking out SlPIF4 blocked the MLT-induced activation of SlTDF1. Furthermore, SlPIF4 directly binds to the canonical E-box sequence in the SlDYT1 promoter. Collectively, these findings suggest that SlPIF4 negatively regulates cold tolerance in anthers by directly interacting with the tapetal regulatory module in a temperature-dependent manner. Our results shed light on the molecular mechanisms underlying the adaptation of anthers to low temperatures.

Background Aedes albopictus originated in the tropical forests of Southeast Asia and can currently be found on all continents. As one of the main arboviral vectors, the control of Ae. albopictus requires novel strategies, informed by a deep knowledge of its biology. Little is known regarding mosquito long noncoding RNAs (lncRNAs), which are transcripts longer than 200 nucleotides that lack protein-coding potential and have roles in developmental regulation. Results Based on RNA-seq data from five developmental time points, eggs, early larvae, late larvae, pupae, and adults (female and male) of Ae. albopictus, 21,414 lncRNAs were characterized in this study. Differential expression analysis revealed that lncRNAs exhibited developmental stage specificity. The expression of most lncRNAs was upregulated at the onset of metamorphosis developmental stages. More differentially expressed lncRNAs were observed between eggs and early larvae. Weighted gene co-expression network analysis (WGCNA) further confirmed that the expression patterns of lncRNAs were obviously correlated with specific developmental time points. Functional annotation using co-expression analysis revealed that lncRNAs may be involved in the regulation of metamorphic developmental transitions of Ae. albopictus. The hub lncRNAs and hub gene clusters were identified for each module that were highly associated with specific developmental time points. Conclusions The results of this study will facilitate future researches to elucidate the regulatory mechanisms of lncRNAs in the development of Ae. albopictus and utilize lncRNAs to assist with mosquito control.

Abstract In the past few decades, higher rates of obesity have coincided with a rising number of individuals affected by allergic disease. Our study objective was to determine if obesity alters the immune cell landscape of the small intestine towards an inflammatory environment that aggravates the development of food allergy. Obesity was induced by feeding male BALB/c mice a western diet for 8 weeks. To induce food allergy, mice were epicutaneously sensitized and orally-challenged with OVA. Compared with lean controls, sensitized obese mice showed a lower body temperature after challenge and increased OVA-specific IgE in serum, suggesting an exacerbated allergic response. Mass cytometry analysis of the intestinal lamina propria revealed that OVA-sensitized obese mice had decreased T regulatory cells, which are critical for the oral tolerance to food allergens. In the mesenteric lymph node, obese mice showed increased frequencies of T follicular helper cells (Tfh) compared with lean controls. Tfh cells are required for the production of allergen-specific IgE, and implicated in allergic disease pathogenesis. To further define the transcriptional mechanisms underpinning the increased inflammatory and allergic responses during obesity, we performed single-cell RNA sequencing of more than 28,000 barcoded CD45+ enriched lamina propria cells from lean and obese mice, with and without OVA sensitization. We provide a transcriptional map of inflammatory cells and define a module of gene expression that distinguishes immune cell-driven allergic responses in the obese intestine. Overall, our findings highlight obesity-induced inflammatory changes in the intestine that may mediate the increased food allergic sensitization during obesity.

Introduction: Patients with leukemic indolent B-cell Non-Hodgkin Lymphomas (LI B-NHL) that do not easily fall into a distinct pathological category with standard diagnostics can pose a challenge as their optimal management is uncertain. Lack of a clear diagnostic label can deny patients access to novel therapies as regulatory approval of drugs is largely granted within histological categories. Moreover, these patients may be excluded from participation in clinical trials. We report findings from a prospective study evaluating targeted next generation sequencing (NGS) of circulating malignant B-cells in this setting. Methods: Over a period of 4 years, 108 patients with LI B-NHL, deemed unclassifiable on standard peripheral blood (PB) diagnostics, were prospectively enrolled from 14 centres around the UK including ours. Patients with non-clonal lymphocytosis, confirmed CLL-type MBL, CLL (Matutes score 4 or 5), MCL, HCL, high grade lymphoma on standard PB diagnostics were excluded. Clinical and morphological characteristics were analysed. PB immunophenotyping was used for characterisation including ROR1 expression. CD15+ cells were positively selected with magnetic beads for germline DNA. Tumor-germline pairs were analysed with theEuroclonality-NGS DNA capture panel, and ARResT/Interrogate and complementary pipelines, to characterise clonal immunoglobulin rearrangements, translocations and somatic mutations (Stewart et al. ASH annual meeting 2019). Germline variants were subtracted to improve somatic mutation detection. Results: Median age was 72yrs with M:F ratio of 2:1. Incidental lymphocytosis on a routine blood count was the commonest mode of presentation with only a third of patients symptomatic at presentation. Median lymphocyte count was 12.4 x 109/L at the time of sampling. Just under 50% of cases had partial or complete CD5 expression, CD19 and 20 expression was moderate to strong in most cases while ROR1 was negative in most cases. NGS was able to assign a diagnostic category in 11/90 (12%) cases based on detection of IGH;CCND1 (6), IGH;BCL2 (2), IGH;BCL3 (2) translocations and in one case a BRAF mutation with confirmation of hairy cell leukaemia on immuno-morphology. These tests were not triggered by standard diagnostic pathways at initial presentation. Two novel translocations of uncertain significance were detected - RAD51B:BIRC3 and IGHMswitch-MICALL2/INTS1. Clonal IGHV-IGHD-IGHJ rearrangements were detected by NGS and/or Sanger sequencing with predominantly IGHV3 and IGHV4 gene usage, IGHV4-34 being the most common with 15 cases. The majority of rearrangements showed somatic hypermutations above 2% and none could be assigned to the 19 major CLL-associated stereotyped subsets (using ARResT/AssignSubsets). Somatic mutations were detected in 74/108 cases. The most frequently mutated genes were TP53 in 16/108 (15%) and the hotspot MYD88 L265P mutation in 14/108 (13%) with VAF of 20-46%. Other variants detected were distributed among genes associated with NFkB signalling and chromatin remodelling (Figure 1). The MYD88 L265P mutation was present in a fifth of cases with mutations detected (14/74) and was the sole change seen in 7 cases. Accompanying mutations in the remaining cases included CD79B (2), POT1 (1), NFKBIE (1). No concomitant CXCR4 mutations were detected. Median age of the MYD88 L265P cohort was 68 years with male predominance (M:F=10:4). 9/14 presented with an incidental lymphocytosis and median count of 11.18 x 109/L. Lymphadenopathy was present in 3/14 (21%) while 7/14 (50%) had splenomegaly. A paraprotein was detected in 6/14 cases (4 IgM, 1 IgG, 1 IgA). CD5 was expressed in 6/14 cases by flow cytometry. CD19 and 20 were uniformly positive, CD79b was variable and 12/14 cases tested did not express ROR1. Conclusion: This prospective study outlines the value of a targeted NGS panel in enhancing the diagnosis of unclassifiable LI B-NHL thereby improving patient access to novel therapies and clinical trials. It highlights recurrent MYD88 mutations in a subset of patients that have splenomegaly as a frequent feature. 5 year clinical follow up data, currently being gathered in both the MYD88 mutated and overall cohort, will be valuable in further characterisation and risk stratification to inform management of these patients. Disclosures Furtado: Abbvie: Other: Conference Support. El-Sharkawi:Abbvie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Innate: Consultancy. Iyengar:Abbvie: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Honoraria; Beigene: Consultancy.

Abstract Abstract 3565 Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, haematologic malignancy. The disease may occur at any age, with strong male predominance. Skin presentation is very common. Histologically, tumour cells constitute a uniform population of round or more pleomorphic medium size elements. Clinically this tumour is very aggressive, with poor outcome in adults (median survival 13 months). Chemotherapy (CT), preferably with acute leukemia-type regimens, is the mainstay of treatment, when possible associated with stem cell transplantation (SCT). Few studies investigated copy number changes by array-based comparative genomic hybridization (array-CGH) and gene expression profiling, highlighting a peculiar set of chromosomal losses involving regulatory genes. The present study focused on 23 cases of BPDCN, that were extensively investigated cytogenetically (array-CGH and SNP6 array) and immunohistochemically. Clinico-pathologic findings and molecular data were analyzed in order to identify prognostic markers and possible clinical subsets. At onset 12 cases had diffuse, 7 multiple non contiguous and 4 localized skin involvement. Cases 16 and 18 were father and his daughter. Specific therapy was administered in 20 cases: 10 ALL-type CT; 4 NHL-type CT; 2 mono-CT with 1 radiotherapy; 2 radiotherapy alone; 2 SCT. Ten patients relapsed, in a median time of 10 months: 9 cases received further CT and 2 underwent allo-SCT. At last follow-up, 11 patients had died of disease and 2 of therapy-related causes. Median overall survival (OS) was 21 months. At last follow-up 10 patients were alive, 6 disease-free (8 to 28 months) and 4 with disease. Immunophenotype was typical (CD4, CD56, BDCA2, Tcl-1, and CD123) in all but three patients: 1 negative both for CD4 and CD56, 1 for CD56 and 1 for CD4. All cases expressed CD45RA, 19 CD68/KP1, 10 CD2 and/or CD7 and, when tested, for BDCA2/CD303. T-cell and B-cell receptor genes were germline with no evidence of EBV infection. We observed chromosomal imbalances in all patients. Loss of CDKN2A locus (9p21.3) occurred in 15/23 patients, and in 5 cases a biallelic loss was found. FISH confirmed 9p21.3 loss in all 5 cases. Of note, patients with biallelic loss of locus 9p21.3, in respect to those with hemizygous loss, had reduced survival probability (hazard ratio 11.98; confidence interval [CI] 1.21–118.96; log-rank test, p=0.0349). Median OS was 20 and 35 months and median time to relapse 11 and 15 months, respectively. Furthermore, loss of RB1 (13q13.1-q14.3) and CDKN1B (12p13.2-p13.1) locus were observed in 13 cases. An additional, yet unreported, cytogenetic alteration was the loss of 7p12 in 5 of our patients. This is the locus of IKZF1 gene, which encodes the transcription factor Ikaros. Of note, biallelic and monoallelic deletions of IKZF1 has been shown to be associated with chemoresistance and very poor outcome in a subset of BCR-ABL1 positive acute lymphoblastic leukemias. Array-CGH and SNP6 in our series confirmed that BPDCN is characterized by a complex pattern of genomic losses, predominantly affecting chromosomes 9 (71%), 13 (61%), 12 (57%), 5 (19%), 7 (19%), 14 (14%) and 15 (14%). In conclusion, this is the largest reported series of BPDCN patients, confirming high aggressiveness of the disease. We found negative correlation between age and time to relapse (p=0.0053) and a lower rate of bone marrow involvement in the group of patients with localized skin disease (2 of whom in CR after local skin radiotherapy). Furthermore, we observed absence of CD4 and/or CD56 expression in 3/23 cases. We are the first to report reduced OS probability among patients with homozygous loss of locus 9p21.3 in comparison to those with hemizygous loss, and the possible loss of 7p12-IKZF1 gene in BPDCN. Disclosures: No relevant conflicts of interest to declare.

Introduction: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by macrocytic anemia, reticulocytopenia, selective deficiency of erythroid precursors, presence of congenital anomalies and an increased risk of cancer. DBA is caused by germline mutations of genes coding for ribosomal proteins (RP). Interestingly, somatic RP mutations have also been found in several malignant diseases (T-ALL, CLL, Hodgkin lymphoma, myelodysplastic syndrome (MDS) and AML). The only representative overview of malignancies in DBA was published in 2012 based on the data from the North American DBA Registry. The malignancies developed in 3% of patients (MDS and AML in 4 patients and solid tumors in 15 patients). The observed-to-expected ratio for all cancers combined was 5.4 (p<0.05). Median age at diagnosis was 41 years and cumulative incidence of solid tumors/leukemias was approximately 20% at the age of 46 years. Five years later, additional solid tumors were reported, in particular gastrointestinal tumors, and an incidence of MDS was alarming. Our aim was to evaluate cancer incidence within Czech National DBA Registry and characterize underlying molecular pathology. Patients and methods: Czech National DBA Registry currently includes 62 patients. In cooperation with 8 national centers of pediatric and adult hematology, we collected data about patients followed with pre-malignant or malignant condition including a detailed analysis of the course of the disease, treatment and type of developed malignant disorder. In patients with an evolving MDS, a classical cytogenetic analysis, flow cytometry, mutational profile (TruSight Myeloid Sequencing Panel (Illumina) containing 54 genes) and commercial TUNEL assay for detection of apoptotic changes of erythroid cells in the bone marrow were performed. Results: Eight of 62 patients from the Registry (13%) had malignant or pre-malignant condition: four females (6.5%) had solid tumors, 3 males (4.8%) had MDS and one female (1.6%) had multiple myeloma. Age of the onset of these disorders ranged between 25-70 years. Three patients harbored RPL5 mutation, 3 patients RPL11 mutation and 2 patients RPS19 mutation. Cancer incidence was significantly higher within the RPL11 and RPL5 subgroups (p=0.0056, Fisher Exact Test). Two female patients were diagnosed with triple-negative breast carcinoma, both of them during pregnancy. The first patient died despite treatment at the age of 29 years, shortly after delivery of her second child. The second patient is currently undergoing neoadjuvant chemotherapy.One patient developed diffuse large B cell lymphoma (DLBCL), underwent chemotherapy and autologous BMT and is alive in second remission.One patient underwent hemicolectomy for colorectal adenocarcinoma at 53 years of age and is in remission 6 years after the surgery.One patient succumbed to multiple myeloma which evolved from monoclonal gammopathy of unknown significance (MGUS) after 14 years of follow-up.Three male patients developed suspected MDS at the age of 25, 28 and 29 years. Two of them had RPL5 mutation and one harbored RPS19 mutation. None of them had decrease of erythroid cells in the bone marrow, but apoptosis of erythroid progenitors was significantly increased in all cases. All 3 patients had bicytopenia in peripheral blood (anemia, leukopenia) and dysplasia in 2 or 3 hematopoietic lineages in the bone marrow, thus fulfilling criteria for MDS with multilineage dysplasia (MDS-MLD). They had no abnormalities detected by flow cytometry or cytogenetics. The patient with RPS19mut harbored ASXL1 mutation; in patient with RPL5 mut, mutational screening is ongoing. Conclusion: Our results confirmed increased incidence of cancer (13%) in patients with DBA at young age. Our cases of DLBCL and MGUS in DBA are the first published to date. In cases with suspected MDS, cytopenia and dysplastic changes in the bone marrow may either reflect specific features of ribosomopathy, or represent a severe disorder of regulatory mechanisms with propensity to clonal proliferation. International collaboration is required to refine the incidence of malignancies in DBA and to issue consensus guidelines for timely detection of solid tumors, leukemias and MDS. The best approach to DBA patients who developed MDS without harboring any mutation considered prognostically significant in MDS, is another subject of discussion. Support: NV16-32105A Disclosures No relevant conflicts of interest to declare.

The quantity of municipal solid waste (MSW) generation is escalating at an alarming rate with every passing year alongside the modernization of our economy. Unfortunately, the majority of this waste remains uncollected or ends up in open dumping and followed by uncontrolled burning. Citing the deep-rooted consequences, open dumping should be absolutely abandoned and scientific interventions should be aggressively exercised to reclaim the municipal brownfields. The present research work undertook the judicial task of assessing the comparative feasibility of biomining and scientific capping as a technology selection for reclamation of about a decade old 120 million tons of waste chunk laying at Jawahar Nagar dump yard. Primary dump samples were collected from various locations, considering depth as a variable. While leachate and groundwater samples were collected from Malkaram lake and preinstalled borewells receptively. Additionally, the ambient air quality and noise level also been ascertained within the buffer zone. The blended representative solid sample was segregated using a 70 mm mesh size trommel into organic and inorganic fractions. The organic fraction was composted using a lab-scale aerobic static pile composting (ASPC) while the trommel reject was processed as refuse derived fuel (RDF). Evidently, the compost lagged quality and depicted nutrient deficiency. While the burning of RDF produced siloxane gas, significantly due to elevated silicon level in the primary waste. Furthermore, due to the prolonged leaching tenure and seasonal dilution, the concentration of legacy leachate was relatively weaker. Borewell samples collected from a depth of 20 feet also portrayed minor contamination up to 500 meters horizontal radius. The issue of leachability can solely be resolved with the capping of the existing dump and the end product quality derived from the biomining process is highly questionable. Thus, handling such large quantity capping is a befitting option over biomining for Jawahar Nagar dumpsite. INTRODUCTION Presently, in India due to rapid urbanization and industrialization, the generation of MSW has been increasing tremendously and also expected to continue a similar trend in the future (Scott, 1995; Bhat et al., 2017; Sethurajan et al., 2018; Sharma et al., 2018). Annually, the comprehensive urban MSW generation in India is more than 62 million tons. Metro cities are the mammoth contributor of the entire chunk and waste production had already reached an alarming figure of 50,000 tonnes/day. While the waste generation from the tier 2 cities is also rigorously escalating and presently contribute up to 20,000 tones/day (Sharma et al., 2018). A study conducted by the central pollution control board (CPCB) revealed MSW generation in India is increasing at a distressing rate of 5 % per annum with a sharp escalation in the quantities of domestic hazardous waste (Sharma et al., 2018). With major financial constraints, inefficacy of collection, treatment, and disposal incurs further reasons to worry. So far India has miserably failed to set up wholesome source segregation and collection method. Presently, the country spends more than 60% of its annual waste management budget only in collection. Besides, only 20% or less of the collected materials are scientifically handled and treated. Citing the statistics, it is evident that the majority of the MSW is simply gets dumped on the low laying grounds located somewhere on the outskirts of the cities. The precipitation, infiltration, surface water runoff, bird menace, rodent interference etc. triggers the vulnerability of waste and leads to mal odor, ground and surface water contamination, human and environmental health deterioration (Jayawardhana et al., 2016). Further, the perseverance of the inorganic and inert fractions leads to soil contamination, poses a fire threat, and also may incur carcinogenicity and acute toxicity among the animals (Mir et al., 2021). There are numerous techniques for the reclamation and remediation of the dumpsites, includes processes such as capping and closure, in-situ vitrification, sub-surface cut-off walls, and waste biomining (Chakrabarti and Dubey, 2015; Thakare and Nandi, 2016). Waste biomining is a stable way to get rid of the entire range of problems associated with open dumping and reclaim valuable land (Kaksonen et al., 2017). There are several instances including reclamation of Mumbai Gorai dump yard by IL & FS Environment, 70 – 80 years old 12,00,000 tons of dump clearance by Nagar Nigam Indore within a minute span of 3 years and many more. But the process of biomining is highly sensitive and case-specific. The success of the process solely depends on factors such as characteristics of the waste, efficacy of the effective microorganism culture, acceptability of the processed end product at the local market etc. (Jerez, 2017; Banerjee et al., 2017; Venkiteela, 2020). Contrarily, though the scientific capping is not an end-to-end solution but still advisable in the cases where the quantity of waste is gigantic, land scarcity is prevalent, no nearby industries to consume the end products etc. Mehta et al. (2018) have also supported the above claim based on the assessment of locations specific MSW dump reclamation case studies. While in another Nagpur-based case study conducted by Ashootosh et al. (2020) reported the superiority of the biominingprocess over simple land capping due to the favorability of the local conditions. Capping eliminates the environmental interference and thereby reduces biosphere contamination and leachate generation. Further, it captivates rodent and vector breeding and thereby curtails the spreading of communicable diseases and improves aesthetics. But right consolidation through compaction and execution is utmost necessary in the above case. As non-compaction and faulty sloping will easily lead to heavy settlement and slope failure (Berkun et al., 2005; Al-Ghouti et al., 2021). The present study has been pursued with the primary objective to run a techno-commercial assessment between scientific capping and biomining. While the secondary objective was to ascertain the level of contamination and propose mitigative measures. MATERIALS AND METHODStudy Area Spanning over 350 acres of a precious piece of land at the outskirts of Hyderabad city, Jawahar Nagar dumping yard was brutally utilized by the Greater Hyderabad Municipal Corporation (GHMC) for open dumping for a prolonged tenure of 10 years. It housed nearly 12 lakh metric tons of heterogeneous solid and domestic hazardous waste and continues polluting until 2015, until the Ramky group was offered to cap the legacy dumping and scientifically handle the site. The present study has been facilitated at Hyderabad Municipal Solid Waste Limited, formerly known as Jawahar Nagar dump yard to analyze and assess the feasibility of bio-mining as handling and management alternate to the existing practice of scientific capping. The epicenter of processing and disposal facility is lying approximately on the cross-section of 17°31'24.45"N and 78°35'23.37"E. As per the contract, the comprehensive legacy dumping to be capped in three phases over about 150 acres of area and Ramky has significantly entered the phase two of the operation only within a span of five years by successfully capping more than half of the legacy footprint. Sampling Methodology The waste pile was divided into three layers namely, base, middle, and top. A uniform amount of sample was collected from the successive layers of all five different corners which cover north, south, east, west, and central of the garbage pile. Sampling inspections were performed using a manual auger besides large samples were collected using a JCB excavator. The top six-inch layer of the pile was removed to avoid any contamination while collecting the samples and 5-10 kg of sample was collected from each of the locations. Further, intermediate and bottom layer samples were collected by digging a 500 mm diameter hole through the heap. A composite was prepared by a homogenized blending of all the fifteen grub samples. The blend was distributed into four equal quadrants and the top and bottom quadrants were eliminated diagonally while the left-over quadrants were mixed thoroughly. This process was repeated until a sample of the required bulk of 20 kg is obtained. Surface and subsurface water samples from borewell were collected in and around the facility. Piezometric monitoring borewells located near the landfills were utilized for the subsurface sample collection. While a rainwater pond turned leachate lake named Malkaram was determined as the primary source for leachate collection. Buffer samples were collected from Ambedkar Nagar, the nearby colony exiting at a distance of only 300 meters. Lab-scale Experimentation The representative sample was characterized for composition and further screened through a 70 mm mesh size trommel. The trommel permeate was considered as the organic fraction while the reject was mostly inorganics and inert. The organics were subjected to ASPC. The quantity of the air required is arrived using the method delineated below (Figure 1). MSW Pile size: 2m x 0.5m x 0.5m Volume of pile: 0.5 m3 Average Density of MSW: 620 Kg/m3 Weight of pile: 310 Kg Nitrogen required for matured compost: 9300 mg/kg dry : 9300 X 310 mg : 2.88 x 106 mg : 2.88 Kg Total air required: 2.88 x 100/76 [as Nitrogen in air is 76% by weight] : 3.79 Kg of dry air : 3.79/1.225 m3 [@ 15 deg C density of air 1.225 kg/m3] : 3.1 m3 This air is to be supplied for 100 min / day for 0.5 m pile Air flow rate required: 3.1 x 60/100 = 1.86 m3/h (for practical purpose a flowrate of 2 m3/h was maintained). The maturation period was considered as 28 days and post-maturation, the stabilized material was further cured for 24 hours and screened using 12 mm and 4 mm trommel respectively to obtain the desired product quality and particle size. Whereas, the trommel reject was evenly spreader on the copper trays and dried in an oven at 1050C for 2 hours. The dried material was micronized to the size of 50 mm or below using a scissor and inert such as glass, sand, stone etc. were segregated manually (Mohan and Joseph, 2020). Concurrently, a bench-scale capped landfill prototype was built using the below-mentioned procedure to evaluate the factors such as settlement and slope stability. A 30 mm thick low permeable soil was laid on the top of the waste, followed by a 60 mm layer of compacted clay liner (CCL). Each join between successive liner material was closely monitored. A 1.5 mm thick HDPE liner was placed on the top of the CCL. A 285 GSM geotextile membrane was placed as the successive above layer followed by a 15 mm thick drainage media layer. A further layer of geotextile membrane was placed on top of the drainage media for better stabilization, grip, and strength. The top vegetative soil layer of 45 mm thickness was laid off on top of the geotextile media and St. Augustine grass was rooted (Cortellazzo et al., 2020; Ashford et al., 2000). 2.4 Sample Analysis pH, Electrical Conductivity (EC) and Turbidity of the samples were analyzed using pH, EC-TDS, and Nephelometer of Mettler Toledo. The pH meter was calibrated with the buffer solution of 4.0, 7.0 & 9.12 at a controlled temperature. EC-TDS meter was calibrated with 0.1 M KCL having 12.8 mS/cm of conductivity. Nephelometer was calibrated with Formazine solution of 10 & 100 NTU. Total Dissolved Solids (TDS), (mg/L) was performed using the gravimetric method at 1800C in the oven. Titrimetric parameters such as Total Alkalinity as CaCO3 (mg/L), Total Hardness as CaCO3 (mg/L), Chloride as Cl- (mg/L), Calcium as Ca2+ (mg/L), Residual Free Chlorine (RFC), (mg/L) were analyzed using APHA (American Public Health Associations) method, 23rd Edition, 2017. Total Kjeldahl Nitrogen (mg/L) and Ammonical Nitrogen (mg/L) were performed through distillation followed by titration with H2SO4 as a titrant. Sulphide as S2- was done with the Iodometric method after distillation. Each titrimetric parameter was analyzed in triplicate after standardizing the titrant with required reagents and crossed checked by keeping a check standard. Sodium as Na (mg/L) and Potassium as K (mg/L) were performed using Flame Photometer. The photometer was calibrated with different standards from 10 to 100 (mg/L) standard solutions. The leachate sample was diluted enough to get the value within the standard range and cross-checked with check standards at the same time. Chemical Oxygen Demand (COD), (mg/L) was performed using the open reflux method for 2 hours at 1500C in COD Digestor. Biochemical Oxygen Demand (BOD), (mg/L) was performed using the alkali iodide azide method for 3 days. The samples were kept in a BOD incubator at 270C for 3 days. It was kept in duplicate to have a check on quality control. Sulphate was analyzed by the gravimetric method instead of turbidimetric or through UV-Visible spectrophotometer as its concentration was found more than 40 mg/L. Nitrate as NO3- was analyzed after filtration at 220-275 nm, while Hexavalent Chromium as Cr6+ was analyzed at 540 nm in the UV-Vis. Parameters like Cyanide as CN-, Fluoride as F-, and Phenolic Compounds were gone through a distillation process followed by UV-Vis. The distillation process ensures the removal of interferences presents either positive or negative. For the parameters like Total Iron or Ferric Iron, the samples were digested properly with the required reagents on the hot plate before analyzing in UV-Vis. For the metal analysis the water samples were digested at a temperature of 1000C using aqua regia as a media. The samples were digested to one-fourth of the volume on a hot plate. The recommended wavelengths as per APHA 3120 B were selected for each of the metals. The standard graph was plotted for each of the metals before analysis and crossed checked with the check standard at the same time. Parameters such as bulk density and particle size were performed through the certified beaker and sieve. The percentage of moisture content was estimated using the oven by keeping the compost sample for 2 hours at 1050C. C/N ratio was estimated through CHNS analyzer keeping sulfanilamide as a check standard. The analysis was performed by extracting the desired component in the desired solution prescribed in the method followed by converting the same from mg/L to mg/Kg. RESULTS AND DISCUSSION An exhaustive bench-study has been pursued and real-time samples were collected and analyzed for all possible parameters to determine the pros and cons attributed to both processes. The investigation begins by collecting the samples and concluded by impact assessment studies inclusive of the buffer zone. Both solid, liquid, and gaseous samples were precisely investigated to opt for the best solution. A detailed finding of the investigation is summarized below. Primarily, the representative solid sample was characterized through a manual separation process and the results are portrayed in Figure 1. Compost Characterization ASPC of the organic fraction has resulted in a recovery of 46.7% of the initial load. While 53.3% of the influent mass were inert and barely degradable fraction contributes to reject, the rest 4.1% is miscellaneous process loss. The processed compost was extensively analyzed including for metal contamination and the same is tabulated in Table 1. The value of C/N ratio, OC, TN, K2O, P2O5, and NPK evidently portrays the shortcoming in terms of nutrient availability. Though it is highly enriched in organic carbon and thus the same can be effectively utilized as a soil preconditioner. Ayilara et al. (2020) also reported a similar finding, where the city compost sourced from MSW lagged major plant nutrients. RDF Characterization Processed trommel rejects constitute cloth, rexine, leather, jute, paper, plastics, coir and other inert contributed to RDF. The fraction of inert was as high as 37.2% of the overall RDF mass and it mostly constituted glass and sand. The combined weight of sand and glass fragments contributed 73.5% of the total inert, while the rest was stone and small brickbats. The higher level of silicon associated with the presence of glass and sand yielded siloxane and triggered the possibility of kiln corrosion. A detailed RDF analysis report is enclosed in Table 2. The values explicitly portray the quality of RDF is moderately lower and higher salts concentration is extremely prevalent. With relatively lower NCV and such high salt concentration, the above specimen will certainly pose a corrosion threat to the kiln and shall be either neglected as kiln feed or can be utilized after dilution with Grade III RDF quality. Further, such high ash generation will also induct high transportation and landfill charges. Leachate Characterization The Malkaram leachate lake is the end result of prolonged, slow, and steady mixing of the legacy leachate through the existing fissure cracks in the sheath rock bottom profile. Apparently, the concentration of leachate is significantly lower due to the dilution. Samples were analyzed in triplicates and the mean value is tabulated here in Table 3. The metal concertation and rest of the parameter values are well within the secondary treatment influent range, except for TDS. Thus, a modular aerobic biological treatment unit such as moving bed biofilm bioreactor (MBBR) or membrane bioreactor (MBR) would be a well-suited pick. However, a reverse osmosis (RO) system needs to be installed to get rid of the high TDS content. The permeate of RO can be reused back into the system. Whereas, the reject can be converted into dried powder through forced evaporation mechanisms. The higher concentration of salts in RDF collaterally justifies the elevated TDS level in leachate. In a leachate impact assessment study performed by El-Salam and Abu-Zuid (2015) the reported BOD/COD ratio of 0.69 is greater than double the value of 0.301 reported in Table 3. Though the difference in both the values are quite high, it is relatable and justifiable by the huge age difference of the source waste. The primarily characterized data is of a fresh leachate generated from regular MSW, while the later one is from a decade old waste that barely has any unstabilized organic content. Groundwater Contamination The obvious reason for downward leachate infiltration and osmotic movement facilitates groundwater contamination. Both surface and subsurface water samples were collected within the dump yard and the buffer zone and analyzed using the standard methods. The results are portrayed in Table 4. The slightly alkaline pH of the borewell sample is an indication of the ongoing anaerobic process. The dissolved oxygen value of 3.5 mg/L further validates the correlation. Higher TDS and hardness values are self-indicative of elevated salt concentration in source waste. Eventually, the same interfered with the RDF quality. Positively in the case of all the parameters, a successive decrement in pollution concentration has been spotted from dump ground towards the buffer zone. In a similar study conducted by Singh et al. (2016) at Varanasi, Uttar Pradesh the reported concentration of the parameters is significantly higher than reported in Table 4. The basic reason behind variation is the dissimilarities of the local soil profile. The sandy and clay loam soil profile of Varanasi allows a greater rate of percolation and infiltration. While the bottom sheath rock profile at Jawahar Nagar permits the only a minute to little percolation rate. The difference in percolation rate is directly correlated to the concentration levels in this case. Contrarily, Kurakalva et al. (2016) have reported much-elevated pollutant concertation both in ground and surface water for a study conducted at the same site in 2016. The higher concentration is relatable to the fact of the non-closure of the open dump back then. Capping activity had at Jawahar Nagar gained its pace 2018 onwards and capping for the primary section of 70 acres got concluded only during mid of 2019. Due to the decrement in runoff and percolation, the quality of both surface and subsurface water has improved drastically. Impact Assessment The odor and groundwater contamination are two of the primary issues that triggered a massive public agitation initially. The root causes of both the issues are identified as rainwater percolation and anaerobic digestion respectively. Eventually, the completion of the capping process would resolve both the problems effectively. Other non-tangential impacts include nausea; headache; irritation of the eye, nasal cavity, and throat; diarrhoeal diseases; vector-borne disease, cattle toxicity etc. Scientific capping can easily cater as the wholesome solution for all (Cortellazzo et al., 2020). Yu et al. (2018) had performed an extensive study to comprehend the relativity of respiratory sickness and MSW borne air pollution. The study made a couple of dreadful revelations such as gases released due to the anaerobic digestion of MSW such as methane, hydrogen sulphide, and ammonia incur detrimental impact on Lysozyme and secretory immunoglobulin A (SIgA). While SO2 was reported as the lung capacity and functionality reducer. Further, a gender-specific study executed by the same research group revealed, air pollution impacts more severely on male children than the female and retards immune functions. Presently, the area of 351 acres has been developed as Asia’s one of the largest state of the art municipal solid waste processing and disposal facility by Ramky Enviro Engineers Limited. This ensured zero dumping and no further environmental interventions. As legal compliance, the facility monitors the quality of groundwater and ambient air quality in and around the facility on monthly basis to assure the biosafety. The variation in concentration of various monitoring parameters between 2012 to 2020 is summarized in Figure 2. The concentration of each of the parameters are showcased in ppm and a standard equipment error was settled at 3% for respirable dust sampler and multi-gas analyzer (Taheri et al., 2014). Despite all parameter values have gradually increased except for methane, the facility still managed to maintain them well under the regulatory limits. The decrement in methane concentration is directly correlated to the practice of aerobic composting and aeration-based secondary treatment that prevented the formation of the anaerobic atmosphere and henceforth methane generation. While for the rest of the parameters the increment in values is quite substantial and predictable due to the sudden escalation in MSW generation in the past decade in correlation with Gross domestic product (GDP) enhancement. The observed and interpreted impacts due to the elevated pollutant level are in-line with the georeferenced findings reported by Deshmukh and Aher (2016) based on a study conducted at Sangamner, Maharashtra. CONCLUSION The study critically analyzed and investigated every techno-environmental and socio-economic aspect correlated to open dumping. The bench-scale experimentation revealed the efficiency of the single liner scientific capping is fair enough to eliminate any further rainwater infiltration, however, it has no control over the generation of leachate due to the inherent moisture. Internal moisture related issue was anyhow compensated with pertinent compaction prior to dispose of the waste. 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Retrotransposons are a subset of DNA sequences that constitute a large part of the mammalian genome. They can translocate autonomously or non-autonomously, potentially jeopardizing the heritable germline genome. Retrotransposons coevolved with the host genome, and the germline is the prominent battlefield between retrotransposons and the host genome to maximize their mutual fitness. Host genomes have developed various mechanisms to suppress and control retrotransposons, including DNA methylation, histone modifications, and Piwi-interacting RNA (piRNA), for their own benefit. Thus, rapidly evolved retrotransposons often acquire positive functions, including gene regulation within the germline, conferring reproductive fitness in a species over the course of evolution. The male germline serves as an ideal model to examine the regulation and evolution of retrotransposons, resulting in genomic co-evolution with the host genome. In this review, we summarize and discuss the regulatory mechanisms of retrotransposons, stage-by-stage, during male germ cell development, with a particular focus on mice as an extensively studied mammalian model, highlighting suppression mechanisms and emerging functions of retrotransposons in the male germline.

AbstractNon-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named “variable chromatin modules” (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

AbstractUnderstanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9-binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF1, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (>=5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.