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1

Cribbs, Adam P., Sebastian Luna-Valero, Charlotte George, Ian M. Sudbery, Antonio J. Berlanga-Taylor, Stephen N. Sansom, Tom Smith, et al. "CGAT-core: a python framework for building scalable, reproducible computational biology workflows." F1000Research 8 (April 4, 2019): 377. http://dx.doi.org/10.12688/f1000research.18674.1.

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In the genomics era computational biologists regularly need to process, analyse and integrate large and complex biomedical datasets. Analysis inevitably involves multiple dependent steps, resulting in complex pipelines or workflows, often with several branches. Large data volumes mean that processing needs to be quick and efficient and scientific rigour requires that analysis be consistent and fully reproducible. We have developed CGAT-core, a python package for the rapid construction of complex computational workflows. CGAT-core seamlessly handles parallelisation across high performance computing clusters, integration of Conda environments, full parameterisation, database integration and logging. To illustrate our workflow framework, we present a pipeline for the analysis of RNAseq data using pseudo-alignment.
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2

Cribbs, Adam P., Sebastian Luna-Valero, Charlotte George, Ian M. Sudbery, Antonio J. Berlanga-Taylor, Stephen N. Sansom, Tom Smith, et al. "CGAT-core: a python framework for building scalable, reproducible computational biology workflows." F1000Research 8 (July 16, 2019): 377. http://dx.doi.org/10.12688/f1000research.18674.2.

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In the genomics era computational biologists regularly need to process, analyse and integrate large and complex biomedical datasets. Analysis inevitably involves multiple dependent steps, resulting in complex pipelines or workflows, often with several branches. Large data volumes mean that processing needs to be quick and efficient and scientific rigour requires that analysis be consistent and fully reproducible. We have developed CGAT-core, a python package for the rapid construction of complex computational workflows. CGAT-core seamlessly handles parallelisation across high performance computing clusters, integration of Conda environments, full parameterisation, database integration and logging. To illustrate our workflow framework, we present a pipeline for the analysis of RNAseq data using pseudo-alignment.
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3

Portet, Anaïs, Eve Toulza, Ana Lokmer, Camille Huot, David Duval, Richard Galinier, and Benjamin Gourbal. "Experimental Infection of the Biomphalaria glabrata Vector Snail by Schistosoma mansoni Parasites Drives Snail Microbiota Dysbiosis." Microorganisms 9, no. 5 (May 18, 2021): 1084. http://dx.doi.org/10.3390/microorganisms9051084.

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Host-parasite interaction can result in a strong alteration of the host-associated microbiota. This dysbiosis can affect the fitness of the host; can modify pathogen interaction and the outcome of diseases. Biomphalaria glabrata is the snail intermediate host of the trematode Schistosoma mansoni, the agent of human schistosomiasis, causing hundreds of thousands of deaths every year. Here, we present the first study of the snail bacterial microbiota in response to Schistosoma infection. We examined the interplay between B. glabrata, S. mansoni and host microbiota. Snails were infected and the microbiota composition was analysed by 16S rDNA amplicon sequencing approach. We demonstrated that the microbial composition of water did not affect the microbiota composition. Then, we characterised the Biomphalaria bacterial microbiota at the individual scale in both naive and infected snails. Sympatric and allopatric strains of parasites were used for infections and re-infections to analyse the modification or dysbiosis of snail microbiota in different host-parasite co-evolutionary contexts. Concomitantly, using RNAseq, we investigated the link between bacterial microbiota dysbiosis and snail anti-microbial peptide immune response. This work paves the way for a better understanding of snail/schistosome interaction and should have critical consequences in terms of snail control strategies for fighting schistosomiasis disease in the field.
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4

Allen, S. J. W., S. H. Krawczyk, L. R. McGee, N. Bischofberger, A. S. Mulato, and J. M. Cherrington. "Inhibition of HIV-1 RNase H Activity by Nucleotide Dimers and Monomers." Antiviral Chemistry and Chemotherapy 7, no. 1 (February 1996): 37–45. http://dx.doi.org/10.1177/095632029600700107.

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Nucleotide dimers and monomers were shown to inhibit human immunodeficiency virus type 1 (HIV) RNase H activity. Several effective inhibitors were identified and placed into three general groups based on biochemical characterization of their inhibition, The first group (group A) inhibited HIV RNase H and the closely related feline immunodeficiency virus (FIV) RNase H, but did not inhibit less related retroviral or cellular RNases H or HIV reverse transcriptase (RT). The second group (group B) inhibited the RNase H activity of several retroviruses as well as the reverse transcriptase function of HIV RT. The third group (group C) inhibited RNases H from retroviral and cellular sources but did not inhibit HIV RT. Kinetic analyses of HIV RNase H inhibition were conducted and all three types of inhibitors exhibited a competitive mode of inhibition with regard to substrate. The small nucleotides described here represent the most potent (Ki values from 0.57 to 16 μM) and selective inhibitors of HIV RNase H reported to date. Further structure - function analyses of these molecules may lead to the discovery of unique, potent antiretroviral therapeutics.
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5

Ahrenfeldt, Johanne, Ditte S. Christensen, Andreas B. Østergaard, Judit Kisistók, Mateo Sokač, and Nicolai J. Birkbak. "The ratio of adaptive to innate immune cells differs between genders and associates with improved prognosis and response to immunotherapy." PLOS ONE 18, no. 2 (February 6, 2023): e0281375. http://dx.doi.org/10.1371/journal.pone.0281375.

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Immunotherapy has revolutionised cancer treatment. However, not all cancer patients benefit, and current stratification strategies based primarily on PD1 status and mutation burden are far from perfect. We hypothesised that high activation of an innate response relative to the adaptive response may prevent proper tumour neoantigen identification and decrease the specific anticancer response, both in the presence and absence of immunotherapy. To investigate this, we obtained transcriptomic data from three large publicly available cancer datasets, the Cancer Genome Atlas (TCGA), the Hartwig Medical Foundation (HMF), and a recently published cohort of metastatic bladder cancer patients treated with immunotherapy. To analyse immune infiltration into bulk tumours, we developed an RNAseq-based model based on previously published definitions to estimate the overall level of infiltrating innate and adaptive immune cells from bulk tumour RNAseq data. From these, the adaptive-to-innate immune ratio (A/I ratio) was defined. A meta-analysis of 32 cancer types from TCGA overall showed improved overall survival in patients with an A/I ratio above median (Hazard ratio (HR) females 0.73, HR males 0.86, P < 0.05). Of particular interest, we found that the association was different for males and females for eight cancer types, demonstrating a gender bias in the relative balance of the infiltration of innate and adaptive immune cells. For patients with metastatic disease, we found that responders to immunotherapy had a significantly higher A/I ratio than non-responders in HMF (P = 0.036) and a significantly higher ratio in complete responders in a separate metastatic bladder cancer dataset (P = 0.022). Overall, the adaptive-to-innate immune ratio seems to define separate states of immune activation, likely linked to fundamental immunological reactions to cancer. This ratio was associated with improved prognosis and improved response to immunotherapy, demonstrating potential relevance to patient stratification. Furthermore, by demonstrating a significant difference between males and females that associates with response, we highlight an important gender bias which likely has direct clinical relevance.
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6

Orlandi, Elisa, Elisa De Tomi, Rachele Campagnari, Francesca Belpinati, Monica Rodolfo, Elisabetta Vergani, Giovanni Malerba, Macarena Gomez-Lira, Marta Menegazzi, and Maria Romanelli. "Human Melanoma Cells Differentially Express RNASEL/RNase-L and miR-146a-5p under Sex Hormonal Stimulation." Current Issues in Molecular Biology 44, no. 10 (October 11, 2022): 4790–802. http://dx.doi.org/10.3390/cimb44100326.

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Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17β-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3’ untranslated region (3′UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17β-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17β-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17β-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17β-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients.
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7

Oczkowicz, Maria, Małgorzata Świątkiewicz, Katarzyna Ropka-Molik, Artur Gurgul, and Kacper Żukowski. "Effects of Different Sources of Fat in the Diet of Pigs on the Liver Transcriptome Estimated by RNA-Seq." Annals of Animal Science 16, no. 4 (October 1, 2016): 1073–90. http://dx.doi.org/10.1515/aoas-2016-0033.

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Abstract In this study, we have attempted to analyse the impact of dietary fats on the liver transcriptome in pigs. Four nutritional groups were created. The animals’ diets differed among groups in terms of the presence of corn dried distillers’ grains with solubles (DDGS) (group I - no DDGS, groups II, III, IV - 20% DDGS) as well as the type of fat (rapeseed oil - groups I and II, beef tallow - group III, coconut oil - group IV) used. Using the RNA-Seq method we identified 39 differentially expressed genes (DEGs) as a result of Cuffdiff analysis of the differences among all groups. Analysis of these genes with Panther Gene Classification System revealed that among identified DEGs, genes responsible for lipid and fatty acids metabolism are overrepresented as well as the genes engaged in oxidoreductase and catalytic activity. The article presents for the first time the RNAseq analysis of the liver transcriptome in pigs fed with different sources of fats. The results may be useful for the elaboration of new therapies for cardiovascular diseases in humans as well as for the preparation of new nutrition strategies in animals.
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8

Penttinen, Jenni, Dwi Ari Pujianto, Petra Sipilä, Ilpo Huhtaniemi, and Matti Poutanen. "Discovery in Silico and Characterization in Vitro of Novel Genes Exclusively Expressed in the Mouse Epididymis." Molecular Endocrinology 17, no. 11 (November 1, 2003): 2138–51. http://dx.doi.org/10.1210/me.2003-0008.

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Abstract Epididymal proteins interact with sperm during their passage through the epididymis and thus contribute to the maturation and fertilizing capacity of the spermatozoa. In the present study we have discovered five novel epididymis-specific genes through in silico analysis of expressed sequence tags (ESTs) at the UniGene library collection. The strategy used is a powerful way to discover novel epididymis-specific genes. The full-length cDNA sequences were determined, and computational tools were used to characterize the genomic structures and to predict putative functions for the encoded proteins. In vitro analyses revealed that all five genes characterized were highly expressed in the defined areas of the epididymis, and they were not expressed at significant levels in any other tissue. Three of the genes were named on the basis of their putative functions: Spint4 (serine protease inhibitor, Kunitz type 4), and Rnase9 and Rnase10 (ribonuclease, Rnase A family 9 and 10), while for the ESTs AV381130 and AV381126 no putative functions could be predicted. The expression of Spint4, Rnase9, and AV381130 was found to be under a direct or indirect regulation by androgens, while the expression of Rnase10 is regulated by a testicular factor(s) other than androgen. None of the genes were expressed in the immature epididymis, while mRNAs were detected from d 17 onward, at the time of maturation of epididymal epithelium. However, the expression of AV381130 was not detected until d 30 after birth, indicating a close connection between gene expression and puberty.
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9

Malvisi, Michela, Nico Curti, Daniel Remondini, Maria Grazia De Iorio, Fiorentina Palazzo, Gustavo Gandini, Silvia Vitali, Michele Polli, John L. Williams, and Giulietta Minozzi. "Combinatorial Discriminant Analysis Applied to RNAseq Data Reveals a Set of 10 Transcripts as Signatures of Exposure of Cattle to Mycobacterium avium subsp. paratuberculosis." Animals 10, no. 2 (February 5, 2020): 253. http://dx.doi.org/10.3390/ani10020253.

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Paratuberculosis or Johne’s disease in cattle is a chronic granulomatous gastroenteritis caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Paratuberculosis is not treatable; therefore, the early identification and isolation of infected animals is a key point to reduce its incidence. In this paper, we analyse RNAseq experimental data of 5 ELISA-negative cattle exposed to MAP in a positive herd, compared to 5 negative-unexposed controls. The purpose was to find a small set of differentially expressed genes able to discriminate between exposed animals in a preclinical phase from non-exposed controls. Our results identified 10 transcripts that differentiate between ELISA-negative, clinically healthy, and exposed animals belonging to paratuberculosis-positive herds and negative-unexposed animals. Of the 10 transcripts, five (TRPV4, RIC8B, IL5RA, ERF, CDC40) showed significant differential expression between the three groups while the remaining 5 (RDM1, EPHX1, STAU1, TLE1, ASB8) did not show a significant difference in at least one of the pairwise comparisons. When tested in a larger cohort, these findings may contribute to the development of a new diagnostic test for paratuberculosis based on a gene expression signature. Such a diagnostic tool could allow early interventions to reduce the risk of the infection spreading.
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10

Ramanauskas, Karolis, and Boris Igić. "The evolutionary history of plant T2/S-type ribonucleases." PeerJ 5 (September 11, 2017): e3790. http://dx.doi.org/10.7717/peerj.3790.

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A growing number of T2/S-RNases are being discovered in plant genomes. Members of this protein family have a variety of known functions, but the vast majority are still uncharacterized. We present data and analyses of phylogenetic relationships among T2/S-RNases, and pay special attention to the group that contains the female component of the most widespread system of self-incompatibility in flowering plants. The returned emphasis on the initially identified component of this mechanism yields important conjectures about its evolutionary context. First, we find that the clade involved in self-rejection (class III) is found exclusively in core eudicots, while the remaining clades contain members from other vascular plants. Second, certain features, such as intron patterns, isoelectric point, and conserved amino acid regions, help differentiate S-RNases, which are necessary for expression of self-incompatibility, from other T2/S-RNase family members. Third, we devise and present a set of approaches to clarify new S-RNase candidates from existing genome assemblies. We use genomic features to identify putative functional and relictual S-loci in genomes of plants with unknown mechanisms of self-incompatibility. The widespread occurrence of possible relicts suggests that the loss of functional self-incompatibility may leave traces long after the fact, and that this manner of molecular fossil-like data could be an important source of information about the history and distribution of both RNase-based and other mechanisms of self-incompatibility. Finally, we release a public resource intended to aid the search for S-locus RNases, and help provide increasingly detailed information about their taxonomic distribution.
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11

Good-Avila, S. V., D. Majumder, H. Amos, and A. G. Stephenson. "Characterization of self-incompatibility in Campanula rapunculoides (Campanulaceae) through genetic analyses and microscopy." Botany 86, no. 1 (January 2008): 1–13. http://dx.doi.org/10.1139/b07-100.

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In this paper, we seek to identify the genetic basis of self-incompatibility (SI) in Campanula rapunculoides L. through diallel analysis of full siblings; to characterize the growth of pollen tubes in vivo after incompatible and compatible pollination; and to determine whether the SI system is based on pistil S-RNases. Pollinations were performed among individuals from five diallel crosses and scored for both fruit set and pollen-tube growth to determine the genetic basis of SI. On a subset of these individuals with known cross-(in)compatibility relationships, additional crosses were performed and pistils collected 1, 3, 6, 12, and 24 h after pollination to assess both the percentage of pollen grains that had germinated on the stigma, and the number of pollen tubes that had grown 20%, 40% 60%, 80%, and 100% of the distance down the pistil over five time intervals. Finally, total pistillate proteins were extracted and subjected to isoelectric focusing and RNase activity staining to find evidence of a highly basic S-RNases associated with SI in the Solanaceae. We found evidence that the SI system was based on the haplotype of the male gametophyte, and was not sporophytic. Protein analyses showed that SI was not based on a pistillate S-RNase. The existence of modifiers of SI and possible polyploidy at the S-locus complicated the expression of SI in this species, and single-gene inheritance could not be determined. This represents the first published characterization of incompatibility in the family Campanulaceae.
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12

Oden, Élise. "La génomique équine : tour d’horizon des outils disponibles pour les applications actuelles et à venir." Le Nouveau Praticien Vétérinaire équine 17, no. 59 (2023): 48–53. http://dx.doi.org/10.1051/npvequi/2024005.

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Depuis quelques décennies, de nombreux outils technologiques initialement destinés à l’étude de la génomique humaine ont été rapidement déployés chez les animaux d’élevage, dont les chevaux. Tout d’abord, le génotypage permet l’analyse des variations génétiques dans l’ADN génomique d’un organisme : par exemple, les marqueurs microsatellites, séquences répétées présentes partout dans les génomes eucaryotes ou bien les SNP (Single Nucleotide Polymorphism) qui correspondent à des variations d’une seule base nucléotidique. En laboratoire, le génotypage est actuellement utilisé pour la réalisation des contrôles de filiation ou pour la recherche d’un caractère d’intérêt et des maladies monogéniques équines. La technologie de séquençage permet, quant à elle, de déterminer la séquence nucléotidique d’un fragment d’ADN ou d’un génome entier : ainsi, Twilight, premier cheval dont le génome a été entièrement séquencé en 2009. D’autres alternatives au séquençage permettent de mesurer l’expression des gènes dans un tissu donné par une approche de transcriptomique (ou RNAseq), de comprendre la régulation de cette expression génique par des études épigénétiques ou bien de connaître le microbiote d’un échantillon par analyse métagénomique. L’ensemble de ces développements génomiques offre de belles perspectives pour le diagnostic équin de demain grâce à une meilleure connaissance des maladies multifactorielles et la mise en place d’une médecine personnalisée. Ces outils apporteront également des éléments nouveaux aux professionnels de la filière en termes d’élevage ou de sélection ainsi qu’une amélioration de la prédiction des aptitudes au sport des chevaux athlètes.
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13

Yang, Qin, Yan Fu, Yalan Liu, Tingting Zhang, Shu Peng, and Jie Deng. "Microscopic and Transcriptome Analysis Reveals that the Self-incompatibility in Rabbiteye Blueberry Belongs to the S-RNase-based Gametophytic Type." J. Amer. Soc. Hort. Sci. 149, no. 4 (July 2024): 179–94. http://dx.doi.org/10.21273/jashs05364-23.

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Berry fruits produced by Vaccinium (Ericaceae) plants are small but have a signature flavor and have become increasingly popular in the 21st century. However, self-incompatibility (SI) results in a relatively low fruit-set ratio and reduced fruit quality in Vaccinium. In this study, using Vaccinium ashei (V. ashei) styles after cross-pollination (CP) and self-pollination (SP) as material, transcriptomics and gene expression analyses were performed using high-throughput RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, evolutionary analysis and conserved sequences analysis of candidate genes were conducted. Among the 135,324 unigenes, 30,863 were shown to be differentially expressed, and eight randomly selected differentially expressed genes were expressed in the styles at 96 hours after SP and CP. The transcriptomics and qRT-PCR results were significantly correlated, which confirmed the reliability of the differentially expressed genes obtained in our study. Compared with SP96, six differentially expressed ribonuclease T2 family genes were obtained in CP96, which were considered candidates for S-RNase. Additionally, the spatiotemporal and organizational expression trends of six candidates for S-RNase were confirmed by qRT-PCR, and the evolutionary and conservative sequence analysis indicated six candidate S-RNases with the typical S-RNase structure. The spatiotemporal and organizational expression results and evolutionary and conservative sequence analyses of the six candidate S-RNases suggest that SI in V. ashei is likely an S-RNase-mediated gametophytic one. This finding suggests the involvement of novel, previously undiscovered components involved in the V. ashei SI system. These findings help elucidate the molecular mechanisms of SI in rabbiteye blueberry and may also benefit breeding, production, and genomics research in V. ashei and other Vaccinium species.
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14

Shlyakhovenko, V. О., І. І. Ganusevich, О. А. Samoylenko, Yu M. Samchenko, А. V. Verbinenko, and O. A. Solovyova. "HUMAN PERIPHERAL BLOOD RIBONUCLEASES REACTIVATION AFTER SORPTION ON NANOPLATELETS OF LAPONITE®." Oncology 25, no. 4 (2023): 302–5. http://dx.doi.org/10.15407/oncology.2023.04.302.

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Summary. Aim: to investigate the possibility of enzymatic reactivation of RNase activity of peripheral blood cells of patients with colorectal cancer (CRC) after sorption on nanoplates Laponite® RD (Lap). Objects and methods: the study was performed with the cell suspension of peripheral blood of CRC patients. Samples of cell lysates were combined with a 1% suspension of Lap nanoplates. Then RNase was extracted with 0,25 N H2SO4 or 2% solution of sodium dodecyl sulfate (DDS). The zymogram technique was used to analyze RNase activity. Results: it was found that RNases bind with nanoplates Lap and form complexes with loss of enzymatic activity. It is known that RNase can be released from the complex by extraction with 0,25 N H2SO4 or 2% sodium DDS solution. RNase is able to restore its enzymatic activity when extraction from the complex with a 2% sodium DDS solution is used. But with the extraction of 0,25 N H2SO4, the enzymatic activity is irreversibly lost. Conclusion: RNase extracted from the nanoplates Lap can be active again as an enzyme that catalyzes the cleavage of RNA and hybrid RNA/DNA molecules, depending on the method of extraction.
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15

Yasuda, T., D. Nadano, H. Takeshita, and K. Kishi. "Two distinct secretory ribonucleases from human cerebrum: purification, characterization and relationships to other ribonucleases." Biochemical Journal 296, no. 3 (December 15, 1993): 617–25. http://dx.doi.org/10.1042/bj2960617.

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Two RNAases from human cerebrum were purified to an electrophoretically homogeneous state and their molecular masses were 22.0 kDa (tentatively called RNAase HB-1) and 19.0 kDa (RNAase HB-2). Analyses of the amino acid compositions, N-terminal amino acid sequences and catalytic properties of these enzymes provided strong evidence that they were strictly related to the secretory (sec) RNAases, such as the pancreatic enzyme, very similar immunologically to urinary sec RNAase, but clearly distinguishable from urinary non-secretory (nonsec) RNAase. There were several differences between HB-1 and HB-2, namely their immunological reactivities with specific antibodies, heat-stabilities, attached carbohydrate moieties and molecular masses. In particular, HB-2 appeared to be nonglycosylated, in view of its lack of affinity for several conjugated lectins, the absence of hexosamine and no change in electrophoretic mobility before and after peptide:N-glycosidase F digestion, whereas HB-1 and human sec RNAases purified from kidney, pancreas and urine all appeared to be glycosylated, as they moved to the same position as HB-2 when electrophoresed after glycosidase digestion. An antibody against urinary sec RNAase inhibited 75% and 20% of the total activity of the crude cerebral extract against RNA at pH 8.0 and 6.0 respectively, whereas an antibody against urinary nonsec RNAase had no such inhibitory effect. These findings suggest that yet another type(s) of cerebral RNAase, which is unable to cross-react immunologically with sec and nonsec RNAases, may exist. Two RNAases corresponding to HB-1 and HB-2 were identified in fresh cerebrospinal fluid.
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Szymańska, Hanna, Krystyna Życzko, and Tadeusz Zabolewicz. "Relationship between RNASE1, ANG and RNASE6 gene polymorphism and the values of blood indices in suckling piglets." Acta Veterinaria Hungarica 67, no. 3 (September 2019): 385–400. http://dx.doi.org/10.1556/004.2019.039.

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The relationship between PcR-restriction fragment length polymorphism in RNASE1 (296 A/G), ANG (149 G/T) and RNASE6 (389 C/T) genes and the values of haematological and biochemical blood indices was analysed in crossbred suckling piglets (n = 473), aged 21 ± 3 days (younger, n = 274) and 35 ± 3 days (older, n = 199), descending from Polish Large White × Polish Landrace sows and Duroc × Pietrain boars. The observed distribution of all genotypes was consistent with the Hardy-Weinberg equilibrium. Anaemia was more common in younger piglets with RNASE1 GA genotype but in the blood of older GA piglets a higher count and percentage of granulocytes were noted. This could be related to the destruction of erythrocytes in younger piglets and enhanced host defence in older ones. ANG gene polymorphism was associated with the severity of iron deficiency in younger piglets. This is supposed to be linked with the different ability to protect immune cells against suppression and degradation during iron deficiency. in older piglets, this mutation differentiated the reactivity of the immune system. Varying levels of iron status and red blood cell indices in RNASE6 genotypes presumably resulted from the coupling of genes involved in iron metabolism and expressed in an age-dependent manner.
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17

Filatov, Dmitry A. "Heterochiasmy and Sex Chromosome Evolution in Silene." Genes 14, no. 3 (February 22, 2023): 543. http://dx.doi.org/10.3390/genes14030543.

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The evolution of a non-recombining sex-specific region is a key step in sex chromosome evolution. Suppression of recombination between the (proto-) X- and Y-chromosomes in male meiosis creates a non-recombining Y-linked region (NRY), while the X-chromosome continues to recombine in females. Lack of recombination in the NRY defines its main properties—genetic degeneration and accumulation of repetitive DNA, making X and Y chromosomes very different from each other. How and why recombination suppression on sex chromosomes evolves remains controversial. A strong difference in recombination rates between the sexes (heterochiasmy) can facilitate or even cause recombination suppression. In the extreme case—complete lack of recombination in the heterogametic sex (achiasmy)—the entire sex-specific chromosome is automatically non-recombining. In this study, I analyse sex-specific recombination rates in a dioecious plant Silene latifolia (Caryophyllaceae), which evolved separate sexes and sex chromosomes ~11 million years ago. I reconstruct high-density RNAseq-based genetic maps including over five thousand genic markers for the two sexes separately. The comparison of the male and female maps reveals only modest heterochiasmy across the genome, with the exception of the sex chromosomes, where recombination is suppressed in males. This indicates that heterochiasmy likely played only a minor, if any, role in NRY evolution in S. latifolia, as recombination suppression is specific to NRY rather than to the entire genome in males. Other mechanisms such as structural rearrangements and/or epigenetic modifications were likely involved, and comparative genome analysis and genetic mapping in multiple Silene species will help to shed light on the mechanism(s) of recombination suppression that led to the evolution of sex chromosomes.
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18

Kanaya, S., and T. Uchida. "Comparison of the primary structures of ribonuclease U2 isoforms." Biochemical Journal 240, no. 1 (November 15, 1986): 163–70. http://dx.doi.org/10.1042/bj2400163.

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The primary structures of the two isoforms of ribonuclease U2, RNAases U2-A and U2-B, were analysed and compared with each other. Among the chymotryptic peptides obtained from the reduced and S-carboxymethylated enzymes, only peptides C-3 were different from each other in terms of chromatographic behaviour on reverse-phase h.p.l.c. On the basis of chemical analyses of these peptides, it was shown that RNAase U2-B had an isopeptide bond in which Asp-32 was linked to Gly-33 through the beta-carboxy group in its side chain instead of the alpha-carboxy group. Deamidation of Asn-32 in RNAase U2-A led to the formation of this unusual linkage. The previously reported sequence of RNAase U2 [Sato & Uchida (1975) Biochem. J. 145, 353-360] was corrected by changing amino acid residues at eight different positions and by inserting an asparagine residue at position 32. The numbering of the positions of amino acid residues located downstream of Asn-32 was therefore shifted by 1. Accordingly, RNAase U2-A was shown to be composed of 114 amino acid residues.
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Watari, Akiko, Toshio Hanada, Hisayo Yamane, Tomoya Esumi, Ryutaro Tao, Hideaki Yaegaki, Masami Yamaguchi, Kenji Beppu, and Ikuo Kataoka. "A Low Transcriptional Level of Se-RNase in the Se -haplotype Confers Self-compatibility in Japanese Plum." Journal of the American Society for Horticultural Science 132, no. 3 (May 2007): 396–406. http://dx.doi.org/10.21273/jashs.132.3.396.

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Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se ) and three SI (Sa , Sb , and Sc ) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe . However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa , SFBb , SFBc , and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se -haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.
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Hegedüs, Attila, Zoltán Szabó, József Nyéki, Júlia Halász, and Andrzej Pedryc. "Molecular Analysis of S-haplotypes in Peach, a Self-compatible Prunus Species." Journal of the American Society for Horticultural Science 131, no. 6 (November 2006): 738–43. http://dx.doi.org/10.21273/jashs.131.6.738.

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The most commercially grown peach [Prunus persica (L.) Batsch.] cultivars do not require cross-pollination for reasonable fruit set; however, self-incompatibility is a well-known feature within the Prunoideae subfamily. Isoelectric focusing and native polyacrylamide gel electrophoresis of S-ribonucleases; PCR analyses of S-RNase and S-haplotype-specific F-box genes as well as DNA sequencing were carried out to survey the self-(in)compatibility allele pool and to uncover the nature of self-compatibility in peach. From 25 cultivars and hybrids with considerable diversity in phenotype and origin, only two S-haplotypes were detected. Allele identity could be checked by exact length determination of the PCR-amplified fragments and/or partial sequencing of the peach S1-, S2-, and Prunus davidiana (Carr.) Franch. S1-RNases. S-RNases of peach were detected to possess ribonuclease activity, and a single nucleotide polymorphism in the S1-RNase was shown, which represents a synonymous substitution and does not change the amino acid present at the position in the protein. A 700-bp fragment of the peach SFB gene was PCR-amplified, which is similar to the fragment size of functional Prunus L. SFBs. All data obtained in this study may support the contribution of genes outside the S-locus to the self-compatible phenotype of peaches.
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Fagagnini, Andrea, Andrea Pica, Sabrina Fasoli, Riccardo Montioli, Massimo Donadelli, Marco Cordani, Elena Butturini, Laura Acquasaliente, Delia Picone, and Giovanni Gotte. "Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in cancer cells*." Biochemical Journal 474, no. 22 (November 6, 2017): 3767–81. http://dx.doi.org/10.1042/bcj20170541.

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Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory ‘pancreatic-type’ RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.
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Potari-gul, L., D. Modos, D. Turei, A. Valdeolivas, M. Madgwick, J. Saez-Rodriguez, and T. Korcsmaros. "P020 Mapping the changing intercellular communication and its downstream effect in Ulcerative Colitis." Journal of Crohn's and Colitis 15, Supplement_1 (May 1, 2021): S138—S139. http://dx.doi.org/10.1093/ecco-jcc/jjab076.149.

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Abstract Background Intercellular communication is essential for growing and differentiating in multicellular organisms by transducing the signal from cell to cell. Despite its importance, the molecular background is less discovered due to the lack of data. This gap has started to be addressed with the appearance of single-cell omics approaches providing an insight among others into the gene expression of individual cells. Methods We have developed a method to predict and compare cell-cell signalling interactions using single-cell RNAseq data from colon biopsies. Transcriptomic data alone is not capable of connecting the cells, a reliable network resource is needed to mediate the signal via protein-protein interactions between the source and target cells. Here we used OmniPath - a resource providing not only intra- and intercellular interactions but also annotations of proteins involved in the interplay of cells - to reconstruct signalling networks. We examined intercellular communication among five cell-types (regulatory T cell, macrophage, dendritic cell, goblet cell and myofibroblast) in healthy colon and during Ulcerative Colitis. Results Our analysis shows that there are significant differences in the type of cell-cell communication (ligand-receptor connections, adherens junctions, etc.) between the healthy and Ulcerative Colitis (UC) conditions, and these differences lead to altered downstream effects in the signal receiving cell. In both conditions, the ligand-receptor and adhesion connections were overrepresented, however cell junctions were less abundant in UC. Regarding the communication among the five cell-types, in healthy condition, cells are tightly connected to dendritic cells while in diseased condition to regulatory T cells. Focusing on ligand-receptor interactions between myofibroblasts and regulatory T cells, our pipeline identified the MAPK, Toll-like receptor (TLR) 2/6 and TLR 7/8 pathways enriched downstream in healthy conditions. In contrast, TLR3 and TLR4 pathways were affected by the myofibroblast in Ulcerative Colitis. Conclusion We found key intercellular mechanisms leading to well-defined differential pathway activation profiles. We showed that in uninflamed UC condition myofibroblasts disrupt the anti-inflammatory effect of regulatory T cells. Our pipeline is able to predict and analyse cell-cell interactions and their downstream effects and to highlight the differences in healthy and diseased states.
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Jones, Eleri, Supatra Marsh, Ryan O'Shaughnessy, Monique Aumailley, John McGrath, Edel O’Toole, and Matthew Caley. "O14 Junctional epidermolysis bullosa: repairing the epidermal lipid barrier." British Journal of Dermatology 189, no. 1 (July 2023): e10-e10. http://dx.doi.org/10.1093/bjd/ljad174.014.

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Abstract Junctional epidermolysis bullosa (JEB) caused by loss of function mutations in basement membrane genes, including that encoding laminin-332, is among the most severe forms of epidermolysis bullosa. Affected individuals suffer from blistering from birth, leading to scarring, granulation tissue and susceptibility to infection. In generalized severe JEB, there is a 50% mortality rate in the first 2 years of life, often due to failure to thrive or overwhelming infection. Using cells with knockdown of laminin-α3 we generated a RNAseq data set and have identified a new pathway altered in JEB, the regulation of cholesterol biosynthesis. It has been shown that loss of laminin-332 results in reduced epidermal lipid barrier specifically due to loss of cholesterol transport within keratinocytes. Cholesterol biosynthesis and cholesterol trafficking are fundamental processes in a wide range of cells and tissues, especially in the formation of the normal epidermal lipid barrier. A cholesterol transport screening assay has been developed and used to analyse the ability of 10 specifically selected compounds to restore cholesterol transport in our in vitro model of JEB. Results of this small-scale screen showed that nine of 10 of the selected drugs were able to restore cholesterol transport up to 50% of control. Six drugs were selected for further analysis within our three-dimensional (3D) skin-equivalent model of JEB. Organotypic skin equivalents of normal skin (siControl) and JEB skin (siLaminin-α3) were treated daily for 7 days with either dimethyl sulfoxide or 1 µmol L–1 of selected drugs. In 3D, drugs 1, 3, 5 and 6 displayed an increase in stratum corneum lipids shown by Nile red staining. Drugs 4 and 7 did not increase stratum corneum lipids above levels seen in the untreated JEB organotypics. Further analysis of skin differentiation by keratin, involucrin and tranglutaminase staining were assessed to narrow drug selection for further testing. These data also suggest that JEB is not just a disorder of epidermal–dermal adhesion, but a disorder of aberrant cholesterol trafficking causing a markedly impaired skin barrier. These findings suggests that drugs to target cholesterol transport within keratinocytes could be a beneficial treatment for JEB, helping to improve the crucial epidermal lipid barrier and prevent water loss and infection at the early stages of life.
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Lamping, Mario, Damian Tobias Rieke, Frederick Klauschen, Korinna Jöhrens, Ioannis Anagnostopoulos, Dido Lenze, Inge Tinhofer, et al. "Clinical impact of comprehensive versus targeted genomic analysis for precision oncology." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13033-e13033. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13033.

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e13033 Background: Panel sequencing (PS) has become a standard-of-care in cancer diagnostics. More comprehensive analyses such as whole-exome (WES) or RNA sequencing (RNAseq) allow for the detection of rare and unknown genetic aberrations that are not covered by predefined assays. The clinical impact of targeted versus comprehensive genomic assays were analyzed in patients presented at the Charité Molecular Tumor Board (MTB). Methods: Patients (pts) with advanced and/or metastatic cancer for whom no standard therapy was available were discussed in the MTB to allocate diagnostic profiling and guide biomarker-based treatment (BBT). Pts had to be < 50 years of age or diagnosed with a rare tumor entity to undergo WES/RNAseq, performed on fresh tissue. If ineligible, standard PS was performed on archival tissue. BBT recommendations, ranked by pre-specified evidence levels, were made by the MTB and pts were followed up. Results: 228 patients (median age 49 years, 108 female and 120 male) were discussed in the MTB between January 2016 and February 2019. We assigned 73 and 155 pts to PS and WES/RNAseq and results were obtained for 78.1% (n = 57/73) and 54.8% (n = 85/155) pts, respectively. Sequencing failed for 11 (PS; 15.1%) and 62 (WES/RNAseq; 40%) pts, most commonly due to insufficient tissue (n = 29). Sequencing was ongoing in 5 (PS) and 8 (WES/RNAseq) pts at the time of analysis. A median of 2 BBTs were recommended for 75.4% (43/57) of PS (range r: 1-3) and 90.6% (77/85) of WES/RNAseq pts (r: 1-6) each. 22% (n = 17/77) of WES/RNAseq pts had ≥4 BBTs made by the MTB. Treatment was initiated in 30.2% (n = 13/43) of PS and 40.2% (n = 31/77) of WES/RNAseq pts. Clinical benefit rates (CBRs) were 23.1% (2 PR, 1 SD) for PS and 45.2% (2 CR, 3 PR, 9 SD) for WES/RNAseq pts. Overall survival data was immature at the time of analysis. Conclusions: Utilizing WES/RNAseq is a feasible approach to perform tumor profiling in a heterogeneous cohort. We here show a higher rate of pts receiving confident evidence-based treatment recommendations in the WES/RNAseq group and a higher rate of treatment initiation. The CBR nearly doubled in the WES/RNAseq cohort when compared to standard PS pts, thus emphasizing the need for larger comparative analyses to guide diagnostic decision-making.
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Miller, Jason R., Kari A. Dilley, Derek M. Harkins, Timothy B. Stockwell, Reed S. Shabman, and Granger G. Sutton. "A host subtraction database for virus discovery in human cell line sequencing data." F1000Research 7 (January 23, 2018): 98. http://dx.doi.org/10.12688/f1000research.13580.1.

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The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.
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Miller, Jason R., Kari A. Dilley, Derek M. Harkins, Timothy B. Stockwell, Reed S. Shabman, and Granger G. Sutton. "A host subtraction database for virus discovery in human cell line sequencing data." F1000Research 7 (July 12, 2018): 98. http://dx.doi.org/10.12688/f1000research.13580.2.

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The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.
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Miller, Jason R., Kari A. Dilley, Derek M. Harkins, Timothy B. Stockwell, Reed S. Shabman, and Granger G. Sutton. "A host subtraction database for virus discovery in human cell line sequencing data." F1000Research 7 (May 21, 2019): 98. http://dx.doi.org/10.12688/f1000research.13580.3.

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The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.
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Mumal, Iqra, Liming Xu, Fupan Yao, Tannu Suwal, Xiaolian Fan, Mei Lu, and Annie Huang. "ETMR-19. SINGLE CELL ANALYSES OF ETMRs REVEAL THAT C19MC+ POPULATION DRIVES CELL CYCLE PROGRESSION AND STEM CELL MAINTENANCE." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii326—iii327. http://dx.doi.org/10.1093/neuonc/noaa222.222.

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Abstract Embryonal tumors with multilayered rosettes (ETMRs) are highly fatal diseases characterized by recurrent amplification of C19MC, an oncogenic miRNA cluster. While C19MC was discovered as a major driver of ETMRs, its direct role in ETMRs remains unknown. As ETMRs exhibit significant heterogeneity in C19MC expression, we employed single cell transcriptomics to investigate features of C19MC+ population. We conducted single-nuclei RNAseq of 23,269 cells from 6 primary and 2 matched recurrent ETMRs. We also conducted single-cell RNAseq of human neural stem cells (hNSC-5miR) and ETMR cell line (A664-5miR) with stable expression of 5 C19MC miRNAs. Bulk RNAseq (n=27), H3K27Ac ChiP-seq (n=5) and ATAC-seq (n=5) corroborated scRNAseq data and identified core transcription factors (TFs) of C19MC+ population. C19MC+ population (24%) mapped to neuro-epithelial cells and exhibited signatures of cell cycle and stem cell maintenance, consistent with bulk-RNAseq data. The C19MC+ population overlaps with MKI67+ cycling (57%) and PROM1+ stem cell population (56%). Interestingly, interrogation of hNSC-5mir and A664-5miR showed a larger MKI67+/PROM1+ population compared to controls. Likewise, hNSC-5miR/A664-5miR in vitro and in vivo experiments showed increased proliferation/stemness. C19MC+ population is characterized by SHH, WNT, mTOR, Hippo and IGF-signalling and driven by MEIS1, SOX11, ZNF521, RFX4 and NR2F2 TFs. Recurrent ETMRs exhibit a persistent but smaller C19MC+ population. Intriguingly, recurrent tumors were more quiescent with a smaller proliferative population. C19MC is directly involved in driving cell cycle and stemness in ETMRs. Cellular and molecular features of primary and recurrent ETMRs were remarkably different, suggesting that C19MC plays a different role upon recurrence.
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Eiteneuer, Constantin, David Velasco, Joseph Atemia, Dan Wang, Rainer Schwacke, Vanessa Wahl, Andrea Schrader, et al. "GXP: Analyze and Plot Plant Omics Data in Web Browsers." Plants 11, no. 6 (March 11, 2022): 745. http://dx.doi.org/10.3390/plants11060745.

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Next-generation sequencing and metabolomics have become very cost and work efficient and are integrated into an ever-growing number of life science research projects. Typically, established software pipelines analyze raw data and produce quantitative data informing about gene expression or concentrations of metabolites. These results need to be visualized and further analyzed in order to support scientific hypothesis building and identification of underlying biological patterns. Some of these tools already exist, but require installation or manual programming. We developed “Gene Expression Plotter” (GXP), an RNAseq and Metabolomics data visualization and analysis tool entirely running in the user’s web browser, thus not needing any custom installation, manual programming or uploading of confidential data to third party servers. Consequently, upon receiving the bioinformatic raw data analysis of RNAseq or other omics results, GXP immediately enables the user to interact with the data according to biological questions by performing knowledge-driven, in-depth data analyses and candidate identification via visualization and data exploration. Thereby, GXP can support and accelerate complex interdisciplinary omics projects and downstream analyses. GXP offers an easy way to publish data, plots, and analysis results either as a simple exported file or as a custom website. GXP is freely available on GitHub (see introduction)
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Belcaid, Zineb, Archana Balan, Christopher Cherry, Mara Lanis, Kristen Marrone, Benjamin Philip Levy, Heather Schneider, et al. "Immunogenomic features of pathologic response to neoadjuvant immune checkpoint blockade in esophageal cancer." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 4042. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.4042.

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4042 Background: Improving immunotherapy efficacy remains an unmet need in esophagogastric cancer and a deeper understanding of tumor and immune system dynamics during therapy may tailor immuno-oncology approaches. Methods: We performed whole exome sequencing (WES) and bulk RNA sequencing (RNAseq) of 70 serial tumor samples from 23 patients with stage II/III esophageal/gastroesophageal junction (E/GEJ) cancer treated on a phase 1B clinical trial with neoadjuvant nivolumab with or without relatlimab (anti-LAG-3) and chemoradiation followed by surgery (NCT03044613; CA209-906). Pathologic response was measured by tumor regression at the time of resection. Median follow up was 23 months post-surgery. Serial tumor samples were collected prior to therapy, after 2 cycles of induction immune checkpoint blockade (ICB), and at the time of resection. Twenty-two baseline tumor/normal DNA pairs were analyzed by WES and 48 serial tumor samples were analyzed by RNAseq. WES data was analyzed to identify somatic mutations, generate tumor mutation burden (TMB) estimates and assess the fraction of expressed mutations in conjunction with RNAseq data. Immune cell subset composition was determined by RNAseq data deconvolution by CIBERSORT and gene set enrichment analyses were performed utilizing GSEA. B-cell density was inferred by immunoglobulin rearrangements detected by RNAseq. Results: Gene set enrichment expression analyses revealed an upregulation of effector pro-inflammatory cytokines after induction ICB. Interferon-gamma, interferon-alpha and TNF-alpha related genes were significantly upregulated after induction ICB compared to baseline (p < 0.0001). In contrast, significant downregulation of E2F targets (p = 0.002), G2M checkpoint genes (p = 0.005) and DNA damage repair genes (p = 0.004) was observed post ICB; enrichment analyses were independent of response to therapy and treatment arm. While TMB was not predictive of pathologic response (p = 0.22), patients with tumors harboring a higher number of expressed mutations were more likely to achieve a pathologic complete response (pCR; p = 0.026). RNAseq deconvolution analyses revealed a higher B-cell density post ICB induction in tumors with pCR (p = 0.018). Furthermore, an increased baseline content of intra-tumoral activated M1 macrophages differentiated tumors from patients achieving a pCR (p = 0.0034), which was further exemplified post induction ICB. Conclusions: Neoadjuvant immunotherapy induces an inflammatory immune response in the tumor microenvironment that is linked with tumor elimination and pathologic response. Our findings highlight the importance of nuanced multi-omics analyses to understand the wiring of response to immunotherapy and guide therapy for E/GEJ cancer.
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Cafournet, Cérane, Sofia Zanin, Anne Guimier, Marie Hully, Zahra Assouline, Giulia Barcia, Pascale de Lonlay, et al. "Novel ELAC2 Mutations in Individuals Presenting with Variably Severe Neurological Disease in the Presence or Absence of Cardiomyopathy." Life 13, no. 2 (February 4, 2023): 445. http://dx.doi.org/10.3390/life13020445.

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Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5′- and 3′-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.
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32

Tollervey, David. "Genetic and biochemical analyses of yeast RNase MRP." Molecular Biology Reports 22, no. 2-3 (1996): 75–79. http://dx.doi.org/10.1007/bf00988709.

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33

Ushijima, Koichiro, Hidenori Sassa, Mihoko Tamura, Makoto Kusaba, Ryutaro Tao, Thomas M. Gradziel, Abhaya M. Dandekar, and Hisashi Hirano. "Characterization of the S-Locus Region of Almond (Prunus dulcis): Analysis of a Somaclonal Mutant and a Cosmid Contig for an S Haplotype." Genetics 158, no. 1 (May 1, 2001): 379–86. http://dx.doi.org/10.1093/genetics/158.1.379.

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Abstract Almond has a self-incompatibility system that is controlled by an S locus consisting of the S-RNase gene and an unidentified “pollen S gene.” An almond cultivar “Jeffries,” a somaclonal mutant of “Nonpareil” (ScSd), has a dysfunctional Sc haplotype both in pistil and pollen. Immunoblot and genomic Southern blot analyses detected no Sc haplotype-specific signal in Jeffries. Southern blot showed that Jeffries has an extra copy of the Sd haplotype. These results indicate that at least two mutations had occurred to generate Jeffries: (1) deletion of the Sc haplotype and (2) duplication of the Sd haplotype. To analyze the extent of the deletion in Jeffries and gain insight into the physical limit of the S locus region, ∼200 kbp of a cosmid contig for the Sc haplotype was constructed. Genomic Southern blot analyses showed that the deletion in Jeffries extends beyond the region covered by the contig. Most cosmid end probes, except those near the Sc-RNase gene, cross-hybridized with DNA fragments from different S haplotypes. This suggests that regions away from the Sc-RNase gene can recombine between different S haplotypes, implying that the cosmid contig extends to the borders of the S locus.
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Kaulen, L. D., E. Denisova, F. Hinz, L. Hai, D. Friedel, O. Henegariu, D. Hoffmann, et al. "P20.12.B INTEGRATED GENETIC ANALYSES OF IMMUNODEFICIENCY-ASSOCIATED EPSTEIN-BARR VIRUS- (EBV) POSITIVE PRIMARY CNS LYMPHOMAS." Neuro-Oncology 26, Supplement_5 (October 2024): v118. http://dx.doi.org/10.1093/neuonc/noae144.399.

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Abstract BACKGROUND Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. MATERIAL AND METHODS In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. Deconvolution of bulk RNA sequencing data was performed using CIBERSORTx to characterize the tumor microenvironment. RESULTS EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV- PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. CONCLUSION In addition to novel insights into the pathobiology of EBV+PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.
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Lee, Seul, Jae-Hwan Kim, Kwangmin Na, Seung Min Yang, Dong Kwon Kim, Sujeong Baek, Seong-san Kang, et al. "Abstract 6780: Characterization of immunological heterogeneity in the tumor microenvironment by integrated analyses using single cell RNAseq, spatial RNAseq and multiplex IHC." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6780. http://dx.doi.org/10.1158/1538-7445.am2023-6780.

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Abstract Heterogeneity in resistant to immunotherapies of tumor microenvironment (TME) has been implicated in immunotherapies to cause immune evasion or drug resistance. This study was conducted to explore the heterogeneity of TME through multiplex IHC, spatial and RNA sequencing analysis. We selected a sample from a lung adenocarcinoma patient without EGFR-activating mutation and expressing 30% of PD-L1. For quantitative analysis by multiplex IHC, various markers including CD4, CD8, FoxP3, granzyme B, CD20 and pan-cytokeratin were stained with 7 different fluorescence dyes, which was imaged with Vectra Polaris (Akoya). For scRNAseq and spatial RNAseq, we used 5’ chromium library kit (10X Genomics) to make library construction. Integrated raw data was generated using Cell ranger, Seurat pipeline and Azimuth package. The tumor area was divided into 16 clusters in which we selected 2 clusters based on CD3/45 expression. There was a noticeable distinction between the two clusters which were defined as the ‘High’ region (CD45highCD3high cluster) and the ‘Low’ region (CD45lowCD3low cluster). By multiplex IHC, percentage of CD8+T cells was higher in the ‘High’ region than in the ‘Low’ region (8.5% vs. 0.8%, respectively). Subsequent analysis of two clusters using spatial and single cell RNA seq, the ‘Low’ region was characterized by increased hypoxia-associated gene expressions including HIF1A, HIF3A and VEGFA. Various immune cells were abundant in the ‘High’ region and CD45 expression level was 11-fold higher in the ‘High’ region compared to the ‘Low’ region. Cytokine/chemokine network analysis via spatial RNAseq revealed that gene expression of tumor necrosis factor (TNF) family-associated factors increased in the 'High' region compared to the ‘Low’ region (TNF, FAS, TRAIL, RANKL and CD40), which is well-known to promotes apoptosis, programmed cell death, or necrosis of certain cancer. Additionally, the ‘High’ region also had elevated levels of the PD-1/PD-L1, CD155, CD122/TIGIT, Siglec10/CD24, LAG3/LAGLS3, and CD47/CD172a axes, suggesting active immune responses. Intriguingly, combined analyses showed that ‘High’ region showed enhanced level of CD44 expression as the leading-edged gene, which suggests the metastatic potential of tumor cells. Furthermore, scRNA analysis confirmed that CD44 expression was mainly higher in macrophages, suggesting that tumor-associated macrophages partially affected tumor cell metastasis in the ‘High’ region. Our finding suggests that understanding the intratumoral immunological heterogeneity of lung adenocarcinoma can help to study the mechanism of tumor heterogeneity by integrated spatial RNAseq and scRNAseq analyses. This type of technique could be applied to understand complex networks of anti-tumor immune activities, drug resistance mechanisms and immunotherapeutic response of cancer. Citation Format: Seul Lee, Jae-Hwan Kim, Kwangmin Na, Seung Min Yang, Dong Kwon Kim, Sujeong Baek, Seong-san Kang, Yu Jin Han, Chun-Bong Synn, Mi hyun Kim, Heekyung Han, Young Taek Kim, Sungwoo Lee, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Characterization of immunological heterogeneity in the tumor microenvironment by integrated analyses using single cell RNAseq, spatial RNAseq and multiplex IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6780.
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36

Coulibaly, Daouda, Feng Gao, Yang Bai, Kenneth Omondi Ouma, Augustine Antwi-Boasiako, Pengyu Zhou, Shahid Iqbal, et al. "Molecular Research Progress on Gametophytic Self-Incompatibility in Rosaceae Species." Horticulturae 10, no. 10 (October 17, 2024): 1101. http://dx.doi.org/10.3390/horticulturae10101101.

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Self-incompatibility (SI) is a complex mechanism that prevents plants from self-fertilizing to preserve and promote genetic variability. The angiosperm species have developed two different SI systems, the sporophytic (SSI) and the gametophytic (GSI) systems. SI is a significant impediment to steady fruit production in fruit tree species of the Rosaceae. In Rosaceae, GSI is genetically regulated via a single locus, named the ‘S-locus’, which includes a minimum of two polymorphic and relatively intercorrelated S genes: a pistil-expressed S-RNase gene and several pollen-expressed SFBB (S-locus F-Box Brothers) or SFB (S haplotype-specific F-box protein). This necessitates the interaction of S-RNases with the male determinants. Although genetic and molecular analyses of S genes have shown that mutations in both pistils and pollen-specific components induce self-compatibility in many species and cultivars, other genes or molecules outside the S-locus can co-participate in the male gamete rejection in GSI. However, we highlight and synthesize the most recent knowledge on different mechanisms of GSI in Rosaceae in this current review.
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37

Cer, Regina Z., J. Enrique Herrera-Galeano, Kenneth G. Frey, Kevin L. Schully, Truong V. Luu, John Pesce, Vishwesh P. Mokashi, Andrea M. Keane-Myers, and Kimberly A. Bishop-Lilly. "Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers." International Journal of Genomics 2017 (2017): 1–13. http://dx.doi.org/10.1155/2017/6489383.

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Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs.
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Lobon-Iglesias, María-Jesús, Arnault Tauziede-Espariat, Mamy Andrianteranagna, Zhiyan Han, Julien Masliah-Planchon, and Franck Bourdeaut. "ATRT-27. COST-EFFECTIVE ASSAYS TO SUBGROUP ATRT IN THE DAILY ROUTINE." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii281. http://dx.doi.org/10.1093/neuonc/noaa222.026.

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Abstract Three atypical teratoid rhabdoid tumors (ATRT) molecular subgroups with different bio-clinical characteristics have been reported (TYR, SHH and MYC). Molecular subgrouping relies on either methylation profiling (reference methods), or expression profiling. However, the cost-effectiveness of such pangenomic screening is questionable. This work aims to study the reliability of alternative techniques for subgroup classification in the daily routine. Illumina EPIC-arrays were performed on 46 samples. Among those cases, expression profiling were analysed by RNAseq (n=30). We designed a 26-gene panel to assess expression profiling using the Nanostring technology; this was applied to 35 tumors. Immunohistochemistry (IHC) was used for 20 samples; it relied on the expression of MITF, TYR, OTX2 and MYC. We first assessed the concordance between DNA methylation and RNAseq based profilings; then, between RNAseq and Nanostring and, finally, between methylation profiling and Nanostring or IHC, the two rapidest and cheapest tools. The concordance between the two expression-based profiling was 19/21. EPIC-arrays and RNAseq or Nanostring were concordant in 26/30 and 30/35 samples, respectively. The concordance was perfect for methylation-defined MYC subtype. Finally, 17/20 tumor samples were classified in the same subgroup by EPIC-arrays and IHC; the 3/20 misclassified tumors were SHH by methylation but consistently MYC by IHC, Nanostring and RNAseq. There was 90–100% of concordance for TYR subgroup (all techniques). We have designed a gene panel-based expression signature that shows promising concordance with RNAseq and methylation profiling. Nanostring assay and IHC well predict ATRT subgroup classification for MYC and TYR subclass, but less so for methylation-defined SHH ones.
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Muhowski, Elizabeth M., and Laura M. Rogers. "Dual TCR-Expressing T Cells in Cancer: How Single-Cell Technologies Enable New Investigation." ImmunoHorizons 7, no. 5 (May 1, 2023): 299–306. http://dx.doi.org/10.4049/immunohorizons.2200062.

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Abstract TCR diversity measures are often used to understand the immune response in cancer. Traditional measures of diversity rely on bulk RNA sequencing (RNAseq) of the β-chain variable regions. However, the full αβ TCR repertoire is a combination of both the α- and β-chains, which are encoded by separate genes. In contrast with bulk RNAseq, single-cell RNAseq (scRNAseq) allows paired chain analyses, yielding a more accurate measure of the repertoire. Interestingly, ∼30% of mature peripheral T cells express multiple TCR alleles (e.g., two α-chains) and may exhibit dual Ag specificity. scRNAseq has become increasingly common, and data from both human and animal studies are publicly available. However, routine workflows discard secondary TCR alleles and focus on a single TCR clone per cell. This perspectives piece emphasizes why this may not be good practice and highlights unanswered questions in the field of T cell dual specificity.
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40

WILHELM, Marcelle, Mansour BOUTABOUT, and François-Xavier WILHELM. "Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities." Biochemical Journal 348, no. 2 (May 23, 2000): 337–42. http://dx.doi.org/10.1042/bj3480337.

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Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl2, NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
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Velichko, Sharlene, Johnathon Anderson, Stephanie Ryan, and Reen Wu. "Global gene expression analysis of Act1’s effects in airway epithelial cells (161.17)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 161.17. http://dx.doi.org/10.4049/jimmunol.186.supp.161.17.

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Abstract Act1/CIKS is an intracellular protein that has been shown to play an important role in mediating IL-17A and IL-25 signaling effects. Recently, defects in Act1 function and/or expression has been implicated in inflammatory disease, such as psoriatic arthritis and atopic dermatitis. We have found that the modulation of Act1 expression levels in human airway epithelial cells changes the expression levels of some genes, in the absence of cytokine stimulation. RNAseq is a powerful new technique to quantitatively measure changes at the transcriptome level. Here we describe the use of RNAseq to globally analyze the effects of modulating Act1 expression in human airway epithelial cells.
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42

Tadokoro, Takashi, Dong-Ju You, Yumi Abe, Hyongi Chon, Hiroyoshi Matsumura, Yuichi Koga, Kazufumi Takano, and Shigenori Kanaya. "Structural, Thermodynamic, and Mutational Analyses of a Psychrotrophic RNase HI†,‡." Biochemistry 46, no. 25 (June 2007): 7460–68. http://dx.doi.org/10.1021/bi7001423.

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43

Zoroddu, Stefano, Luca Sanna, Valentina Bordoni, Weidong Lyu, Gabriele Murineddu, Gerard A. Pinna, Sonia Vanina Forcales, Arturo Sala, David J. Kelvin, and Luigi Bagella. "RNAseq Analysis of Novel 1,3,4-Oxadiazole Chalcogen Analogues Reveals Anti-Tubulin Properties on Cancer Cell Lines." International Journal of Molecular Sciences 24, no. 14 (July 9, 2023): 11263. http://dx.doi.org/10.3390/ijms241411263.

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1,3,4-Oxadiazole derivatives are among the most studied anticancer drugs. Previous studies have analyzed the action of different 1,3,4-oxadiazole derivatives and their effects on cancer cells. This study investigated the characterization of two new compounds named 6 and 14 on HeLa and PC-3 cancer cell lines. Based on the previously obtained IC50, cell cycle effects were monitored by flow cytometry. RNA sequencing (RNAseq) was performed to identify differentially expressed genes, followed by functional annotation using gene ontology (GO), KEGG signaling pathway enrichment, and protein–protein interaction (PPI) network analyses. The tubulin polymerization assay was used to analyze the interaction of both compounds with tubulin. The results showed that 6 and 14 strongly inhibited the proliferation of cancer cells by arresting them in the G2/M phase of the cell cycle. Transcriptome analysis showed that exposure of HeLa and PC-3 cells to the compounds caused a marked reprograming of gene expression. Functional enrichment analysis indicated that differentially expressed genes were significantly enriched throughout the cell cycle and cancer-related biological processes. Furthermore, PPI network, hub gene, and CMap analyses revealed that compounds 14 and 6 shared target genes with established microtubule inhibitors, indicating points of similarity between the two molecules and microtubule inhibitors in terms of the mechanism of action. They were also able to influence the polymerization process of tubulin, suggesting the potential of these new compounds to be used as efficient chemotherapeutic agents.
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44

Mora-Márquez, Fernando, José Luis Vázquez-Poletti, and Unai López de Heredia. "NGScloud2: optimized bioinformatic analysis using Amazon Web Services." PeerJ 9 (April 16, 2021): e11237. http://dx.doi.org/10.7717/peerj.11237.

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Background NGScloud was a bioinformatic system developed to perform de novo RNAseq analysis of non-model species by exploiting the cloud computing capabilities of Amazon Web Services. The rapid changes undergone in the way this cloud computing service operates, along with the continuous release of novel bioinformatic applications to analyze next generation sequencing data, have made the software obsolete. NGScloud2 is an enhanced and expanded version of NGScloud that permits the access to ad hoc cloud computing infrastructure, scaled according to the complexity of each experiment. Methods NGScloud2 presents major technical improvements, such as the possibility of running spot instances and the most updated AWS instances types, that can lead to significant cost savings. As compared to its initial implementation, this improved version updates and includes common applications for de novo RNAseq analysis, and incorporates tools to operate workflows of bioinformatic analysis of reference-based RNAseq, RADseq and functional annotation. NGScloud2 optimizes the access to Amazon’s large computing infrastructures to easily run popular bioinformatic software applications, otherwise inaccessible to non-specialized users lacking suitable hardware infrastructures. Results The correct performance of the pipelines for de novo RNAseq, reference-based RNAseq, RADseq and functional annotation was tested with real experimental data, providing workflow performance estimates and tips to make optimal use of NGScloud2. Further, we provide a qualitative comparison of NGScloud2 vs. the Galaxy framework. NGScloud2 code, instructions for software installation and use are available at https://github.com/GGFHF/NGScloud2. NGScloud2 includes a companion package, NGShelper that contains Python utilities to post-process the output of the pipelines for downstream analysis at https://github.com/GGFHF/NGShelper.
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45

Ubertini, Valentina, Sarah Hall, Frida Ponthan, and James Wilson. "Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity." Cancer Research 84, no. 6_Supplement (March 22, 2024): 2125. http://dx.doi.org/10.1158/1538-7445.am2024-2125.

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Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.
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46

Norero, Natalia, María Rey Burusco, Sebastián D’Ippólito, Cecilia Décima Oneto, Gabriela Massa, Martín Castellote, Sergio Feingold, and María Guevara. "Genome-Wide Analyses of Aspartic Proteases on Potato Genome (Solanum tuberosum): Generating New Tools to Improve the Resistance of Plants to Abiotic Stress." Plants 11, no. 4 (February 18, 2022): 544. http://dx.doi.org/10.3390/plants11040544.

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Aspartic proteases are proteolytic enzymes widely distributed in living organisms and viruses. Although they have been extensively studied in many plant species, they are poorly described in potatoes. The present study aimed to identify and characterize S. tuberosum aspartic proteases. Gene structure, chromosome and protein domain organization, phylogeny, and subcellular predicted localization were analyzed and integrated with RNAseq data from different tissues, organs, and conditions focused on abiotic stress. Sixty-two aspartic protease genes were retrieved from the potato genome, distributed in 12 chromosomes. A high number of intronless genes and segmental and tandem duplications were detected. Phylogenetic analysis revealed eight StAP groups, named from StAPI to StAPVIII, that were differentiated into typical (StAPI), nucellin-like (StAPIIIa), and atypical aspartic proteases (StAPII, StAPIIIb to StAPVIII). RNAseq data analyses showed that gene expression was consistent with the presence of cis-acting regulatory elements on StAP promoter regions related to water deficit. The study presents the first identification and characterization of 62 aspartic protease genes and proteins on the potato genome and provides the baseline material for functional gene determinations and potato breeding programs, including gene editing mediated by CRISPR.
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47

Skoczek, Halina, and Michał Borys. "Rybonukleaza w korzeniach i węzłach krzewienia dwóch odmian jęczmienia [Ribonuclease in roots and nodes of two barley cultivars]." Acta Agrobotanica 32, no. 2 (2015): 173–83. http://dx.doi.org/10.5586/aa.1979.016.

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Cultivar Union which, compared with cultivar Lubuski has a larger root system, was found to show a significantly higher RNase activity. The nodes of cv. Union show a lower RNase activity per gram of tissue fresh weight and a higher specific activity, in comparison with cv. Lubuski. The number of RNase isoenzymes in the roots of both examined cultivars is the same. No difference in the number of RNase isoenzymes between the nodes of the examined cultivars was found either. On the other hand, certain differences between the examined cultivars in some RNase isoenzymes activities were observed in the analysed parts of the plants.
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48

Pruefer, Franz, K. Vazquez-Santillan, L. Muñoz-Galindo, J. L. Cruz-Colin, V. Maldonado, and Jorge Melendez-Zajgla. "TIMP4 Modulates ER-α Signalling in MCF7 Breast Cancer Cells." Folia Biologica 62, no. 2 (2016): 75–81. http://dx.doi.org/10.14712/fb2016062020075.

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Tissue inhibitor of metalloprotease 4 (TIMP4) contributes to poor prognosis in breast and other tumours. However, the mechanisms of how TIMP4 influences breast cancer cell behaviour are unknown. Our aim was to explore the signalling pathways modulated by TIMP4 in breast cancer cells. Human recombinant TIMP4 was added to MCF7 breast cancer cells and RNASeq was performed. TIMP4 RNASeq results were validated by RT-PCR. Network analyses of TIMP4-exposed cells showed that ER-α, HIF1A and TGF-β signalling were activated, whereas FOXO3 signalling was downregulated. ER-α protein levels were increased and concordantly, promoters of TIMP4-upregulated genes were significantly enriched in oestrogen-binding sites. We concluded that TIMP4 modulates multiple signalling pathways relevant in cancer in MCF7 cells, including the ER-α cascade.
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Erster, S. H., L. A. Finn, D. A. Frendewey, and D. M. Helfman. "Use of RNase H and primer extension to analyze RNA splicing." Nucleic Acids Research 16, no. 13 (July 11, 1988): 5999–6014. http://dx.doi.org/10.1093/nar/16.13.5999.

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50

Laalami, Soumaya, Philippe Bessières, Anna Rocca, Léna Zig, Pierre Nicolas, and Harald Putzer. "Bacillus subtilis RNase Y Activity In Vivo Analysed by Tiling Microarrays." PLoS ONE 8, no. 1 (January 10, 2013): e54062. http://dx.doi.org/10.1371/journal.pone.0054062.

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