Littérature scientifique sur le sujet « TIL, Lipocalin »

La evaluación de la función renal se ha realizado históricamente en base a la medición de la tasa de filtración glomerular, la cual fue reemplazada, posteriormente, por la determinación de creatinina sérica debido a su amplia disponibilidad y accesibildad. Sin embargo, en los trabajos actuales, cuando es comparada con el aumento de los biomarcadores de injuria renal aguda (IRA), se observa que la elevación desde su valor basal ante una lesión renal repentina sucede horas más tarde que el Neutrophil Gelatinase-Associated Lipocalin (NGAL) urinario. El objetivo de la presente revisión sobre NGAL, y sus alcances como marcador biológico temprano de injuria renal aguda, es conocer su validación como tal y su aplicabilidad en la práctica médica de pacientes caninos, ya que la identificación temprana de un proceso que altere la funcionalidad y viabilidad de los riñones, permite intervenir de manera terapéutica y anticiparse a un grado irreversible de lesión renal. El método utilizado para la revisión fue consultar revistas científicas internacionales y, sobre estas, seleccionar trabajos de la última década que demuestran resultados estadísticamente significativos sobre el aumento de NGAL entre dos (2) y doce (12) horas antes que la creatinina. Si bien los datos aportados por los trabajos de investigación son claros, aún queda un camino por recorrer en relación a los tipos de injuria renal aguda y sus diferentes causas. NGAL, como biomarcador renal, presente un espectro favorable en cuanto a utilidad clínica, dada la accesibilidad a los métodos complementarios que permiten valorarlo en medicina veterinaria.

Abstract Seminal plasma (SP) proteins participate in the process of fertilization by binding to the sperm membrane, particularly to the phosphorylcholine-containing lipids. This study aimed to identify and characterize non-phosphorylcholine-binding and phosphorylcholine-binding proteins (nPch- BPs and PchBPs, respectively) of canine SP. The nPchBPs and PchBPs were isolated from canine SP by affinity chromatography. Electrophoretic studies revealed that the nPchBPs and PchBPs occurred in their native state as high-molecular-weight aggregates. Immunofluorescent staining showed preferential binding of nPchBPs to the sperm acrosome membrane, whereas PchBPs coating was uniformly distributed on the sperm post-acrosomal membrane, mid-piece and tail regions. Analysis with mass spectrometry confirmed that canine prostate specific esterase (CPSE) is a component of the nPchBPs and PchBPs, which is implicated in key mechanisms of protein-coating on the sperm plasma membrane surface. In addition, proteins of known binding properties such as prostaglandin-H2 D-isomerase and lipocalin-like 1 protein, identified in canine SP, might have a specific role in the fertilization-associated processes.

SummaryNitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≈35 and ≈155%, at doses of 8 and 80 μg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≈60% reduction at 400 μg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.

The argasid tick Ornithodoros moubata is the main vector of human relapsing fever (HRF) and African swine fever (ASF) in Africa. Salivary proteins are part of the host-tick interface and play vital roles in the tick feeding process and the host infection by tick-borne pathogens; they represent interesting targets for immune interventions aimed at tick control. The present work describes the transcriptome profile of salivary glands of O. moubata and assesses the gene expression dynamics along the trophogonic cycle using Illumina sequencing. De novo transcriptome assembling resulted in 71,194 transcript clusters and 41,011 annotated transcripts, which represent 57.6% of the annotation success. Most salivary gene expression takes place during the first 7 days after feeding (6,287 upregulated transcripts), while a minority of genes (203 upregulated transcripts) are differentially expressed between 7 and 14 days after feeding. The functional protein groups more abundantly overrepresented after blood feeding were lipocalins, proteases (especially metalloproteases), protease inhibitors including the Kunitz/BPTI-family, proteins with phospholipase A2 activity, acid tail proteins, basic tail proteins, vitellogenins, the 7DB family and proteins involved in tick immunity and defence. The complexity and functional redundancy observed in the sialotranscriptome of O. moubata are comparable to those of the sialomes of other argasid and ixodid ticks. This transcriptome provides a valuable reference database for ongoing proteomics studies of the salivary glands and saliva of O. moubata aimed at confirming and expanding previous data on the O. moubata sialoproteome.

Abstract Background The enteric microbiota has been recognized as an essential regulator of both gut and brain physiology, a complex interaction generally termed the microbiota-gut-brain axis. Disturbances to gastrointestinal physiology lead to alterations in the composition of the enteric microbiota, whereas dysbiosis can also contribute to pathophysiology. Inflammatory bowel diseases (IBD) are chronic, relapsing inflammatory conditions of the gastrointestinal tract, associated with microbial dysbiosis. Interestingly, IBD patients exhibit an increased incidence of mental illness (i.e. anxiety and depression), often termed “sickness behavior”, even during the remitting phase of their disease. Nevertheless, it is unclear if alterations in the enteric microbiota associated with IBD are responsible for the observed modification in brain function and behavior. Here, we hypothesized that sickness behavior is driven by alterations in microbial composition, which occur in the context of intestinal inflammation. Aims We sought to determine whether transfer of the microbiota from colitic mice, exhibiting sickness behaviour, into healthy counterparts would induce behavioral changes. Methods Male mice (C57Bl/6J; 8 weeks old) were used in all experiments. Colitis was induced by administration of 2.5% dextran sodium sulfate (DSS) in the drinking water for 5 days. Colonic inflammation was assessed by measuring fecal lipocalin-2 and the expression of pro-inflammatory mediators via qPCR. Cecal matter from donor mice (control or DSS treated) were collected for fecal microbiota transplant (FMT). FMT was performed via oral gavage in antibiotic-treated recipient mice. Gut bacteria were evaluated by 16S rRNA sequencing in cecal samples. Anxiety- and depression-like behavior were assessed by elevated plus maze and tail suspension test, respectively. Results DSS-treated mice exhibited clinical disease, reflected by body weight loss, increased fecal lipocalin-2 and elevated colonic pro-inflammatory cytokine transcripts. An increase in anxiety-like behavior was observed in mice with colitis, although no alterations in depression-like behavior were detected. Colitic mice exhibited a unique microbial community. Transferring cecal material from colitic mice into recipient, antibiotic-treated mice, recapitulated alterations in behavior seen in colitic donors, as shown by increased anxiety-like behavior and unexpectedly, increased depression-like behavior. These behavioral changes occurred in the absence of colonic or brain inflammation in the recipient mice, but were associated with changes in stress-related gene expression (i.e. Crh). Conclusions Colitis-associated sickness behavior can be transmitted to antibiotic-treated recipient mice via FMT, which occurs in the absence of overt intestinal or brain inflammation. Funding Agencies CIHRNational Council for Scientific and Technological Development (CNPq-Brazil)

Abstract Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending limb (TAL) epithelial cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. We aimed to dissect the role of dietary salt in renal UMOD excretion in normotension and chronic hypertension. Normotensive Wistar–Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) (n=8/sex/strain) were maintained on 1% NaCl for 3 weeks. A subset of salt-loaded SHRSP was treated with nifedipine. Salt-loading in SHRSP increased blood pressure (ΔSBP 35 ± 5 mmHg, P<0.0001) and kidney injury markers such as kidney injury marker-1 (KIM-1; fold change, FC 3.4; P=0.003), neutrophil gelatinase-associated lipocalin (NGAL; FC, 2.0; P=0.012) and proteinuria. After salt-loading there was a reduction in urinary UMOD excretion in WKY and SHRSP by 26 and 55% respectively, compared with baseline. Nifedipine treatment reduced blood pressure (BP) in SHRSP, however, did not prevent salt-induced reduction in urinary UMOD excretion. In all experiments, changes in urinary UMOD excretion were dissociated from kidney UMOD protein and mRNA levels. Colocalization and ex-vivo studies showed that salt-loading increased intracellular UMOD retention in both WKY and SHRSP. Our study provides novel insights into the interplay among salt, UMOD, and BP. The role of UMOD as a cardiovascular risk marker deserves mechanistic reappraisal and further investigations based on our findings.

Adriamycin (ADR) administration in susceptible rodents such as the BALB/c mouse strain produces injury to the glomerulus mimicking human chronic kidney disease due to primary focal segmental glomerulosclerosis. The goal of the present study was to use this model to investigate antiproteinuric actions of the (pro)renin receptor decoy inhibitor PRO20. BALB/c mice were pretreated for 1 day with PRO20 at 500 μg·kg−1·day−1 via an osmotic minipump followed by a single injection of vehicle or ADR (10 mg/kg) via the tail vein. Albuminuria and renal function were analyzed at the fourth week post-ADR administration. ADR-treated mice exhibited severe proteinuria, hypoalbuminemia and hyperlipidemia, glomerulosclerosis, podocyte loss, tubulointerstitial fibrosis, and oxidative stress, accompanied by elevated urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, all of which were significantly attenuated by PRO20. Urinary and renal renin activity and angiotensin II were elevated by ADR and suppressed by PRO20. In parallel, urinary and renal H2O2 levels and renal NADPH oxidase 4 (Nox4) and transient receptor potential channel C6 (TRPC6) expression in response to ADR were all similarly suppressed. Taken together, the results of the present study provide the first evidence that PRO20 can protect against podocyte damage and interstitial fibrosis in ADR nephropathy by preventing activation of the intrarenal renin-angiotensin system and upregulation of Nox4 and TRPC6 expression. PRO20 may have a potential application in the treatment of ADR nephropathy.

Cisplatin is one of the most effective antitumor agents, but its clinical use is highly limited by its severe side effects, especially nephrotoxicity. Recently, the active form of gasdermin D (GSDMD), termed GSDMD-N, was identified to mediate pyroptotic inflammatory cell death in several diseases. However, the role of the GSDMD-N fragment in cisplatin-induced acute kidney injury (AKI) remains unclear. In the present study, we found that pyroptosis was induced by cisplatin in both mouse kidney tissues and renal tubular epithelial cells, accompanied by increased expression of the GSDMD-N fragment. In GSDMD knockout mice with cisplatin-induced AKI, we found that cisplatin-induced loss of renal function, renal tubular injury, and inflammation was significantly attenuated compared with wild-type mice. Furthermore, the GSDMD-N fragment was overexpressed by an established rapid plasmid tail vein injection approach to evaluate the role of this cleaved form of GSDMD in AKI. As expected, mice with GSDMD-N fragment overexpression in the kidney were more susceptible to cisplatin-induced AKI than control mice, as evidenced by further elevated serum levels of blood urea nitrogen and creatinine, aggravated renal pathology, increased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and enhanced renal inflammatory cytokine secretion, which indicates a pathogenic role of GSDMD-N in cisplatin-induced AKI by triggering cell pyroptosis. Similar results were also observed in renal tubular epithelial cells overexpressing the GSDMD-N fragment. Thus these findings suggested that the activation of GSDMD contributes to cisplatin-induced AKI, possibly through triggering pyroptosis.

Contrary to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order to shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride. We are showing here that the effect of crowding agents on the structure and conformational stability of the recombinant bOBP depends on the size of the crowder, with the smaller crowding agents being more effective in the stabilization of the bOBP native dimeric state against the guanidine hydrochloride denaturing action. This effect of the crowding agents is concentration dependent, with the high concentrations of the agents being more effective.

The fossil energy resources decrease and climate changes, caused by carbon dioxide (CO2) emissions, have led most industrialized countries to undertake policies aimed at the development and use of renewable energy sources. Among the renewable energies, vegetal biomasses play a key role because widely available and potentially able to cover up to 200% of the global energy demand. Vegetal biomasses can be used mainly as raw materials for the production of chemicals, biofuels and energy, in the increasingly important green economy concept based on biorefineries creation. Although the vegetal biomasses result widely available, rising costs of food raw material such as wheat, corn and sugar beet have raised a serious ethical problem using these resources. To avoid the use of such raw materials, the exploitation of lignocellulosic biomasses plays a fundamental role in the industry. However, for an efficient utilization of lignocellulosic biomasses, new technologies are required in order to transform the starting biomass into simple molecules, such as pentoses and hexoses sugars, more easily to use by the microrganism, which will have the task of producing both fine chemicals and bulk chemicals in an economically and environmentally sustainable processes. In this regard, industrial biotechnologies should be able to develop new microrganisms capable to face the harsh environmental conditions that occur during an industrial production process. For many of these productions the yeast Saccharomyces cerevisiae is largely used, not only because of its naturally ability to produce large ethanol amount, but also is widely known both at genetic and metabolic level, outlining a good starting point for the development of producers strains with high tolerance against different stresses occur during an industrial process. This is the view adopted by NEMO project (Novel high performance Enzymes and Microrganisms for conversion of lignocellulosic biomass to ethanol), belonging to the European Union seventh framework program, where it become of primary importance the development of microrganisms, especially S.cerevisiae, for the second generation ethanol production. Microrganisms must be, on the one hand able to efficiently utilize all the sugars released from lignocellulosic biomass pre-treatment, on the other hand should be more tolerant against process conditons, such as inhibitory compounds and environmental stresses. A point of relevant importance is the ability to utilize pentose sugars, like D-xylose, released in large amount after lignocellulose pre-treatment. Currently, worldwide researches are focused on the development of yeast strains engineered with xylose degradation pathways involving the pentose phosphate pathway. In fact the fungal pathway exploits xylose reductase and the xylitol dehydrogenase while the bacterial pathway exploits xylose isomerase; both pathways degrade D-xylose into D-xylulose, which will enter into pentose phosphate pathway. In addition to these two pathways studied since the ‘80s of the last century, there also two other poorly known metabolisms, described for the first time in the ‘70s, which produce alpha-ketoglutarate or pyruvate and glycolaldehyde through an oxidative xylose degradation. These pathways are composed of 5 enzymatic reactions by the Weimberg’s pathway and of 4 enzymatic reactions by the Dahms’ pathway, however they share the first 3 enzymatic reactions. After bioinformatics we were identified the presence of Weimberg’s pathway into Burkholderia xenovorans, while the reaction that characterizes the Dahms’ pathway has been identified in Escherichia coli. The encoding genes for these enzymatic activities were expressed in S.cerevisiae, and the capacity to grow on D-xylose as carbon source are evaluated. The reconstruction of these two pathways showed a poorly growth capacity on xylose. Such growth limitation seems to be related to several factors: the presence of bottlenecks associated to enzymes functionality, like D-xylonate dehydratase activity; the yeast ability to internalize xylose efficiently; the involved genes optimization. Another important aspect is the yeast ability to face and overcome environmental stresses encountered during an industrial process. The cytoplasmic membrane plays a key role in cellular homeostasis, being at the interface between the cell and the external environment, and reacting at environmental changes. The plant membrane protein TIL gives particular strength to the yeast cells when these are subjected to environmental stresses of industrial relevance, such as the presence of oxidative agents or during temperature changes. However, when TIL is expressed in an industrial and/or in an engineered laboratory strains, for industrial use, the protective effect against prolonged stress exposure and process conditions disappear. Finally, a further important aspect during an industrial process is the S.cerevisiae ability to tolerate the growth inhibitory compounds presence into pre-treated lignocellulose. In fact has been largely described how chemical compounds like aldehydes, organic acids and phenolic compounds, released during lignocellulose pre-treatment process, are toxic at certain concentration, inhibiting S.cerevisiae growth or causing yeast death. The growth performance of different wild type or engineered yeast strains are evaluated on spruce and giant cane lignocellulose pre-treated: in addition the same strains were tested on minimal formulated medium according to the spruce pre-treated composition. The results showed that the combination of low pH and the presence of organic acids, especially acetic acid and formic acid, are dramatically harmful for growth of both industrial strain, naturally more tolerant, and engineered strain, for the production and recycle of L-ascorbic acid. However, the behavior of engineered strain for production and recycle of L-ascorbic acid is interesting at low pH, because showed higher tolerance than other strains in terms of growth rate and ethanol production and productivity. Despite the positive results obtained by engineering microrganisms, especially S.cerevisiae, in laboratory, their industrial uses still remain limited. Therefore, appears extremely important the construction of more robustness strains, able to withstand different environmental conditions along an entire industrial process, with consequent influence on yields, production and productivity. For these reasons, the research is aimed to combine these aspects to provide the best microrganism possible to industry productions.