Disruption of hypoxia-inducible factor-2α in neutrophils decreases colitis-associated colon cancer.

Neutrophils are abundant immune cells in the colon tumor microenvironment. Studies have shown that neutrophils are recruited into hypoxic foci in colon cancer. However, the impact of hypoxia signaling on neutrophil function and its involvement in colon tumorigenesis remain unclear. To address this, we generated mice with a deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α in neutrophils driven by the MRP8Cre (HIF-1αΔNeu) or (HIF-2αΔNeu) and littermate controls. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colon cancer, the disruption of neutrophils-HIF-1α did not result in any significant changes in body weight, colon length, tumor size, proliferation, or burden. However, the disruption of HIF-2α in neutrophils led to a slight increase in body weight, a significant decrease in the number of tumors, and a reduction in tumor size and volume compared with their littermate controls. Histological analysis of colon tissue from mice with HIF-2α-deficient neutrophils revealed notable reductions in proliferation as compared with control mice. In addition, we observed reduced levels of proinflammatory cytokines, such as TNF-α and IL-1ß, in neutrophil-specific HIF-2α-deficient mice in both the tumor tissue as well as the neutrophils. Importantly, it is worth noting that the reduced tumorigenesis associated with HIF-2α deficiency in neutrophils was not evident in already established syngeneic tumors or a DSS-induced inflammation model, indicating a potential role of HIF-2α specifically in colon tumorigenesis. In conclusion, we found that the loss of neutrophil-specific HIF-2α slows colon tumor growth and progression by reducing the levels of inflammatory mediators.NEW & NOTEWORTHY Despite the importance of hypoxia and neutrophils in colorectal cancer (CRC), the contribution of neutrophil-specific HIFs to colon tumorigenesis is not known. We describe that neutrophil HIF-1α has no impact on colon cancer, whereas neutrophil HIF-2α loss reduces CRC growth by decreasing proinflammatory and immunosuppressive cytokines. Furthermore, neutrophil HIF-2α does not reduce preestablished tumor growth or inflammation-induced colitis. The present study offers novel potential of neutrophil HIF-2α as a therapeutic target in CRC.

CDK5: an oncogene or an anti-oncogene: location location location.

Recent studies have uncovered various physiological functions of CDK5 in many nonneuronal tissues. Upregulation of CDK5 and/or its activator p35 in neurons promotes healthy neuronal functions, but their overexpression in nonneuronal tissues is causally linked to cancer of many origins. This review focuses on the molecular mechanisms by which CDK5 recruits diverse tissue-specific substrates to elicit distinct phenotypes in sixteen different human cancers. The emerging theme suggests that CDK5's role as an oncogene or anti-oncogene depends upon its subcellular localization. CDK5 mostly acts as an oncogene, but in gastric cancer, it is a tumor suppressor due to its unique nuclear localization. This indicates that CDK5's access to certain nuclear substrates converts it into an anti-oncogenic kinase. While acting as a bonafide oncogene, CDK5 also activates a few cancer-suppressive pathways in some cancers, presumably due to the mislocalization of nuclear substrates in the cytoplasm. Therefore, directing CDK5 to the nucleus or exporting tumor-suppressive nuclear substrates to the cytoplasm may be promising approaches to combat CDK5-induced oncogenicity, analogous to neurotoxicity triggered by nuclear CDK5. Furthermore, while p35 overexpression is oncogenic, hyperactivation of CDK5 by inducing p25 formation results in apoptosis, which could be exploited to selectively kill cancer cells by dialing up CDK5 activity, instead of inhibiting it. CDK5 thus acts as a molecular rheostat, with different activity levels eliciting distinct functional outcomes. Finally, as CDK5's role is defined by its substrates, targeting them individually or in conjunction with CDK5 should create potentially valuable new clinical opportunities.

Mapping of molecular interactions between human E3 ligase TRIM69 and Dengue virus NS3 protease using hydrogen-deuterium exchange mass spectrometry.

Tripartite motif (TRIM) E3 ligases target specific substrates, including viral proteins, for proteasomal degradation, and are thus essential regulators of the innate antiviral response. TRIM69 ubiquitinates the non-structural NS3 protein of Dengue virus for its degradation by the host machinery. This antiviral strategy abrogates the immunosuppression mediated by the NS2B-NS3 protease complex. To understand how this host-driven antiviral response against Dengue virus, we sought to define the mode of interaction between human TRIM69 and Dengue NS2B-NS3 and the subsequent polyubiquitination of the protease by the E3 ligase. We show that NS2B-NS3Δpro is sufficient as a substrate for ubiquitination by TRIM69 using ELISA and in vitro assays. Using hydrogen-deuterium exchange mass spectrometry (HDXMS), we mapped the interface of the interaction between TRIM69 and NS2B-NS3Δpro, and propose a rationale for the binding and subsequent ubiquitination process. Furthermore, through sequence analysis, we showed that the regions targeted by TRIM69 on the DENV-2 NS3 protease (NS3Δpro) are well conserved across DENV serotypes and other flaviviruses, including Zika virus, West Nile virus, and Japanese encephalitis virus. Our results show the direct interactions of TRIM69 with viral proteins, provide mechanistic insights at a molecular level, and highlight the functional relevance of TRIM69 interacting with the Dengue viral protein. Collectively, our findings suggest that TRIM69 may act as a pan-antiflaviviral restriction factor.

Expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes in relation to lactation performance in Deoni and Holstein Friesian crossbred cows.

The study was designed with the objective of expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes on lactation performance in Bos indicus and HF crossbred cows during early lactation. BAX/BCL-2 mRNA expression ratio in HF crossbreds showed a steady increase from 30th day to 90th day, but in Deoni cows the ratio exhibited a different pattern, which increased from day 30 to day 60, decreased on day 75, and then increased on day 90. BAX/BCL-2 expression ratio in Deoni and HF crossbreds were lowest on day 30 and highest on day 90. On contrary, the milk yield was highest on day 30 and lowest on day 90 suggesting BCL-2 gene favors milk production and BAX gene oppose milk production. In comparison to HF crossbreds, Deoni cows exhibited highest BAX/BCL-2 ratio at the end of early lactation, indicating Bos indicus cows were more sensitive to apoptosis than HF crossbreds. Comparison of daily milk yield with BAX/BCL-2 mRNA expression ratio revealed significant negative correlation with a correlation coefficient of -0.98 (P < 0.01) and -0.95 (P < 0.05) in Deoni and HF crossbred cows, respectively. Our study provides new insights into understanding the genetic control of mammary apoptosis between Bos indicus and HF crossbreds.

Genome-wide association mapping reveals genes underlying population-level metabolome diversity in a fungal crop pathogen.

BACKGROUND: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood. RESULTS: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation. CONCLUSIONS: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies.

Effect of α-tocopherol in the vitrification medium on the viability, lipid peroxidation, expression of key developmental, apoptotic and stress-related genes in ovine secondary follicles.

The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 µm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.

Exploring the gut microbiota composition of Indian major carp, rohu (Labeo rohita), under diverse culture conditions.

Gut microbiota of freshwater carps are often investigated for their roles in nutrient absorption, enzyme activities and probiotic properties. However, little is known about core microbiota, assembly pattern and the environmental influence on the gut microbiota of the Indian major carp, rohu. The gut microbial composition of rohu reared in different culture conditions was analysed by 16S rRNA amplicon sequencing. There was variation on gut microbial diversity and composition. A significant negative correlation between dissolved oxygen content (DO) and alpha diversity was observed, thus signifying DO content as one of the key environmental factors that regulated the diversity of rohu gut microbial community. A significant positive correlation was observed between phosphate concentration and abundance of Actinobacteria in different culture conditions. Two phyla, Proteobacteria and Actinobacteria along with OTU750868 (Streptomyces) showed significant (p < 0.05) differences in their abundance among all culture conditions. The Non-metric multidimensional scaling ordination (NMDS) analysis using Bray-Curtis distances, showed the presence of unique gut microbiota in rohu compared to other herbivorous fish. Based on niche breadth, 3 OTUs were identified as core generalists, persistent across all the culture conditions whereas the specialists dominated in the rohu gut microbiota assembly. Co-occurrence network analysis revealed positive interaction within core members while mutual exclusion between core and non-core members. Predicted microbiota function revealed that different culture conditions affected the metabolic capacity of gut microbiota of rohu. The results overall indicated the significant effect of different rearing environments on gut microbiota structure, assembly and inferred community function of rohu which might be useful for effective manipulation of gut microbial communities of rohu to promote better health and growth under different husbandry settings.

Effect of cryopreservation and capacitation on expression patterns of ATP synthesis associated genes in bubaline spermatozoa.

This study investigated the expression patterns of certain ATP synthesis associated genes (GAPDHS, PGK2, ENO4 and MDH2) in fresh and frozen-thawed bubaline spermatozoa vis-à-vis capacitation. Eight adult Murrah buffalo bulls, aged 5-7 years, were randomly selected from a bull farm and fresh semen was collected once a week for four weeks (n = 32) from the selected bulls. Simultaneously, frozen straws of the same bulls were collected (n = 32, four straws were pooled per sample). The samples were divided into four groups i.e., fresh non capacitated (control/group 1), fresh capacitated (group 2), frozen non capacitated (group 3) and frozen capacitated (group 4). In all groups mRNA extraction was done. The mRNA expression of GAPDHS was significantly higher in group 3 compared to the other groups, whereas the expression of ENO4 was significantly up-regulated in group 2 and 3 compared to the group 4. Similarly, the mRNA expression of PGK2 was significantly up-regulated in group 3 whereas in case of MDH2, it was significantly higher in group 2, 3 and 4 compared group 1. It can be concluded from our study that freezing and capacitation has a significant effect on the expression dynamics of the ATP synthesis-associated genes warranting necessary interventions in handling procedures to minimise the adverse effect of freezing on ATP synthesis process to ensure persistence of fertilizing ability of sperm.

Alteration of gut microbiota composition and function of Indian major carp, rohu (Labeo rohita) infected with Argulus siamensis.

Gut microbiome homeostasis is critical in preventing diseases. However, the effect of disease on gut microbiota assembly remains unclear. At present, there are no reports on the composition and functional analysis of intestinal microbiota of Indian major carp, rohu (L. rohita) infected with ectoparasite, Argulus. In this study, we analysed and compared the intestinal microbiota of healthy and Argulus-infected rohu by 16S rRNA amplicon sequencing. Argulus infection could significantly influence the diversity and richness of the gut microbiota. However, abundance of Actinobacteria and Patescibacteria were enriched significantly in Argulus-infected fish. Venn diagram revealed that there were many more unique genera in the infected group as compared to control fish. The genera, Stenotrophomonas and Pirellula were significantly increased in infected fish while the abundance of Reyranella was decreased. LEfSe analysis showed a significant enrichment in abundances of 11 taxa in healthy group and 17 taxa in infected group. Furthermore, genera Rubellimicrobium, Dielma, Hyphomicrobium, Reyranella, Streptomyces and Cloacibacterium performed the best in differentiating between both the groups. Predicted microbiota function by PICRUSt revealed that the gut microbiota of infected fish was mainly associated with enriched synthesis of chitinases, chitin binding proteins, osmoprotectant proteins and sulfatases enzymes. There was a positive association between the structural and functional composition of the gut microbiota. The results indicated that the Argulus infection could affect the intestinal microbiota composition and function of rohu.

Solvent controlled synthesis of 2,3-diarylepoxy indenones and α-hydroxy diarylindanones and their evaluation as inhibitors of DNA alkylation repair.

Herein, we report a novel and unexpected metal-free oxygenation of 2,3-diphenyl-1-indenones, under an oxygen atmosphere (air), to either 2,3-epoxy-2,3-diphenyl-1-indenone or 2-hydroxy-2,3-diphenyl-1-indanone, depending on the conditions. Several bioactive epoxy indenones and one-pot α-hydroxy indanones (α-acyloin) were synthesized from 2,3-diaryl dihydroindanone and 2,3-diarylindenone, respectively. A plausible reaction mechanism is also proposed, where oxygenation would take place at the α-position and further proton abstraction from the ß-position leads to epoxy indenone derivatives. A one-pot cis-hydroxy indanone protocol is also achieved directly from biaryl indenone via reduction, epimerization, and oxygenation. The synthesized compounds were evaluated for inhibitory activity against the DNA repair protein AlkB. Among the screened (17 tested) compounds, one epoxide derivative was found to be a specific inhibitor of AlkB enzyme function.

Regulatory role of Wnt signal in the oestradiol synthesis of different size categories of ovarian follicles in buffalo (Bubalus bubalis).

The aim of the present study was to understand the role of Wnt signal in ovarian oestradiol synthesis in various size categories of ovarian follicles. A six-day cell culture system was adopted to test the effect of a Wnt inhibitor i.e. Inhibitor of Wnt response (IWR) on the ovarian granulosa cell oestradiol synthesis and associated genes related to oestradiol synthesis and Wnt signalling (CYP19A1, CCND2, WNT2, FZD6, DVL1, APC, AXIN2, CTNNB1) in buffalo. It was conducted with four groups: Group 1: control, Group 2: control + FSH, Group 3: IWR, Group 4: IWR + FSH. No significant effect of IWR was observed on the ovarian granulosa cell proliferation. No significant difference in the oestradiol levels was found in the spent media harvested after six days of in vitro culture among different groups in small and large-sized ovarian follicles. However, the oestradiol level varied significantly (p < .05) among different treatment groups in medium-sized follicles. The oestradiol level was significantly lower (p < .05) in IWR group compared with the control group and was also significantly lower in IWR + FSH group compared with the FSH group. The Wnt inhibitor had significantly (p < .05) reduced the gene expression of CYP19A1 in large ovarian follicles. Varied effects of IWR-1 and FSH on the expression of other genes were observed. The results indicated that there is a positive role of Wnt signal in oestradiol synthesis in buffalo, but the positive role was more discernible in medium- and large-sized follicles.

Rapid sequence evolution driven by transposable elements at a virulence locus in a fungal wheat pathogen.

BACKGROUND: Plant pathogens cause substantial crop losses in agriculture production and threaten food security. Plants evolved the ability to recognize virulence factors and pathogens have repeatedly escaped recognition due rapid evolutionary change at pathogen virulence loci (i.e. effector genes). The presence of transposable elements (TEs) in close physical proximity of effector genes can have important consequences for gene regulation and sequence evolution. Species-wide investigations of effector gene loci remain rare hindering our ability to predict pathogen evolvability. RESULTS: Here, we performed genome-wide association studies (GWAS) on a highly polymorphic mapping population of 120 isolates of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe. We identified a major locus underlying significant variation in reproductive success of the pathogen and damage caused on the wheat cultivar Claro. The most strongly associated locus is intergenic and flanked by genes encoding a predicted effector and a serine-type endopeptidase. The center of the locus contained a highly dynamic region consisting of multiple families of TEs. Based on a large global collection of assembled genomes, we show that the virulence locus has undergone substantial recent sequence evolution. Large insertion and deletion events generated length variation between the flanking genes by a factor of seven (5-35 kb). The locus showed also strong signatures of genomic defenses against TEs (i.e. RIP) contributing to the rapid diversification of the locus. CONCLUSIONS: In conjunction, our work highlights the power of combining GWAS and population-scale genome analyses to investigate major effect loci in pathogens.

Phosphorylation-dependent regulation of SPOP by LIMK2 promotes castration-resistant prostate cancer.

BACKGROUND: SPOP, an E3 ubiquitin ligase adaptor, can act either as a tumour suppressor or a tumour promoter. In prostate cancer (PCa), it inhibits tumorigenesis by degrading several oncogenic substrates. SPOP is the most altered gene in PCa (~15%), which renders it ineffective, promoting cancer. The remaining PCa tumours, which retain WT-SPOP, still progress to castration-resistant (CRPC) stage, indicating that other critical mechanisms exist for downregulating SPOP. SPOP is reduced in ~94% of WT-SPOP-bearing prostate tumours; however, no molecular mechanism is known for its downregulation. METHODS: SPOP was identified as a direct target of LIMK2 using an innovative technique. The reciprocal relationship between SPOP and LIMK2 and its consequences on oncogenicity were analysed using a variety of biochemical assays. To probe this relationship in vivo, xenograft studies were conducted. RESULTS: LIMK2 degrades SPOP by direct phosphorylation at three sites. SPOP promotes LIMK2's ubiquitylation, creating a feedback loop. SPOP's degradation stabilises AR, ARv7 and c-Myc promoting oncogenicity. Phospho-resistant SPOP completely suppresses tumorigenesis in vivo, indicating that LIMK2-mediated SPOP degradation is a key event in PCa progression. CONCLUSIONS: While genomically altered SPOP-bearing tumours require gene therapy, uncovering LIMK2-SPOP relationship provides a powerful opportunity to retain WT-SPOP by inhibiting LIMK2, thereby halting disease progression.

The complex genomic basis of rapid convergent adaptation to pesticides across continents in a fungal plant pathogen.

Convergent evolution leads to identical phenotypic traits in different species or populations. Convergence can be driven by standing variation allowing selection to favour identical alleles in parallel or the same mutations can arise independently. However, the molecular basis of such convergent adaptation remains often poorly resolved. Pesticide resistance in agricultural ecosystems is a hallmark of convergence in phenotypic traits. Here, we analyse the major fungal pathogen Zymoseptoria tritici causing serious losses on wheat and with fungicide resistance emergence across several continents. We sampled three population pairs each from a different continent spanning periods early and late in the application of fungicides. To identify causal loci for resistance, we combined knowledge from molecular genetics work and performed genome-wide association studies (GWAS) on a global set of isolates. We discovered yet unknown factors in azole resistance including a gene encoding membrane associated functions. We found strong support for the "hotspot" model of resistance evolution with convergent changes in a small set of loci but additional loci showed more population-specific allele frequency changes. Genome-wide scans of selection showed that half of all known resistance loci were overlapping a selective sweep region. Hence, the application of fungicides was one of the major selective agents acting on the pathogen over the past decades. Furthermore, loci identified through GWAS showed the highest overlap with selective sweep regions underlining the importance to map phenotypic trait variation in evolving populations. Our population genomic analyses highlighted that both de novo mutations and gene flow contributed to convergent pesticide adaptation.

Synthesis and antibacterial activities of marine natural product ianthelliformisamines and subereamine synthetic analogues.

Marine sponges of the genusSuberea produce variety of brominated tyrosine alkaloids which display diverse range of biological activities including antiproliferative, antimicrobial and antimalarial activities. In continuation of our search for biologically active marine natural products for antibacterial compounds, we report here the synthesis and evaluation of biological activity of panel of ianthelliformisamines and subereamine analogues using the literature known acid-amine coupling reaction. Several derivatives of Ianthelliformisamine were achieved by the coupling of Boc-protected polyamine chain with brominated aromatic acrylic acid derivatives by varying the bromine substituents on aromatic acid derivatives, amine spacer as well as geometry of the double bond, and then Boc-deprotection using TFA. Similarly, subereamine analogues were also synthesized employing coupling reaction between various brominated phenyl acrylic acids with commercially available chiral amino ester derivatives followed by ester hydrolysis. We screened these synthetic analogues for antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. One of the compound 7c showed bactericidal activity against Staphylococcus aureus with an IC50 value of 3.8 µM (MIC = 25 µM).

Copper and Selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: possible involvement of AKT and WNT signalling pathways.

The role of copper and selenium on activation of estradiol synthesis pathways viz. PKA/AKT/WNT is not clearly elucidated. On this background we attempt to elcuiated the role of copper and selenium on mRNA expression of genes associated with estradiol synthesis in caprine ovarian granulose cell models. Ovarian granulosa cells from medium (3-5 mm) sized follicles were aspirated and distributed separately to different groups. Group I: control, Group II: cupric chloride (Cu: 0.5 mM), Group III: sodium selenite (Se: 100 ng/ml), Group IV: Cu + Se. The cells (105/well) were cultured in 96 well plate in the base culture medium of MEMα comprising of nonessential amino acids (1.1 mM), FSH (10 ng/mL), transferrin (5 µg/mL), IGF-I (2 ng/mL), androstenedione (10-6 M), penicillin (100 IU/mL), streptomycin (0.1 mg/mL) and fungizone (0.625 µl/mL) and insulin (1 ng/mL). The cells were incubated in a carbondioxide incubator (38 °C, 5% CO2, 95% RH). The medium was changed on alternate days and cells were harvested on day 6. Day 6 media was used for estimation of estradiol. The RNA isolated form harvested cells was used for qPCR assay. There was no significant (p > 0.05) difference in estradiol concentration between groups. The mRNA expression of AKT1, CYP19A1, WNT2 & 4, FZD6 and APC2 were significantly (p < 0.05) higher in Cu and Cu + Se groups compared to control. Whereas, the mRNA transcript of DVL1 and CSNK1 was significantly (p < 0.05) higher in Cu + Se group compared to control. Incontrast, no significant difference in mRNA expression of PRKAR1A and CTNNB1 was noticed. Our study support a key role of copper and selenium in activation of AKT and WNT signalling pathway that further lead to increase in the mRNA expression of CYP19A1.

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis).

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1-Control fresh preantral follicles; Group 2-Vitrification treatment (Vitrification solution 1 (VS1) -TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3-vitrification treatment +5 µM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 µM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.

The Cdk5-Mcl-1 axis promotes mitochondrial dysfunction and neurodegeneration in a model of Alzheimer's disease.

Cdk5 deregulation is highly neurotoxic in Alzheimer's disease (AD). We identified Mcl-1 as a direct Cdk5 substrate using an innovative chemical screen in mouse brain lysates. Our data demonstrate that Mcl-1 levels determine the threshold for cellular damage in response to neurotoxic insults. Mcl-1 is a disease-specific target of Cdk5, which associates with Cdk5 under basal conditions, but is not regulated by it. Neurotoxic insults hyperactivate Cdk5 causing Mcl-1 phosphorylation at T92. This phosphorylation event triggers Mcl-1 ubiquitylation, which directly correlates with mitochondrial dysfunction. Consequently, ectopic expression of phosphorylation-dead T92A-Mcl-1 fully prevents mitochondrial damage and subsequent cell death triggered by neurotoxic treatments in neuronal cells and primary cortical neurons. Notably, enhancing Mcl-1 levels offers comparable neuroprotection to that observed upon Cdk5 depletion, suggesting that Mcl-1 degradation by direct phosphorylation is a key mechanism by which Cdk5 promotes neurotoxicity in AD. The clinical significance of the Mcl-1-Cdk5 axis was investigated in human AD clinical specimens, revealing an inverse correlation between Mcl-1 levels and disease severity. These results emphasize the potential of Mcl-1 upregulation as an attractive therapeutic strategy for delaying or preventing neurodegeneration in AD.

The Aurora-A-Twist1 axis promotes highly aggressive phenotypes in pancreatic carcinoma.

We uncovered a crucial role for the Aurora kinase A (AURKA)-Twist1 axis in promoting epithelial-to-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer. Twist1 is the first EMT-specific target of AURKA that was identified using an innovative screen. AURKA phosphorylates Twist1 at three sites, which results in its multifaceted regulation - AURKA inhibits its ubiquitylation, increases its transcriptional activity and favors its homodimerization. Twist1 reciprocates and prevents AURKA degradation, thereby triggering a feedback loop. Ablation of either AURKA or Twist1 completely inhibits EMT, highlighting both proteins as central players in EMT progression. Phosphorylation-dead Twist1 serves as a dominant-negative and fully reverses the EMT phenotype induced by Twist1, underscoring the crucial role of AURKA-mediated phosphorylation in mediating Twist1-induced malignancy. Likewise, Twist1-overexpressing BxPC3 cells formed large tumors in vivo, whereas expression of phosphorylation-dead Twist1 fully abrogated this effect. Furthermore, immunohistochemical analysis of pancreatic cancer specimens revealed a 3-fold higher level of Twist1 compared to that seen in healthy normal tissues. This is the first study that links Twist1 in a feedback loop with its activating kinase, which indicates that concurrent inhibition of AURKA and Twist1 will be synergistic in inhibiting pancreatic tumorigenesis and metastasis.

Quality characterization and effect of sonication time on bioactive properties of honey from North East India.

The North-Eastern region of India is known for its rich flora and has great potential for honey production. Honey samples collected from local markets of different regions of North-East India viz. Shillong, Sohra, Mawsynram, Jorhat and Tezpur were analyzed for physicochemical properties, bioactivity and mineral content. Effect of sonication time on the bioactive properties of honey was determined. All the honey samples had good bioactive properties and high content of potassium, sodium, calcium and iron. The Jorhat sample had the highest phenolic content (296.68 ± 2.16 mg GAE/100 g) and flavonoid content (155.26 ± 2.90 µg quercetin/100 g) whereas highest DPPH radical scavenging activity with an IC50 value of 29.8 ± 0.20 g and a FRAP value of 7291.60 ± 584.6 µM Fe(II)/100 g were noted in Shillong sample. Sonication exhibited various effects on the bioactive properties of the selected honey samples based on their source and treatment time. Honey from Jorhat and Sohra exhibited good quality standards with HMF content less than 80 mg/kg.