Biology subjects

Gupta, S. K.

Publications and source records attributed to Gupta, S. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Genotype imputation improves GWAS resolution and facilitates candidate gene identification in pearl millet

Pearl millet (Cenchrus americanus; syn. Pennisetum glaucum (L.) R. Br.) is an important cereal crop mainly grown in semi-arid areas. However, in pearl millet, genes underlying many agronomic traits remain unknown. Our previous genome-wide association study (GWAS) was based primarily on genotype data derived from restriction site-associated DNA sequencing (RAD-seq), which contained a high proportion of missing genotypes and may have limited mapping resolution. To address this limitation, we used whole-genome sequencing (WGS)-derived genotype data from 21 elite lines as a reference panel to impute missing genotypes in the RAD-seq-derived dataset. Genotype imputation reduced the proportion of missing genotypes from 82.42% to 0% and increased the mean density of non-missing genotypes from 569.61 to 1,158.09 genotypes/Mb. Leave-one-out validation yielded a mean genotype concordance of 89.9% (SD = 4.6%). GWAS using the imputed dataset showed more localized association signals for several traits. Homologs of rice SHORT INTERNODES1, Arabidopsis FLOWERING LOCUS Y, and rice EPIDERMAL PATTERNING FACTOR 2 were identified as candidates for genes regulating culm height, days to panicle emergence, and awn growth, respectively. These results demonstrate that reference-panel-based genotype imputation can increase the utility of reduced-representation genotype datasets and facilitate candidate gene identification in pearl millet.

genomics↗

bioRxiv2025

A highly efficient method to differentiate CGRP-expressing peptidergic nociceptors from human induced pluripotent stem cells

Human cellular models of disease developed using induced pluripotent stem cells (iPSC) have enabled wide-ranging research from investigation of human disease mechanisms to phenotypic drug screens. Pain disorders such as neuropathic pain and headache remain areas of considerable unmet need and are considered high risk by pharma. Human iPSC-derived sensory neurons have already been used to accelerate translational research but the current differentiation protocols produce non-peptidergic nociceptors. We demonstrate for the first time the robust differentiation of hiPSC into peptidergic nociceptor lineage with high yield. These nociceptors express CGRP and TRPV1 whilst the non-peptidergic marker RET was completely absent. Functionally, the nociceptors show functional maturity including the expression of TTX-resistant currents and responding to TRPV1 and TRPA1 agonists. Importantly, they were able to release CGRP basally and upon stimulation by an inflammatory soup, which was inhibited upon the application of the 5-HT1B/1D/1F agonist, sumatriptan and topiramate, a migraine prophylactic drug. Overall, we report the successful generation of a novel in vitro functional peptidergic nociceptor model which will allow investigation of disease mechanisms in pain and for the development of new effective pain therapies.

neuroscience↗

bioRxiv2025

Loss-of-Function RUP-Variants Influence Ubiquitin-Proteasome System and Enhance Carotenoid and Folate Levels in Tomato

REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) negatively regulates UV- B signalling, yet its broader physiological roles in crop plants remain largely unexplored. We compared two tomato accessions bearing truncated RUP proteins--rup-1 and rup-2 (rup- variants)--with the cultivar Arka Vikas (AV), which harbours the native RUP protein. Seedlings of rup-variants exhibited enhanced tolerance to supplemental UV-B light. Red ripe (RR) fruits of rup-variants showed significantly elevated carotenoid and folate levels compared to AV. Introgression of rup-variants into AV confirmed that the increased carotenoid accumulation is genetically linked to RUP truncation. Metabolomic profiling of rup-variants revealed a substantial shift in primary metabolic homeostasis, particularly at the breaker stage, marked by a pronounced reduction in sugars and amino acids. Proteomic analyses of rup- variants across ripening stages identified that a significant proportion of differentially expressed proteins belonged to chaperones and ubiquitin-proteasome system (UPS). Upregulation of four key enzymes in the carotenoid biosynthesis pathway likely contributed to increased lycopene content in rup-variants. Elevated folate levels in rup-variants were associated with the upregulation of folate biosynthesis and C1 metabolism enzymes. Despite widespread metabolic reprogramming in rup-variants, hormonal regulatory pathways remained largely unaltered. Our results suggest that RUP modulates metabolic pathways during fruit ripening, and its loss triggers metabolic reprogramming associated with elevated folate/carotenoid levels.

plant biology↗

bioRxiv2025

M6A reader protein YTHDF3 regulates cardiomyocyte death and atrophy by modulating the alternative splicing program

BackgroundThe functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. ObjectiveWe aim to uncover the function of Ythdf3 in the hearts. Methods& ResultsHere, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKII{delta}, and its knockdown leads to an increase in CaMKII{delta}A and CaMKII{delta}C isoforms while a decrease in CaMKII{delta}9 isoform. ConclusionCollectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program.

cell biology↗

bioRxiv2024

Salmonella effector SseL induces PD-L1 up-regulation and T cell inactivation via beta-catenin signalling axis

The upregulation of PD-L1 by various pathogens is a recognized strategy to evade the adaptive immune response. Salmonella infection also upregulates PD-L1 levels causing culling of the activated T-cell; however, the underlying mechanism behind this upregulation is not known. Our findings indicate that the upregulation of PD-L1 is through Salmonella pathogenicity island 2 (SPI-2) encoded effectors since PFA-fixed STM WT and STM{Delta}ssaV (which is unable to secrete effector proteins) did not alter PD-L1 levels. We have further investigated the role of the SPI-2 effector SseL (a deubiquitinase known to affect the NF-B pathway) in PD-L1 upregulation. Our study identifies SPI-2 effector SseL to be crucial for upregulating PD-L1 in vitro as well as in vivo murine models. The increase in PD-L1 levels induced by STM WT facilitates colonization in secondary infection sites in C57BL/6 mice, including the liver and spleen, while the STM{Delta}sseL strain exhibits significant colonization defects. Notably, despite the reduced colonization capacity of STM{Delta}sseL, infected mice exhibit earlier mortality associated with heightened inflammation. We further elucidated the molecular mechanism behind PD-L1 upregulation and observed that bacterial effector SseL helps in the stabilization of {beta}-catenin inside the cell. {beta}-catenin thus translocates into the nucleus and directly regulates the transcriptional levels of PD-L1, which is abrogated upon using {beta}-catenin/TCF inhibitor FH535. Collectively, our study elucidates the mechanism by which Salmonella mediates immune suppression through PD-L1 upregulation. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/620790v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ba486org.highwire.dtl.DTLVardef@3e8b40org.highwire.dtl.DTLVardef@28546corg.highwire.dtl.DTLVardef@1bea897_HPS_FORMAT_FIGEXP M_FIG Abstract figure: Schematic representation of SseL mediated PDL1 upregulation and further affecting the T cell proliferation C_FIG

immunology↗

bioRxiv2024

Knockout of the tomato HAIRY MERISTEM 4 alters phloem-characteristics and impairs development

The HAIRY MERISTEM (HAM) gene family encodes Type I and II GRAS domain transcription factors in plants. Type II HAMs, predominantly expressed in meristems and regulated by microRNA171, are essential for maintaining undifferentiated meristems, a role conserved across various species. Conversely, the functions of Type I HAMs have been less characterized. In this study, we investigated the role of SlHAM4, a Type I HAM in tomato. Using publicly available expression data and a GUS reporter gene driven by the native SlHAM4 promoter, we determined that SlHAM4 is predominantly expressed in phloem tissues. CRISPR-induced SlHAM4 loss-of- function mutations (slham4CR) resulted in a range of shoot and fruit abnormalities, which were fully reversed by reintroducing SlHAM4 under its native promoter in the mutant background. Mutant abnormalities included increased anthocyanin pigmentation in the leaf and sepal primordia, reminiscent of the phenotypes observed in certain Arabidopsis mutants with compromised phloem, and development of simpler leaves, which was associated with reduction in external phloem area in the leaf rachis. In addition, slham4CR plants produced significantly smaller fruits of which a fraction of them exhibited catface-like scars, attributed to tears which occurred in the pericarp of mutant ovaries following fruit set. Transcriptome analysis of the wild-type looking mutant ovaries at anthesis revealed specific downregulation of genes implicated in phloem development and functions, in particular those expressed in companion cells (CC). We propose that SlHAM4 is necessary for proper phloem function in part by regulating the expression of a suite of CCs genes that encode essential phloem proteins. One-sentence summarySlHAM4 is predominantly expressed in the phloem and its knockout alters phloem- characteristics and impair development highlighting its requirement for proper phloem functionality.

plant biology↗

bioRxiv2024

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence in Mycobacterium tuberculosis

The long-term survival of Mtb mandates judicious utilization of the available resources inside the host. Uninterrupted access to host-derived nutrients holds the key to the success of Mtb. Phosphoglucomutase enzyme besides synthesizing glycogen, which serves as a nutrient reservoir, also helps modulate the carbon flux in different pathogens. Studies on the role of glycogen metabolism in disease progression, reactivation, and drug susceptibility in tuberculosis are severely lacking. To investigate this, we generated an Mtb strain ({Delta}pgmA) devoid of the gene that encodes for the enzyme phosphoglucomutase A (pgmA). The absence of pgmA impedes the ability of the pathogen to survive under nutrient-limiting and reactivation conditions. In the current study, we demonstrate that the absence of cell membrane-associated glycolipids in {Delta}pgmA compromised the cell wall integrity and increased the susceptibility of {Delta}pgmA to various stresses. Interestingly, in comparison to the wild type, low cAMP levels in {Delta}pgmA imparted an enhanced growth phenotype on cholesterol. Differential gene expression and carbon flux analysis suggest that stored carbon in the form of glycogen is essential for the survival of Mtb under nutrient-limiting conditions. Finally, we demonstrate that the pgmA gene of Mtb is essential for the growth of Mtb inside the host. Overall, this study unveils the significance of pgmA-mediated regulation of membrane glycolipids and its implication on antibiotic and disease persistence in tuberculosis. Additionally, information derived from this study will help design anti-TB strategies that are novel, short, and more efficient.

microbiology↗

bioRxiv2024

Dynamin-1 is a potential mediator in Cancer-Related Cognitive Impairment

Dynamin-1 (DNM1) consolidates memory through synaptic transmission and modulation and has been explored as a therapeutic target in Alzheimers disease. Through a two-prong approach, this study examined its role in cancer-related cognitive impairment (CRCI) pathogenesis using human and animal models. The human study recruited newly diagnosed, chemotherapy-naive adolescent and young adult cancer and non-cancer controls to complete a cognitive instrument (FACT-Cog) and blood draws for up to three time points. Concurrently, a syngeneic young-adult WT (C57BL/6 female) mouse model of breast cancer was developed to study DNM1 expression in the brain. Samples from eighty-six participants with 30 adolescent and young adult (AYA) cancer and 56 non-cancer participants were analyzed. DNM1 levels were significantly lower among cancer participants compared to non-cancer prior to treatment. While receiving cancer treatment, cognitively impaired patients were found with a significant downregulation of DNM1, but not among those without impairment. In murine breast cancer-bearing mice receiving chemotherapy, we consistently found a significant decline in DNM1 immunoreactivity in the hippocampal CA1 and CA3 subregions. Observed in both human and animal studies, the downregulation of DNM1 is linked with the onset of CRCI. Future research should explore the potential of DNM1 in CRCI pathogenesis and therapeutics development.

pathology↗

bioRxiv2023

A tomato ethylene-insensitive mutant displays altered growth and higher β-carotene levels in fruit

The mutants insensitive to ethylene are helpful in deciphering the role of ethylene in plant development. We isolated an ethylene-insensitive tomato (Solanum lycopersicum) mutant by screening for acetylene-resistant (atr-1) seedlings. The atr-1 mutant displayed resistance to kinetin, suggesting attenuation of the ethylene sensing response. atr-1 also exhibited resistance to ABA- and glucose-mediated inhibition of seed germination. Unlike the Never- ripe (Nr) mutant, atr-1 seedlings were resistant to glucose, indicating ethylene sensing in atr-1 is located in a component distinct from Nr. Metabolically, atr-1 seedlings had lower levels of amino acids but higher levels of several phytohormones, including ABA. atr-1 plants grew faster and produced more flowers, leading to a higher fruit set. However, the atr- 1 fruits took a longer duration to reach the red-ripe (RR) stage. The ripened atr-1 fruits had higher {beta}-carotene levels, retained high {beta}-carotene and lycopene levels post-RR stage. The metabolome profiles of post-RR stage atr-1 fruits revealed increased levels of sugars. The atr-1 had a P279L mutation in the GAF domain of the ETR4, a key ethylene receptor regulating tomato ripening. Our study highlights that novel alleles in ethylene receptors may aid in enhancing the nutritional quality of tomato.

plant biology↗

bioRxiv2023

Hypothetical gene Rv0495c regulates redox homeostasis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has evolved sophisticated surveillance mechanisms to regulate and neutralize redox imbalances and associated lethal consequences. Failing this, the accumulated ROS induces toxicity by oxidizing a variety of biological molecules including proteins, nucleic acids and lipids. In the present study we identified Mtbs Rv0495c gene as an important regulator of oxidized cytosolic environment. Compared to wild type Mtb strain lacking the Rv0495c gene, {Delta}Rv0495c, had increased ROS and NAD+/NADH ratio creating a highly oxidized intracellular environment. {Delta}Rv0495c strain demonstrated slow growth phenotype under in vitro and ex-vivo growth conditions and demonstrated enhanced susceptibility to drugs, oxidative, nitrosative and hypoxic growth conditions. In addition, the increase in the superoxide radicals triggered a Fenton-like reaction rendering the {Delta}Rv0495c susceptible to free iron. The increase in the intracellular ROS levels of the {Delta}Rv0495c was further corroborated by an increase in the expression of proteins involved in antioxidant defense and enhanced ROS-mediated oxidation and degradation of mycobacterial lipids. This superoxide-induced lipid degradation resulted in altered colony morphology and loss of membrane integrity in the {Delta}Rv0495c. Surprisingly, despite showing a growth defect phenotype in an ex-vivo macrophage infection model, the absence of the Rv0495c gene in Mtb enhanced the pathogenicity and augmented the ability of the Mtb to grow inside the host. Gene expression analysis revealed a Rv0495c mediated immunomodulation of the host controls inflammation and helps creates a favorable niche for long-term survival of Mtb inside the host. In summary, the current study underscores the fact that the truce in the war between the host and the pathogen favors long-term disease persistence in tuberculosis. We believe targeting Rv0495c could potentially be explored as a strategy to potentiate the current anti-TB regimen.

microbiology↗

bioRxiv2023

Upregulated GRP78 and sFlt-1 in preeclampsia induces IRE1 and ATF6 arms of UPR leading to ER stress in placental cells

IntroductionAmongst vivid and diversified stresses which placenta in preeclampsia undergoes, ER stress has been a hal of the fame which is an insinuation of ill-fated UPR. In our previous study, we have already reported activation of PERK arm due to sFlt-1 present in preeclamptic mothers. The present study is an attempt to interpret rest of the two arms (IRE1 and ATF6) of UPR and ER stress subsequently by in vitro study using BeWo cells after upregulation of master regulator of UPR (GRP78) in placental tissue. Materials and MethodsPart I: Serum analysis of circulating levels of GRP78 and sFlt-1 [in 50 pairs of preeclamptic and normotensive, non-proteinuric (control) pregnant women)] using ELISA. Part II: Correlation analysis of levels of GRP78 and sFlt-1 in preeclamptic and control groups. Part III: Status of expression of GRP78 in placentae (n=10 each, preeclamptic and control groups) was reported using immunofluorescence. Part IV: In vitro experiments using BeWo cells were carried out to analyse the effect of GRP78 and sFlt-1 on IRE1 and ATF6 arms of UPR at protein (immunofluorescence and western blot) and transcript (qRT-PCR) levels. ResultsProtein expressions of GRP78 and sFlt-1 were found significantly elevated in preeclamptic patients as compared to controls. Enhanced expression of master regulator of UPR (GRP78) in placental tissue of preeclamptic women was reported. Up-regulated expression of XBP1 (IRE1 arm) and ATF6 markers of UPR was observed in trophoblast cells. ConclusionThe upregulated expression of GRP78 in preeclamptic placentae and enhanced expression of XBP1 and ATF6 markers in BeWo cells at both protein and transcript levels connote role played by raised circulating levels of GRP78 and sFlt-1 in preeclampsia.

developmental biology↗

bioRxiv2023

Abnormal trophoblast invasion in early onset preeclampsia: involvement of cystathionine β-synthase, specificity protein 1 and miR-22

IntroductionImpaired trophoblast invasion has been observed in early onset Preeclampsia patients (EOPE). Trophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having EOPE is not known. Aims and ObjectivesTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 in EOPE patients and normotensive, non-proteinuric controls. Materials and methods100 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=50) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=50) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 5 ml of venous blood was collected from all the recruited women (2.5 ml in each EDTA and sera vial) followed by plasma and sera separation. Plasma samples were used subsequently to determine gene expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 by qRT-PCR and sera samples were used to estimate their protein levels by ELISA. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated expression of MMPs 2, 9, CBS and Sp1 whereas up regulated expression for that of TIMPs 1, 2 was observed in EOPE patients as compared to healthy pregnant women at both transcription and translation levels. Expression of miR-22 (pre miR-22 and miR-22-3p) was found to be significantly elevated in EOPE patients as compared to normotensive, non-proteinuric controls. ConclusionThis is the very first study of its kind which implicates that down regulated MMPs 2, 9, CBS, Sp1 levels and simultaneously upregulation of miR-22 expression in EOPE patients could have some association. In vitro experiments are needed to prove their association which if proven may provide a new, potential therapeutic target to treat early onset Preeclampsia.

developmental biology↗

bioRxiv2023

MicroRNA-22, Specificity protein-1 and Cystathionine β-synthase in early onset Preeclampsia: significance in trophoblast invasion

IntroductionTrophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having early onset preeclampsia is not known. Aim of the studyTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp-1 and miR-22 in early onset preeclamptic patients and normotensive, non-proteinuric controls at both transcription and translation levels. Materials and methods30 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=15) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=15) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 30 caesarean delivered placentae (15 each of patients and controls) were collected to analyse the mRNA and protein expression/levels of MMPs 2, 9, TIMPs 1, 2, CBS, and Sp1. Protein activity of MMP-2 and 9 was determined. Gene expression of miR-22 was observed in placentae of the recruited patients and controls. Data were analysed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly reduced mRNA and protein expression/levels of MMPs 2, 9, CBS and Sp1 whereas elevated for those of TIMP-1 and TIMP-2 was observed in EOPE patients as compared to controls. Gelatinolytic activity of MMP-2 and 9 was also found significantly reduced in placentae from EOPE patients whereas gene expression of miR-22 was found significantly up regulated in the early onset preeclamptic patients in comparison to controls. ConclusionThis is the first study of its kind which implicates that insufficient trophoblastic invasion may be because of down regulation of MMPs 2, 9, CBS, Sp1 and concomitant up regulation of TIMPs 1, 2 and miR-22 in the early onset preeclamptic patients as compared to controls.

developmental biology↗

bioRxiv2023

Role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine beta-synthase (CBS) axis in regulating trophoblast invasion: an in vitro study

IntroductionPlacenta expresses many ubiquitous as well as specific miRNAs which regulate trophoblast cell differentiation, proliferation, apoptosis, invasion, migration, and angiogenesis. Aberrant miRNAs expression (miR-22) has been linked to pregnancy complications, such as preeclampsia. Specificity protein 1 (Sp1) was identified a direct target of miR-22 and an inverse linear correlation between expression of miR-22 and Sp1 mRNA in gastric tumour has been reported. Also, Sp1 has a critical and indispensable role in the regulation of cystathionine {beta}-synthase (H2S synthesizing enzyme). Exogenous H2S could promote cell proliferation and invasion by upregulating the expression of MMP-2 and MMP-9 in human bladder cancer EJ cells. Worldwide studies reported significant role of MMP-2 and MMP-9 in the regulation of trophoblast invasion. Aims and ObjectivesTo determine the role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine {beta}-synthase (CBS) axis in regulating trophoblast invasion in the first-trimester trophoblast cells. Materials and methodsThe human first trimester trophoblast cell line (HTR-8/SVneo Cell line) was procured from American Type Culture Collection. Gain/Loss of function studies were conducted on HTR-8/SVneo cells and the expression of miR-22 (pre miR-22 and miR-22-3p), Sp1, CBS, MMP-2 and MMP-9 was observed. Invasion capacity of the cells was assessed by transwell invasion assay. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated regulated mRNA and protein expression of Sp1, CBS and and MMPs 2, 9 was observed when the cells were transfected with miR-22-3p mimic in comparison to miR-22-3p inhibitor transfected cells. Expression of CBS and MMPs 2, 9 was found to be significantly up regulated when the cells were transfected with Sp1 plasmid as compared to Sp1 inhibitor (2,4,5 trifluoroaniline) treated cells at both transcription and translation levels. MMP-2 and MMP-9 levels were found to be significantly elevated when the cells were subjected to NaHS [sodium hydrogen sulphide (H2S donor)] treatment as compared to AOAA (aminooxyacetic acid) treated cells. ConclusionThis is the first study which implies that miR-22 via targeting Sp1 must have influenced the mRNA and protein expression of CBS, MMP-2 and MMP-9 in order to regulate the trophoblast invasion.

developmental biology↗

bioRxiv2023

Altered expression of MMP9 and TIMP1 leads to impaired placentation in early onset preeclamptic patients: a case control study

IntroductionMatrix metalloproteinases (MMPs) specifically MMP-9 is a key regulator of vascular and uterine spiral artery remodelling and its activity is controlled at multiple levels, including gene transcription, activation of its latent forms and endogenous inhibition by tissue inhibitors of metalloproteinases [TIMPs (specifically TIMP-1)]. Alteration in MMP-9 and TIMP-1 expression may contribute to uterine and vascular dysfunction leading to adverse pregnancy outcomes such as Preeclampsia (PE). Depending on time, PE is classified as early-onset preeclampsia, which is diagnosed before 34 weeks of gestation, or late-onset preeclampsia, which is diagnosed after 34 weeks. Early-onset preeclampsia (EOPE) is a severe obstetrics disease which threatens mother and foetus. Therefore, in the present study, we aimed to investigate the levels of MMP-9 and its inhibitor TIMP-1 in the placentae of EOPE patients and their maternal age matched normotensive, non-proteinuric controls at both mRNA and protein levels. MethodsA total of 30 caesarean delivered placentae (15 EOPE patients and 15 controls) were collected from Department of Obstetrics and Gynaecology, AIIMS, New Delhi after taking permission from Institute Ethics Committee. MMP-9 and TIMP-1 protein expression was observed by immunohistochemistry and immunofluorescence stainings. Gelatin Gel Zymography was done to evaluate MMP-9 activity and Western Blot was done for the quantification of MMP-9 and TIMP-1 proteins. mRNA levels of MMP-9 and TIMP-1 were determined by qRT-PCR ResultsImmunohistochemistry and Immunofluorescence staining discerned stronger expression of MMP-9 in normotensive placentae as compared to EOPE placentae whereas stronger expression of TIMP-1 was seen in EOPE placentae in comparison to those of normotensive placentae. Gelatin Gel Zymography reflected that MMP-9 activity was found elevated in maternal placental side in normotensive placentae as compared to EOPE placentae. Western Blot analysis revealed that protein expression of MMP-9 was elevated whereas for that of TIMP-1 was reduced in normotensive placentae in comparison to EOPE placentae. mRNA expression of MMP-9 was found up-regulated whereas for that of TIMP-1 was down-regulated in normotensive placentae as compared to EOPE placentae. ConclusionThe present study implies that aberrant functioning of MMP-9 and TIMP-1 in EOPE patients contribute to impaired placentation which might be relevant for possible future screening programs in order to predict and to design therapies for early onset preeclamptic patients.

developmental biology↗

bioRxiv2023

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗

bioRxiv2023

Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

Intracellular Salmonella resides and multiplies in cholesterol-rich specialized compartment called Salmonella-containing vacuoles (SCVs) and avoids fusion with acidic lysosomes. Given, lysosomes are primary organelle that redistributes LDL derived cholesterol to other organelles; we questioned how lysosomal cholesterol can be transported to SCV. We demonstrate here that peroxisomes are recruited to SCVs in human primary macrophages, epithelial cells and functions as pro-bacterial organelles. Further, this interaction is assisted by SseI, a Salmonella effector protein containing mammalian peroxisome targeting sequence. SseI localizes to peroxisome, interacts and activates a host Ras GTPase, ARF-1 on the peroxisome membrane. Activation of ARF-1 leads to recruitment of phosphatidylinsolitol-5- phosphate-4 kinase to generate phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. Accordingly, the{Delta} sseI strain showed reduced virulence in cell lines and during mice infection. Taken together, our work identified a fascinating mechanism by which a pathogen targets host organelles via its secretory effectors and exploits host metabolic intermediates for its intracellular proliferation.

cell biology↗

bioRxiv2023

Artificial Intelligence uncovers Evolutionarily Conserved Intracellular Allosteric Modulators of GPCR-Gα interface.

Endogenous intracellular allosteric modulators of GPCRs remain largely unexplored, with limited binding and phenotype data available. This gap arises from the lack of robust computational methods for unbiased cavity identification, cavity-specific ligand design, synthesis, and validation across GPCR topology. Here, we developed Gcoupler, an AI-driven generalized computational toolkit that leverages an integrative approach combining de novo ligand design, statistical methods, Graph Neural Networks, and Bioactivity-based ligand prioritization for rationally predicting high-affinity ligands. Using Gcoupler, we interrogated intracellular metabolites that target and regulate the GPCR-G interface (Ste2p-Gpa1p), affecting pheromone-induced programmed cell death in yeast. Our computational analysis, complemented by experimental validations, including genetic screening, multi-omics, site-directed mutagenesis, biochemical assays, and physiological readouts, identified endogenous hydrophobic metabolites, notably sterols, as direct intracellular allosteric modulators of Ste2p. Molecular simulations coupled with biochemical signaling assessment in site-directed Ste2p mutants further confirmed that metabolites binding to GPCR-G obstruct downstream signaling, possibly via a cohesive effect. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed that elevated metabolite levels attenuate hypertrophic response, reinforcing the evolutionary relevance of this mechanism.

bioinformatics↗