Evaluation of the Crosstalk Between the Host Mycobiome and Bacteriome in Patients with Chronic Pancreatitis.

The human microbiome is a diverse consortium of microbial kingdoms that play pivotal roles in host health and diseases. We previously reported a dysbiotic bacteriome in chronic pancreatitis patients with diabetes (CPD) compared with patients with it's nondiabetic (CPND) phenotype. In this study, we extended our exploration to elucidate the intricate interactions between the mycobiome, bacteriome, and hosts' plasma metabolome with the disease phenotypes. A total of 25 participants (CPD, n = 7; CPND, n = 10; healthy control, n = 8) were recruited for the study. We observed elevated species richness in both the bacterial and fungal profiles within the CP diabetic cohort compared to the nondiabetic CP phenotype and healthy control cohorts. Notably, the CP group displayed heterogeneous fungal diversity, with only 40% of the CP nondiabetic patients and 20% of the CP diabetic patients exhibiting common core gut fungal profiles. Specific microbial taxa alterations were identified, including a reduction in Bifidobacterium adolescentis and an increase in the prevalence of Aspergillus penicilloides and Klebsiella sp. in the disease groups. In silico analysis revealed the enrichment of pathways related to lipopolysaccharide (LPS), apoptosis, and peptidase, as well as reduced counts of the genes responsible for carbohydrate metabolism in the CP groups. Additionally, distinct plasma metabolome signatures were observed, with CPD group exhibiting higher concentrations of sugars and glycerolipids, while the CPND cohort displayed elevated levels of amino acids in their blood. The fatty acid-binding protein (FABP) concentration was notably greater in the CPD group than in the HC group (4.220 vs. 1.10 ng/ml, p = 0.04). Furthermore, compared with healthy controls, disease groups exhibited fewer correlations between key fungal taxa (Aspergillus sp., Candida sp.) and bacterial taxa (Prevotella copri, Bifidobacteria sp., Rumminococcaceae). Our study unveils, for the first time, a dysbiotic mycobiome and emphasizes unique host bacterial-mycobial interactions in CP patient with diabetes, potentially influencing disease severity. These findings provide crucial insights for future mechanistic studies aiming to unravel the determinants of disease severity in this complex clinical context. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01207-8.

Starch spectra of Ampelopteris prolifera (Retz.) Copel, a new addition to the existing lexicon and its comparison with a local potato cultivar (Solanum tuberosum L. cv. Kufri Jyoti).

Novel kinds of starch spectra were generated from a lesser-known plant, making this investigation unique. The recent trend of starch characterization shows the establishment of novel bioresources from nonconventional unexplored databases. The present endeavor was made to obtain the starch fingerprint of Ampelopteris prolifera (rhizome) belonging to seedless vascular plants. For comparison, a commercial local cultivar of potato (Kufri Jyoti) was taken. The starch particle of A. prolifera shows much uniqueness depicting its novelty viz., crystallinity index of 60.04 %, powder diffractogram at (2θ scale)17.57° to 39.78°; this diffractogram pattern is reported from this study as newer one i.e. R type(whereas potato starch is CB type); characteristic peak at 2θ = 20.07° suggests starch-lipid complex formation and V type crystallinity (i.e. RS 5 type); FTIR spectra showing the presence of more short chain branching; high gelatinization temperature(84.62 ±â€¯0.10), particle size and zeta value of A. prolifera is 4.00 ±â€¯0.81 µm and - 18.91 ±â€¯3.58 mV respectively. Bragg's peak from the single crystal X-ray diffraction has been generated for the first time of A. prolifera. Extraction of the starch particle was performed in chilled water. Therefore, the present study suggests wide-spectrum commercial utility and cost-effective production.

Glucose to lactate shift reprograms CDK-dependent mitotic decisions and its communication with MAPK Sty1 in Schizosaccharomyces pombe.

Cell cycle regulation in response to biochemical cues is a fundamental event associated with many diseases. The regulation of such responses in complex metabolic environments is poorly understood. This study reveals unknown aspects of the metabolic regulation of cell division in Schizosaccharomyces pombe. We show that changing the carbon source from glucose to lactic acid alters the functions of the cyclin-dependent kinase (CDK) Cdc2 and mitogen-activated protein kinase (MAPK) Sty1, leading to unanticipated outcomes in the behavior and fate of such cells. Functional communication of Cdc2 with Sty1 is known to be an integral part of the cellular response to aberrant Cdc2 activity in S. pombe. Our results show that cross-talk between Cdc2 and Sty1, and the consequent Sty1-dependent regulation of Cdc2 activity, appears to be compromised and the relationship between Cdc2 activity and mitotic timing is also reversed in the presence of lactate. We also show that the biochemical status of cells under these conditions is an important determinant of the altered molecular functions mentioned above as well as the altered behavior of these cells.

Phytotherapeutic targeting of the mitochondria in neurodegenerative disorders.

Neurodegenerative diseases are characterized by degeneration or cellular atrophy within specific structures of the brain. Neurons are the major target of neurodegeneration. Neurons utilize 75-80% of the energy produced in the brain. This energy is either formed by utilizing the glucose provided by the cerebrovascular blood flow or by the in-house energy producers, mitochondria. Mitochondrial dysfunction has been associated with neurodegenerative diseases. But recently it has been noticed that neurodegenerative diseases are often associated with cerebrovascular diseases. Cerebral blood flow requires vasodilation which to an extent regulated by mitochondria. We hypothesize that when mitochondrial functioning is disrupted, it is not able to supply energy to the neurons. This disruption also affects cerebral blood flow, further reducing the possibilities of energy supply. Loss of sufficient energy leads to neuronal dysfunction, atrophy, and degeneration. In this chapter, we will discuss the metabolic modifications of mitochondria in aging-related neurological disorders and the potential of phytocompounds targeting them.

Kynurenine pathway alteration in acute pancreatitis and its role as a biomarker of infected necrosis.

INTRODUCTION: Infected pancreatic necrosis (IPN) is a major cause of mortality in acute pancreatitis (AP). Currently, no specific strategies are available to predict the development of IPN. Earlier we reported that persistent down-regulation of HLA-DR increases risk of developing IPN. Altered kynurenine pathway (KP) metabolites showed poor prognosis in sepsis. Here we evaluated the role of HLA-DR and KP in IPN. METHODS: Patients with ANP and healthy controls were enrolled. Demographic and clinical parameters were recorded. Circulating interleukin (IL)-8, 6, 1ß, 10, Tumor necrosis factor-α were quantified using flowcytometry. Plasma procalcitonin, endotoxin, and KP (tryptophan, kynurenine) concentrations were estimated using ELISA. qRT-PCR was conducted to evaluate mRNA expression of HLA-DR, IL-10, Toll like receptor-4 (TLR-4), and kynurenine-3-monooxygenase (KMO) genes on peripheral blood mononuclear cells. Plasma metabolites were quantified using gas chromatography mass spectrometry (GC-MS/MS). Standard statistical methods were used to compare study groups. Metaboanalyst was used to analyse/visualize the metabolomics data. RESULTS: We recruited 56 patients in Cohort-1 (IPN:26,Non-IPN:30), 78 in Cohort-2 (IPN:57,Non-IPN:21), 26 healthy controls. Increased cytokines, endotoxin, and procalcitonin were observed in patients with IPN compared to Non-IPN. HLA-DR and KMO gene expressions were significantly down-regulated in IPN groups, showed positive correlation with one another but negatively correlated with IL-6 and endotoxin concentrations. Increased IDO and decreased plasma tryptophan were observed in IPN patients. Metabolome analysis showed significant reduction in several essential amino acids including tryptophan in IPN patients. Tryptophan, at a concentration of 9 mg/ml showed an AUC of 91.9 (95%CI 86.5-97.4) in discriminating IPN. CONCLUSION: HLA-DR downregulation and KP alteration are related to IPN. The KP metabolite plasma tryptophan can act as a potential biomarker for IPN.

Multi-Omics Analysis Demonstrates the Critical Role of Non-Ethanolic Components of Alcoholic Beverages in the Host Microbiome and Metabolome: A Human- and Animal-Based Study.

It is known that alcoholic beverages alter the human gut microbiome. This study focused on the potential impact of non-ethanolic ingredients in whisky on the gut bacteriome. A pilot study was carried out on 15 whisky drinkers, 5 rice beer drinkers, and 9 non-drinkers to determine the effect of alcoholic beverages on the host microbiome and metabolome. Additionally, a mouse model was used to assess the differential impact of three whisky brands (each with an equal ethanol concentration). The results indicate that the non-ethanolic components have an impact on the gut microbiome, as well as on the metabolites in blood and feces. The amount of Prevotella copri, a typical core Indian gut bacterium, decreased in both the human and mouse groups of whisky type 1, but an increase in abundance of Helicobacteriaceae (p = 0.01) was noticed in both groups. Additionally, the alcohol-treated cohorts had lower levels of short-chain fatty acids (SCFAs), specifically butyric acid, and higher amounts of lipids and stress marker IL1-ß than the untreated groups (p = 0.04-0.01). Furthermore, two compounds, ethanal/acetaldehyde (found in all the whisky samples) and arabitol (unique to whisky type 1), were tested in the mice. Similar to the human subjects, the whisky type 1 treated mouse cohort and the arabitol-treated group showed decreased levels of Prevotella copri (p = 0.01) in their gut. The results showed that non-ethanolic compounds have a significant impact on host gut bacterial diversity and metabolite composition, which has a further vital impact on host health. Our work further emphasizes the need to study the impact of non-ethanolic ingredients of alcoholic beverages on host health.

Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage.

Multiplex protein imaging technologies enable deep phenotyping and provide rich spatial information about biological samples. Existing methods have shown great success but also harbored trade-offs between various pros and cons, underscoring the persisting necessity to expand the imaging toolkits. Here we present PACIFIC: photoactive immunofluorescence with iterative cleavage, a new modality of multiplex protein imaging methods. PACIFIC achieves iterative multiplexing by implementing photocleavable fluorophores for antibody labeling with one-step spin-column purification. PACIFIC requires no specialized instrument, no DNA encoding, or chemical treatments. We demonstrate that PACIFIC can resolve cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells. To further highlight how PACIFIC assists discovery, we integrate PACIFIC with live-cell tracking and identify phosphor-p70S6K as a critical driver that governs U87 cell mobility. Considering the cost, flexibility, and compatibility, we foresee that PACIFIC can confer deep phenotyping capabilities to anyone with access to traditional immunofluorescence platforms.

Hydroxyl-Rich Hydrophilic Endocytosis-Promoting Peptide with No Positive Charge.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

Pain, depression, and poor quality of life in chronic pancreatitis: Relationship with altered brain metabolites.

BACKGROUND: To evaluate if altered brain metabolites are connected to pain, depression and affective responses in CP. METHODS: In this prospective study we evaluated pain characteristics, QOL (EORTC QLQc30+PAN28), depression (Beck depression inventory [BDI] II) in 558 patients with CP and 67 healthy controls. Brain metabolites were evaluated using magnetic resonance spectroscopy (MRS) in 49 patients and 5 healthy controls. We measured plasma metabolites using gas chromatography-mass spectrometry (GC-MS/MS). Relationship between metabolomic alterations, pain, depression and QOL components were assessed using statistical/bioinformatics methods. Benjamini-Hochberg FDR correction was applied for multiple testing. RESULTS: 261 (46.8%) patients had depression compared to 5 (7.5%) among healthy controls [n = 67](p < 0.0001). Risk [OR (95% CI) of developing depression in the presence of pain was 1.9 (1.33-1.68); p = 0.0004. The depression scores correlated negatively with functional components and positively with symptom components of EORTC QLQ30. Significant negative correlation, though based on a small sample size, was observed between N-acetyl aspartate in the left hippocampus and choline in the left prefrontal cortex with emotional and cognitive functions. PLS-DA modelling revealed significant alteration in the plasma metabolomic profile among patients with CP who had depression. Six metabolites were significantly different between CP with depression and healthy controls, of which glycine contributed most significantly to the PLS-DA model (VIP score of 3.5). CONCLUSIONS: A significant proportion of patients with CP develops depression that correlate with poor QOL functions. Pain, depression, and emotional components of QOL in patients with CP correlated with N-acetyl aspartate and choline in the left hippocampus and left prefrontal cortex of the brain.

Dataset describing the genome wide effects on transcription resulting from alterations in the relative levels of the bZIP transcription factors Atf1 and Pcr1 in Schizosaccharomyces pombe.

Schizosaccharomyces pombe has been used as an excellent model for studying eukaryotic cell cycle regulation and stress responses. The bZIP transcription factors Atf1(ATF2 homolog) and Pcr1(CREB homolog) have been shown to be important for regulating the expression of genes related to both stress response and cell cycle. Pcr1 has in fact been implicated as a determining factor in the segregation of the cell cycle and stress response related functions of Atf1. Interestingly Atf1 and Pcr1 levels are known to vary during the cell cycle thus giving rise to the possibility that their relative levels can influence the periodic transcriptional program of the cell. Here we report our observations on the changes in transcriptome of S. pombe cells which have been genetically manipulated to create relative differences in the levels of Atf1 and Pcr1. These results highlight new information regarding the potential role of Atf1 and Pcr1 in orchestrating the integration of the transcriptional programs of cell cycle and stress response.

Real-Time Analysis of AKT Signaling Activities at Single-Cell Resolution Using Cyclic Peptide-Based Probes.

Here we present a protocol for interrogating AKT signaling activities in living single cells, using a pair of cyclic peptide-based fluorescent probes. These probes are encapsulated in liposomes and delivered into cells, where they continuously report on AKT signaling activities through a Föster resonance energy transfer mechanism. We describe the use of a microwell chip to achieve single-cell resolution and demonstrate the procedure for on-chip immunostaining. Finally, we provide a method for data extraction, correction, and processing.

Zederone Improves the Fecal Microbial Profile in Dementia Induced Rat Model: A First Report.

BACKGROUND: Dementia correlates with Alzheimer's disease, Parkinson's disease, frontotemporal and cerebrovascular diseases. There are supporting shreds of evidence on the pharmacological activity of curcuma caesia (Zingiberaceae family) for its antioxidant, antidepressant, analgesic, anticonvulsant, and anti-acetylcholinesterase effect. OBJECTIVE: This study aims to analyze the fecal microbial profile in Zederone treated demented rat model. METHODS: In our study, isolation and characterization of Zederone were carried out from curcuma caesia rhizomes, followed by estimation of its memory-enhancing effect on Aluminium-induced demented rat, which was evaluated by behavioural study on radial 8 arm maze. Moreover the detection of amyloid plaque formation was carried out using fluorescent microscopy of the congo red-stained rat brain tissues of the cerebral neocortex region. This study included eighteen female Wistar Albino rats that were divided into three groups that consisted of six rats in each group. The study of fecal microbial profile by metagenomic DNA extraction followed by next-generation sequencing was carried out to establish the correlation between gut microbes and amyloid plaques in dementia. RESULTS: Zederone could be characterized as pale yellow colored, needle-shaped crystals with 96.57% purity. This compound at 10 mg/kg body weight showed cognition improving capacity (p ≤ 0.0001) with a reduction of accumulated amyloid plaques that were detected in the demented group in fluorescence microscope and fecal microbiome study divulged an increased shift towards Lactobacillus genera in the treated group from Bacteroides in the demented group. CONCLUSION: This sesquiterpenoid compound would assist in the modulation of gut bacterial dysbiosis and act as a better therapeutic drug for dementia and other neurological disorders.

Digitonin-facilitated delivery of imaging probes enables single-cell analysis of AKT signalling activities in suspension cells.

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.

Single-Cell Profiling of Fatty Acid Uptake Using Surface-Immobilized Dendrimers.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

In Silico Analysis to Link Insulin Resistance, Obesity and Ageing with Alzheimer's Disease.

The process of ageing accompanies several metabolic diseases. With ageing, fats accumulate to increase the visceral and abdominal adiposity leading to hyperinsulinemia, insulin resistance, obesity and several other diseases. Drosophila melanogaster is often used to study the ageing process and its related disorders. Therefore, in this study, we performed an in silico analysis to relate the process of ageing and insulin resistance. We analysed the data of insulin-resistant Drosophila from the GEO database and compared it with the data from the literature survey. We observed that 98 genes were common in both the models, and they showed gene modulations related to metabolic pathways, fatty acid metabolism, insulin resistance and neural receptor-ligand binding pathways. Analysis of the REACTOME database against human data revealed that the TRKB signalling pathway is commonly affected. The TRKB-mediated BDNF pathway is a major regulator of memory loss. We further analysed the common genes in Alzheimer's disease and compared the fly data with human data to identify the diseases related to these common genes. Then, we performed a literature survey to provide protective mechanisms for the TRKB signalling pathway activation, mediated through polyphenols. We treated the flies with sesamol-conjugated lipoic acid derivative (a phenolic compound) at hormetic doses to evaluate its effect on the memory of flies.

The gut microbiome in pancreatogenic diabetes differs from that of Type 1 and Type 2 diabetes.

We hypothesized that the gut microbiome in patients with diabetes secondary to chronic pancreatitis (Type 3c) is different from those with Type 1 and Type 2 diabetes. This was a cross-sectional preliminary study that included 8 patients with Type 1, 10 with Type 2, 17 with Type 3c diabetes and 9 healthy controls. Demographic, clinical, biochemical, imaging and treatment data were recorded and sequencing of the V3-V4 region of the bacterial 16SrRNA was done on fecal samples. Bioinformatics and statistical analyses was performed to evaluate the differences in the diversity indices, distance matrices, relative abundances and uniqueness of organisms between the types of diabetes. There was significant difference in the species richness. Beta diversity was significantly different between patients with Type 3c diabetes and the other groups. 31 genera were common to all the three types of diabetes. There was significant differences in the species level taxa between Type 3c diabetes and the other groups. The unique bacterial species signature in Type 3c diabetes compared to Type 1 and Type 2 diabetes included Nesterenkonia sp. AN1, Clostridium magnum, Acinetobacter lwoffii, Clostridium septicum, Porphyromonas somerae, Terrabacter tumescens, and Synechococus sp.

Analysing Curcuma caesia fractions and essential oil for neuroprotective potential against anxiety, depression, and amnesia.

Scientific pieces of evidence support the pharmacological activity of Curcuma caesia for its antidepressant, analgesic, anticonvulsant and antioxidant effect. Here, we evaluate the bioactivity of essential oil and the various polarity-based solvent partitioned fractions obtained from Curcuma caesia for anti-amnesia, anxiolytic and antidepressant activities using Elevated plus maze and Morris water maze models. The cold maceration technique using methanol was adopted for extraction from dried powdered rhizomes and essential oil was extracted by hydrodistillation method. Partitioning of the methanolic extract based on solvent polarity by hexane, ethyl acetate, and methanol was continued, followed by column chromatography of the ethyl acetate fraction. Suspensions were prepared for fractions (dissolved in distilled water) and essential oil (dissolved in tween 20) at 200 mg/kg and 400 mg/kg after acute toxicity study and were orally administered to Wistar albino female rats after the orientation of hypoxia by sodium nitrite (50 mg/kg) and amnesia by scopolamine (1 mg/kg). Behavioural observations, biochemical and histopathological examinations were carried out for all the treated groups. Diazepam (12 mg/kg) and galantamine (3 mg/kg) were used as standard drugs for this study against hypoxia and amnesia. Data acquired from behavioural, biochemical (acetylcholinesterase, myeloperoxidase, superoxide dismutase, reduced glutathione, catalase) and histopathological studies have illustrated that fraction II acquires highly significant memory-enhancing, anxiolytic and antidepressant effects. Rest fractions (I and III) and essential oil showed moderate efficacy. In prospects, identification of active molecules from the most active fraction (fraction II) and further studies on a molecular basis would substantiate its specific mechanism of neuroprotective action.

A cyclic peptide antenna ligand for enhancing terbium luminescence.

We present here a cyclic peptide ligand, cy(WQETR), that binds to the terbium ion (Tb3+) and enhances Tb3+ luminescence intensity through the antenna effect. This peptide was identified through screening a cyclic peptide library against Tb3+ with an apparent EC50 of 540 µM. The tryptophan residue from the peptide directly interacts with the Tb3+ ion, which provides access to a low-lying triplet excited state of the tryptophan. Direct excitation of this triplet state enables energy transfer to the Tb3+ ion and enhances Tb3+ luminescence intensity by 150 fold. We further showcase the application of this cy(WQETR)-Tb3+ system by demonstrating the detection of tromethamine with a detection limit of 0.5 mM.

In silico explanation for the causalities of deleterious RNF213 SNPs in Moyamoya disease and insulin resistance.

Moyamoya disease (MMD), a cerebrovascular disorder caused by the RNF213 gene, is a cerebrovascular, neurological disorder leading to ischemic strokes. Our previous work suggested that RNF213 might be involved in the pro-inflammatory TNFα-mediated insulin-resistance pathway in adipocytes. Insulin resistance can lead to cerebrovascular diseases and ischemic strokes. Though p. R4810 K has been reported as the founder mutation for Asian population with this disease, there are several mutations continuously reported in clinical diagnosis. We are interested to know whether these mutations can modulate insulin resistance. Also, we are intended to understand the causalities of RNF213 and its associated mutations in MMD. For this, we have adopted a computational approach to characterize RNF213 and its naturally occurring SNPs. Clinically reported SNPs and the predicted SNPs were analyzed for their pathogenicity and effect on the biological function of the protein. To increase accuracy, this was performed through three different analysis software (PROVEAN, SIFT, and SNAP2). The mutations that were found to be deleterious in all the three platforms were further analyzed for their effect on the thermal stability of the protein through I-mutant and iStable. It was found that R4810 K and other mutations decreased the thermodynamic stability of the protein. Loss of function of RNF213 was suggested in some reports. Contrary to this, some studies reported a gain of function state due to the R4810K mutation. To understand this we have measured the ligand-binding ability of this mutated protein through COFACTOR and COACH. An increase in ligand binding is always related to the functional stability of a protein. We have observed that the R4810K mutation might increase the iron-binding efficiency of the amino acid residues. This increase in binding was further validated by analyzing the binding efficiencies by docking. Since RNF213 was previously reported as a target for Protein Tyrosine Phosphatase 1B (PTP1B), we have also analyzed whether PTP1B-binding positions are susceptible to mutations. We have re-analyzed our earlier report on the differential expression pattern of RNF213 in cancer and obese samples. We have provided a detailed analysis of the most deleterious SNPs related to RNF213. Also, we provide a prediction for the loss of function and gain of function attributes of RNF213 and its predicted causalities in MMD and insulin resistance.

New insights into TNFα/PTP1B and PPARγ pathway through RNF213- a link between inflammation, obesity, insulin resistance, and Moyamoya disease.

Diabetic patients are always at a higher risk of ischemic diseases like coronary artery diseases. One such ischemic carotid artery disease is Moyamoya disease (MMD) associated with diabetes Type I and II, but the causality was unclear. Ring Finger Protein 213 (RNF213) is the major susceptible gene for MMD. To understand the association between diabetes mellitus and MMD we chose the major players from both of the anomalies: insulin and RNF213. But before establishing the role of RNF213 in the insulin-regulating pathway we had to understand the involvement of RNF213 within different biological systems. For this, we have adopted a preliminary computational approach to find the prominent interactions of RNF213. Our first objective was to construct an interactome for RNF213. We have analyzed several curated databases and adapted a list of RNF213 interacting partners to develop its interactome. Then to understand the involvement of this interactome in biological functions we have analyzed major biological pathways, biological processes, and prominent clusters related to this interactome through a computational approach. Then to develop a pathway that might give clues for RNF213 involvement in the insulin regulatory pathway we have validated the intercluster and intracluster predictions and identified a regulatory pathway for RNF213. RNF213 interactome was observed to be involved in adaptive immunity with 4 major clusters; one of the clusters involved TNFα. The immune system involves several pathways, and therefore at this point, we have chosen an event-based strategy to obtain an explicit target. Immunity is mediated by pro-inflammatory cytokines like TNFα. TNFα-mediated inflammation, obesity, and insulin resistance are associated. Therefore we chose to explore the role of RNF213 in TNFα-mediated inflammation in macrophages and inflammation-mediated insulin-resistance in adipocytes. We have observed an enhancement of RNF213 gene expression by LPS mediated pro-inflammatory stimuli and suppression by PPARγ-mediated anti-inflammatory, insulin-sensitizing stimuli in macrophages, and also in adipocytes. Administration of the pro-inflammatory cytokine TNFα was able to impede the reduction in RNF213 expression during adipogenesis and this effect was observed to be mediated by PTP1B. Inactivation of PTP1B abolished RNF213 expression which in turn enhanced the adipogenesis process through enhanced PPARγ. Constitutive expression of RNF213 suppressed the adipocyte differentiation by the inhibition of PPARγ. We could show the regulation of RNF213 by TNFα/PTP1B pathway and PPARγ. The constitutive expression of RNF213 during adipogenesis appears to be an adipostatic measure that obese patients acquire to inhibit further adipogenesis. This is verified in silico by analyzing the gene expression data obtained from the Gene Expression Omnibus database, which showed a higher expression of RNF213 in adipose tissue samples of obese people. Overall this study gives new insights into the TNFα-mediated pathway in adipogenesis and suggests the role of RNF213 in adipogenesis via this pathway.