Biology subjects

Ghosh, A.

Publications and source records attributed to Ghosh, A..

At least 163 records · Page 9Linked to original sources

bioRxiv2022

A mechanistic insight into Nigella sativa mediated anticancer effect on breast cancer through regulation of various miRNAs: An in vitro & in vivo study

BackgroundCancer continues to threat the mortal alongside scientific community with its burgeoning grasp. Although efforts have been directed to tame cancer by radiotherapy, photodynamic therapy, chemotherapy it came at a cost of fatal side effects. PurposePlant derived bioactive compounds carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. This study analyzed anti-cancerous potential of volatile oil, extracted from Nigella sativa, in-vitro against MDA-MB-231, MCF-7 & in-vivo on tumor growth in mice after successful oral administration. Study DesignOur strategic approach employed solvent extraction of black seed oil (BSO) to highlight orchestrated use of its potent integrants - TQ, Carvacrol & TA which in modest amounts show anti-cancerous properties compared to their individual treatment. We attempted to show this cost effective, safe & bioavailable form of dealing with the atrocities of breast cancer by means of MTT, Apoptotic, Western Blot Assays besides Transwell & Wound healing Assay. Reduction in the solid tumour in-vivo & near normalcy restoration of tissue section architecture from the BSO treated tumour sets are indicative of the better anti-tumorigenic potential of BSO. MethodsBSOs Solvent extraction was performed followed by its characterization. MTT aided cytotoxicity study of BSO alongside major components in PBMC & cancer cells while its efficacy was presented by flow cytometric ROS analysis, cell cycle arrest & apoptosis assessment. Anti-migratory potential evaluated by Wound Healing, Transwell Migration & Western Blot while the expression study of a wide range of proteins, miRNAs & the in-vivo studies undertaken climaxed the confirmation of the anti-cancerous potential of BSO. ResultsComparatively reduced concentrations of TQ, TA & Carvacrol in BSO played a synergistic role to enhance apoptotic potential via Caspase 7 & 9, through enhanced ROS & expression of apoptotic family of proteins, miRNAs besides uplifting the anti-migratory perspective by effectively enhancing E-cad & downregulating lamellipodia, filopodia assembly & MMPs in MCF-7 & MDA-MB-231. Similar observations in-vivo outlined the therapeutic potential of BSO. ConclusionThis study culminates isolation & processing of BSO in a simplified procedure, thereby aiming at a more lucrative paradigm to be accepted in contemporary phytomedicine research.

cancer biology↗

bioRxiv2022

Initiator AUGs are discriminated from Elongator AUGs predominantly through mRNA accessibility in C. crescentus

Translation initiation in bacteria is thought to occur upon base-pairing between the Shine-Dalgarno site in the mRNA and anti-Shine-Dalgarno site in the rRNA. However, in many bacterial species, such as Caulobacter crescentus, a minority of mRNAs have Shine-Dalgarno sites. To examine the functional importance of Shine-Dalgarno sites in C. crescentus, we analyzed the transcriptome and found more Shine-Dalgarno sites exist in the coding sequence than preceding start codons. To examine the function of Shine-Dalgarno sites in initiation we designed a series of mutants with altered ribosome accessibility and Shine-Dalgarno content in translation initiation regions (TIRs) and elongator AUG regions (EARs). A lack of mRNA structure content is required for initiation in TIRs, and when introduced into EARs, can stimulate initiation, suggesting that low mRNA structure content is a major feature required for initiation. SD sites appear to stimulate initiation in TIRs, which generally lack structure content, but SD sites only stimulate initiation in EARs if RNA secondary structures are destabilized. Taken together, this suggests that the difference in secondary structure between TIRs and EARs directs ribosomes to start codons where SD base pairing can tune the efficiency of initiation, but SDs in EARs do not stimulate initiation as they are blocked by stable secondary structures. This highlights the importance of studying translation initiation mechanisms in diverse bacterial species.

microbiology↗

bioRxiv2022

Nanoscale infrared spectroscopy identifies parallel to antiparallel beta sheet transformation of Aβ fibrils

Spontaneous aggregation of amyloid beta (A{beta}) proteins leading to the formation of oligomers and eventually into fibrils has been identified as a key pathological signature of Alzheimers disease. Structure of late stage aggregates have been studied in depth by conventional structural biology techniques including Nuclear Magnetic Resonance, X-ray crystallography and Infrared Spectroscopy; however the structure of early-stage aggregates is less known due to their transient nature. As a result, the structural evolution of amyloid aggregates from its early oligomers to mature fibril is still not fully understood. Here we have applied AFM-IR nanospectroscopy to investigate the aggregation of A{beta} 16-22, which spans the amyloidogenic core of the amyloid beta peptide. Our results demonstrate that A{beta} 16-22 involves a structural transition from oligomers with parallel beta sheets to antiparallel fibrils through disordered and possibly helical intermediate fibril structures, contrary to the known aggregation pathway of full-length A{beta}.

biophysics↗

bioRxiv2022

Common multi-day rhythms in smartphone behavior

The idea that abnormal human activities follow multi-day rhythms spans ancient beliefs centered on the moon to modern clinical observations in epilepsy and mood disorders. Multi-day rhythms remain obscure in normal human activities. To address multi-day rhythms in healthy human behavior we analyzed over 300 million smartphone touchscreen interactions logging up to 2 years of day-to-day activities (N = 401 subjects). By using non-negative matrix factorization and data-driven clustering of [~]1 million periodograms, we captured a range of multi-day rhythms spanning periods from 7 to 52 days - cutting across age and gender. Despite their common occurrence, any given multi-day rhythm was observed in different parts of the smartphone behavior from one person to the next. There was little support in the data for ubiquitous rhythm drivers like the moon. We propose that multiple multi-day rhythms are a common trait, but their consequences may be uniquely experienced in day-to-day behavior.

animal behavior and cognition↗

bioRxiv2022

Neural processing of goal and non-goal-directed movements on the smartphone

The discrete behavioral events captured on the smartphone touchscreen may help unravel real-world neural processing. We find that neural signals (EEG) surrounding a touchscreen event show peculiarly contralateral motor preparation, visual processing, and the consolidation of information. We leveraged these events in conjunction with kinematic recordings of the thumb and an artificial neural network to separate highly similar movements according to whether they resulted in a smartphone touch (goal-directed) or not (non-goal-directed). Despite their kinematic similarity underscored by the model, the signatures of neural control of movement and the post-movement processing were substantially dampened for the non-goal-directed movements, and these movements uniquely evoked error-related signals. We speculate that these unnecessary movements are common in the real world and although inconsequential the brain still provides limited motor preparation and tracks the action outcome. Real-world behavior is composed of neural processes that are difficult to capture in conventional laboratory-based tasks.

neuroscience↗

bioRxiv2022

Structural rationale to understand the effect of disease-associated mutations on Myotubularin

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1 gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains- a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerization of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silico and in vitro studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature. Author SummaryX-linked myotubular myopathy is a rare paediatric disorder and affected males suffer neonatal death or may live on only with ventilatory support. In this study, we employed a range of approaches to understand the molecular level effects of patient-derived mutations on an enzyme directly linked to the congenital muscular disorder. Using three-dimensional modelling and simulations, the effect of these mutations on the structure of the enzyme and its ability to bind its substrate was studied. To complement theoretical observations, experiments were performed with cells expressing this enzyme and its mutants. These studies reveal that each part of the protein may directly or indirectly affect its enzyme activity and most of the patient-derived mutations are expressed insufficiently in the cell. With the advent of genome sequencing technology, identification of congenital mutations is easier; computational studies of molecular consequences of mutations on a protein function such as this will prove immensely useful in understanding the disease and its prognosis.

bioinformatics↗

bioRxiv2022

Enhanced epithelial to mesenchymal transition and chemoresistance in advanced Retinoblastoma tumors is driven by miR-181a

Advanced retinoblastoma (Rb) tumors can infiltrate distant tissues and cause a potent threat to vision and life. Through transcriptomic profiling, we discovered key epithelial to mesenchymal transition (EMT) and chemotherapy resistance genes at higher expression levels in advanced Rb tumors. Rb-/- tumor cells acquire metastasis-like phenotype through the EMT program that critically contributes to chemoresistance. We demonstrate that prolonged chemo-drug exposure in Rb cells elicits an EMT program through ZEB1 and SNAI2 that further acquires therapeutic resistance through cathepsin L and MDR1 mediated drug efflux mechanisms. Further, 16 significantly differentially expressed miRNAs were identified in patient tumors, of which miR-181a-5p was significantly reduced in advanced Rb tumors and associated with altered EMT and drug resistance genes. Enhancing miR-181a-5p levels in Rb-/- cells and Rb-/- chemo-resistant sublines controls EMT transcription factors ZEB1 and SNAI2 and halts the transition switch, thereby reversing drug resistance. We thus identify miR-181a-5p as a potential therapeutic target for EMT triggered drug-resistant cancers that can halt their invasion and sensitize them to low dose chemotherapy drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/501381v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@114edeorg.highwire.dtl.DTLVardef@50da16org.highwire.dtl.DTLVardef@1df21borg.highwire.dtl.DTLVardef@1ea79_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Nodulin 16 of Lotus japonicus (Nlj16) regulates the recruitment of Leghemoglobin (LegH) to the infected nodule cell membrane during symbiotic nitrogen fixation

Nitrogenase that catalyses anaerobic symbiotic nitrogen fixation (SNF) in legumes is synthesized by rhizobium. Legume root cells express nodulin proteins after infection with rhizobia. Nodulins have been classified as early and late, reflecting the time points of their expression. Leghemoglobin (LegH), which is a classic example of a late nodulin, sequesters oxygen inside the nodule to protect the nitrogenase from oxygen toxicity to sustain SNF. Previous data from our laboratory demonstrated that phosphorylated LegH at S45 showed compromised oxygen sequestration in vitro due to structural disruption of the porphyrin binding pocket responsible for its oxygen binding. Moreover, we have demonstrated by using co-immunoprecipitation that LegH interacts both in vitro with Nodulin 16 of Lotus japonicus (Nlj16), another late nodulin. Fluorescence Immunohistochemistry (IHC) data shows that both LegH and Nlj16 are localized in the membrane and cytosol of infected cells. Notably, serine phosphorylation of LegH and interaction of Nlj16 with LegH respectively reduces and increases its in vitro oxygen sequestration ability. In this report, to further elucidate the spatio-temporal regulation of this interaction, we generated hairy root transgenic Lotus japonicus plants where Nlj16 has been knocked down by using siRNA. Most interestingly, our data shows that the membrane localization of LegH is obliterated in the Nlj16 knocked-down root cells infected with the cognate Rhizobia suggesting a distinct role of Nlj16 in its recruitment to the membrane of nodule cells.

plant biology↗

bioRxiv2022

Two point mutations in protocadherin-1 disrupt Andes hantavirus recognition and afford protection against lethal infection

Andes virus and Sin Nombre virus are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we used computational and experimental approaches to delineate the binding surface of the ANDV glycoprotein complex on PCDH1s first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influenced the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this, and a neighboring residue, substantially protected Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

microbiology↗

bioRxiv2022

Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre;Pitpnaflox/flox mice leads to hyperglycemia resulting from decreased glucose-stimulated insulin secretion (GSIS) and reduced pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirmed its role in PtdIns-4-P synthesis and led to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reversed these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

cell biology↗

bioRxiv2022

Sub-operon promoter arrangement of disA facilitates c-di-AMP homeostasis and selective stress responses in M. smegmatis

Bacterial second messenger signaling often plays an important role in cellular physiology. In this study, we have attempted to understand how c-di-AMP synthesis and degradation are transcriptionally regulated in M. smegmatis. We have discovered that, although c-di-AMP synthesis gene disA exists in a multi-gene operon; a sub-operon promoter arrangement plays a key role under various stress conditions, keeping its dual function property intact. Further, we learned that c-di-AMP plays a role in the autoregulation of the disA promoter to limit intracellular c-di-AMP concentration. We also identified an alternate start codon within the disA gene which can lead to the synthesis of truncated DisA protein at times using an independent stress-inducible promoter. All in all, this study was helpful to understand how c-di-AMP synthesis is regulated under normal and stress conditions linked to its physiological relevance in M. smegmatis.

microbiology↗

bioRxiv2022

PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

Phosphatidylinositol 3-phosphate (PI3P) and Phosphatidylinositol 5-phosphate (PI5P) are low abundant phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P, in vitro. In this study, we report a novel role for PIP4K in regulating PI3P levels in Drosophila tissues. Loss-of-function mutants of the only PIP4K gene in Drosophila (dPIP4K29) show reduced cell size in larval salivary glands. We find that PI3P levels are elevated in dPIP4K29 tissues and that reverting PI3P levels back towards wild type, without changes in PI5P levels, can also rescue the reduced cell size phenotype. dPIP4K29 mutants also show an upregulation in autophagy and the reduced cell size can be reverted by decreasing Atg8a, that is required for autophagosome maturation. Lastly, increasing PI3P levels in wild type salivary glands can phenocopy the reduction in cell size and associated upregulation of autophagy seen in dPIP4K29. Thus, our study reports for the first time, a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size regulation that may explain the reported role of PIP4K in regulating neurodegeneration and tumour growth.

cell biology↗

bioRxiv2022

The extracellular RNA pool within Zea mays apoplast: composition and differential expression during Ustilago maydis infection

The existence of an extracellular pool of RNA (exRNA) has been documented in both animal and plant cells in a number of instances. These exRNA species play important role in host response against different environmental stimuli. The mechanism of their function however remains largely unknown. In this study we report the composition of the exRNA pool within the leaf apoplast of Z. mays under normal growth condition. We could detect RNA transcripts originating from both the genic as well as the intergenic regions of the nuclear, mitochondrial and chloroplast genomes of maize in our exRNA sequencing data. Our data showed increased abundance of about 75% of the exRNA transcripts during infection with a basidiomycete smut fungi, Ustilago maydis. Functional classification of the differentially abundant exRNA transcripts within U. maydis SG200 WT infected maize apoplast with respect to uninfected apoplast revealed significant enrichment of the exRNA transcripts corresponding to the ribosome biogenesis pathway. Data related to the effect of two extracellular T2 type ribonucleases, Nuc1 and Nuc2 from U. maydis on the composition of exRNA pool of maize is also presented.

plant biology↗

bioRxiv2022

Structural heterogeneity of amyloid aggregates identified by spatially resolved nanoscale infrared spectroscopy

Amyloid plaques, composed of aggregates of the amyloid beta (A{beta}) protein, are one of the central manifestations of Alzheimers disease pathology. Aggregation of A{beta} from amorphous oligomeric species to mature fibrils has been extensively studied. However, significantly less in known about early-stage aggregates compared to fibrils. In particular, structural heterogeneities in prefibrillar species, and how that affects the structure of later stage aggregates are not yet well understood. Conventional spectroscopies cannot attribute structural facets to specific aggregates due to lack of spatial resolution, and hence aggregates at any stage of aggregation must be viewed as having the same average structure. The integration of infrared spectroscopy with Atomic Force Microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. In this report, we use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra, ranging from ordered beta structure to disordered conformations with predominant random coil and beta turns. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show for the first time that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains can exist in the same fibril. We further show the implications of these results in amyloid plaques in Alzheimers tissue using infrared imaging, where these structural heterogeneities manifest themselves as lack of expected beta sheet structure.

biophysics↗

bioRxiv2022

Understanding the role of c-di-AMP signaling in determining antibiotic tolerance in Mycobacterium smegmatis: generation of resistant mutants and regrowth of persisters

In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment.

microbiology↗

bioRxiv2022

Autonomous untethered microinjectors for gastrointestinal delivery of insulin

The delivery of macromolecular drugs via the gastrointestinal (GI) tract is challenging. Macromolecular drugs display low stability and poor absorption across the intestinal epithelium. While permeation-enhancing drug delivery methods can increase the bioavailability of low molecular weight drugs, the effective delivery of high molecular weight drugs across the tight epithelial cell junctions remains a formidable challenge. Here, we describe autonomous microinjectors that can efficiently penetrate the GI mucosa and deliver insulin systemically. In addition, we performed in vitro studies to characterize insulin release and the penetration capacity of microinjectors and measure in vivo release of insulin in live rats. We found that the microinjectors administered within the luminal GI tract could deliver insulin trans-mucosally to the systemic circulation at similar levels to intravenously administered insulin. Due to their small size, tunability in sizing and dosing, wafer-scale fabrication, and parallel, autonomous operation, we anticipate that these novel microinjectors could significantly advance drug delivery across the GI tract mucosa to the systemic circulation.

bioengineering↗

bioRxiv2022

Deciphering the mRNA-lncRNA-miRNA interaction landscape in human pluripotency

Human embryonic stem cells offer a huge potential for the study of early human development and for application in biomedical sciences. Growing evidence has shown that besides the protein coding genes, the non-coding elements of the human genome play a crucial role in maintaining its property of self renewal and in cell fate determination. However, a clear understanding of this regulatory mechanism and the landscape of interactions between the coding and non-coding elements was still lacking. To fill in this void, we use transcriptomic data from RNA-seq and small RNA-seq experiments to reconstruct the core pluripotency circuitry involving mRNAs, lncRNAs and miRNAs. The overall interaction landscape revealed an alternate circuit for the maintenance of pluripotency devoid of the classic pluripotency transcription factors NANOG, SOX2 and POU5F1. We also identified networks specific to the naive and primed states of human pluripotency revealing a new set of transcriptomic markers that could not only be used to differentiate pluripotent state from non-pluripotent state but also to identify the intra-pluripotency state. The lncRNA DANT1 was found to be crucial in determination to the two pluripotency states as it formed a bridge between the naive and primed state specific pluripotency networks. Further, we also identified and computationally validated putative ceRNA mechanism involving DANT1, the miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p, and several key pluripotency related genes including PTPRZ1, SALL2, TOX3, ZNF695, and ZYG11A which warrants further experimental validation. HIGHLIGHTSO_LIReconstructed the key MLMi circuitry underlying human pluripotency by combining RNA-Seq data and known interaction information. C_LIO_LIIdentified an alternate pluripotency circuit devoid of classic pluripotency transcription factors NANOG, SOX2 and POU5F1. C_LIO_LIPredicted a new set of markers that can not only distinguish between pluripotent and non-pluripotent states but also identify the intra-pluripotency state. C_LIO_LIIdentified novel putative function of lncRNA DANT1 as ceRNA in formation of naive and primed state pluripotency. C_LIO_LIDANT1 harbours binding sites for miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p. C_LI

bioinformatics↗

bioRxiv2022

N-terminal helices and A domain of archaeal FtsY facilitate SRP54 binding and the association with cell membrane

The process of protein translocation is essential to the maintenance of cellular life and has been critically addressed in eukaryotes and bacteria. However, little information is available regarding protein translocation across archaeal membranes. The signal recognition particle (SRP) plays an important role in this process. It binds the signal peptide at the N-terminus of the polypeptide chain and interacts with the cognate SRP receptor (FtsY) located on the target membrane to form a targeting complex (TC). Concomitant GTP hydrolysis by SRP and FtsY delivers the polypeptide to the adjacent protein-conducting channel. The present study aims to characterize the structural domains of FtsY contributing to the targeting complex (TC) formation in Sulfolobus acidocaldarius, a thermo-acidophilic crenarchaeon. The contacting residues between SRP54 and FtsY were mapped along the N1-N3 helices. Interestingly, the previously reported crystal structure did not take the N-terminal A domain into account - a region rich in negatively charged residues. Such observation led us to investigate the contribution of each of the three participating helices (N1-3) in terms of membrane association and functional TC formation. Through biophysical analyses of SRP-FtsY and FtsY-membrane interaction, and biochemical characterization of the reciprocal GTPase activity, this work sought to elucidate the minimal structural motif controlling the archaeal TC assembly.

biochemistry↗