Biology subjects

Yadav, K.

Publications and source records attributed to Yadav, K..

16 recordsLinked to original sources

bioRxiv2026

Dynamics of calcium oxalate monohydrate in high and low temperature phases using 17O solid-state NMR

Calcium oxalates are naturally occurring minerals, with the monohydrate form CaC2O4xH2O (COM) being the most stable. COM contains two crystallographically different water sites. We investigate the details of water internal dynamics in the high and low temperature phases of COM using 17O central transition solid-state NMR line shapes, as well as laboratory and rotating frame relaxation rates. The measurements were performed either under static or magic angle spinning conditions and in a wide temperature range from 343 to 180 K. The combination of all measurements allows for precise constraints on motional mechanisms, rate constants, and amplitudes of motions. The high temperature phase is dominated by large-angle fluctuations with an amplitude of about 100 degrees, identical in both sites. During the phase transition between 323 to 300 K, these large-angle jumps freeze out in one of the water sites, while remaining active in the other. In the low temperature phase from 280 to 180 K, small-angle fluctuations of 2-8 degrees in amplitude dominate the relaxation. Transverse relaxation rates also point to the existence of a very slow collective rocking motion down to about 220-200 K.

biophysics↗

bioRxiv2026

Recombinant measles virus equipped with BNiP3, a pro-apoptotic gene, targets β-catenin pathway in triple negative breast cancer cells

Oncolytic virotherapy is an emerging cancer therapy using genetically modified viruses. We previously reported engineering of measles virus with BNiP3, a proapoptotic gene for oncolytic purposes. The recombinant virus had shown promising results in breast cancer cells with a bias towards TNBC, an invasive and an aggressive subtype. Here, we investigated the mechanistic insights of anti-tumor effects induced by the recombinant virus. Initially, TNBC and non-TNBC tumor cell lines were compared bioinformatically using the available gene expression data through protein-protein interaction network using different topological properties. Four hub genes involved in tumor development and progression were identified to be the top genes in both the data sets. Of which, CTNNB1 gene encoding {beta}-catenin was found to be the significant one; as {beta}-catenin pathway is known to be a driver of tumor cell invasion and migration, the impact of the virus on this pathway was investigated in breast tumor cells. The results had demonstrated a notable decrease in {beta}-catenin expression and its downstream targets, cyclin D1, MMP7 reducing the migration potential of TNBC cells following virus infection. These findings suggest that the recombinant measles virus could be one of the effective treatment modalities to target invasive TNBC tumors. In vivo validation of engineered virus is ongoing to explore the therapeutic application of this virus. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/718830v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@19d321dorg.highwire.dtl.DTLVardef@1450ebeorg.highwire.dtl.DTLVardef@cb191eorg.highwire.dtl.DTLVardef@1de2d16_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIUse of recombinant measles virus with a pro-apoptotic gene, BNiP3 to target breast cancer cells C_LIO_LIIdentification of top regulatory genes in breast cancer development and progression C_LIO_LIReduction of {beta}-catenin expression encoded by CTNNB1 gene in TNBC cells following virus infection C_LIO_LIDownregulation of {beta}-catenin downstream targets in TNBC cells with virus infection C_LIO_LIInhibition of migratory potential of TNBC cells following infection C_LI

cancer biology↗

bioRxiv2026

Diet-conditioned microbiota enhances fecal microbiota transplantation efficacy in alcoholic liver disease through caproic acid-PPARα signaling

Background and AimFecal microbiota transplantation (FMT) in Alcohol-related liver disease (ALD) has shown therapeutic potential, with variable efficacy and unclear mechanism. Because dietary protein influences gut microbiota composition, we hypothesized that donor dietary preconditioning could enhance FMT efficacy. We therefore examined in a murine ALD model if high-protein donor diet improves FMT outcome. MethodsALD was induced in C57BL/6N mice using a Lieber-DeCarli ethanol diet combined with thioacetamide administration for 12 weeks. FMT was performed using stool from diet-modulated donors, and recovery was assessed on day7 post-FMT. Multi-omics analysis using 16s rRNA and mass spectroscopy was performed for Gut microbiota composition, plasma- and stool-metabolome, and hepatic proteomes. Multi-omics outcomes were validated in ALD animal and Huh7 hepatocytes. ResultsBoth protein-based FMTs improved ALD recovery; Veg-FMT demonstrated superior efficacy, significantly reducing hepatic injury (AST 1.2-fold, p=0.002; bilirubin 1.2-fold, p=0.03; steatosis 1.7-fold,p=0.01) and restoring gut barrier integrity (occludin 1.5-fold,p=0.04; mucin 2 2.2-fold, p=002; and plasma endotoxin 1.7-fold, p=0.02). A significant 2-fold increase was observed in Lachnospiraceae NK4A136, Coriobacteriaceae UCG-002, and short-chain fatty acids, particularly caproic acid. Functional validation confirmed that caproic acid promoted hepatic fatty acid {beta}-oxidation through PPAR-dependent mechanisms, reducing triglyceride accumulation and lipogenesis in both cellular and animal models. ConclusionDonor preconditioning with a plant-protein enriched diet enhances FMT efficacy in ALD by gut microbiota modulation with increased metabolites like caproic acid. These findings highlight a microbiota-metabolite-host axis through which diet-modulated FMT improves hepatic lipid metabolism and injury, and identifies a pathway via which FMT imparts its effect. SignificanceThis study identifies a mechanistic basis for improving fecal microbiota transplantation (FMT) efficacy in alcohol-related liver disease (ALD) by demonstrating that dietary preconditioning of donor microbiota improves therapeutic outcomes. We show that plant protein-modulated donor microbiota supplements abstinence-associated recovery through increased production of the microbial metabolite caproic acid, which promotes hepatic fatty acid {beta}-oxidation via PPAR signaling. These findings highlight donor dietary conditioning and microbiota-derived metabolites, rather than microbial composition alone, as important determinants of FMT efficacy. The results suggest that microbial metabolites such as caproic acid may represent potential therapeutic targets or biomarkers to enhance and standardize microbiota-based interventions in ALD. Although the current work is based on a murine model, the identified microbiota-metabolite-host metabolic axis provides a framework for future translational studies aimed at optimizing FMT strategies in liver disease.

microbiology↗

bioRxiv2025

Urinary Exosomal miRNA Profiling Reveals Sensitive Non-Invasive Diagnosis of Bladder Cancer

UnstructuredmiRNAs represent a transformative advancement in both research and clinical management of urinary bladder cancer (UBC), emerging as clinically significant molecular targets with the potential to revolutionize existing diagnostic standards. This study exploited the robust stability of miRNAs and profiled urinary miRNAs in UBC patients and controls through miRNA sequencing, revealing an expanded and altered miRNA repertoire in cancer samples. Validation in an independent cohort revealed fold changes for miR-6724-5p, miR-1273h-5p, miR-7704, miR-200-5p, and miR-10400-5p ranged from [~]46 to [~]2,777 (p < 0.01). Stage-specific analyses highlighted dynamic miRNA expression linked to tumor progression. Diagnostic evaluation identified an optimal three-miRNA panel comprising miR-6724-5p, miR-10400-5p, and miR-7704, achieving diagnostic accuracies (AUC > 70%) and high sensitivity (>90%). These data demonstrate the potential of urinary mature miRNAs as robust biomarkers for non-invasive detection and monitoring of UBC in early stages, supporting their translation into clinical diagnostic assays pending larger prospective studies. StructuredO_ST_ABSBackgroundC_ST_ABSUrinary microRNAs (miRNAs) are promising candidates due to their molecular stability and disease-specific expression. miRNAs represent a transformative advancement in both research and clinical management of urinary bladder cancer (UBC), emerging as actionable molecular targets with the potential to revolutionize existing diagnostic standards. MethodsSmall RNA sequencing was performed on urine samples from UBC patients and healthy controls to profile miRNA expression. Mature miRNAs were prioritized by exclusion of precursor forms using computational filtering. Validation by quantitative RT-PCR was conducted on an independent, age-matched cohort (n = 45). Diagnostic potential of candidate miRNAs was assessed by receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses. ResultsSequencing identified 865 known and 11 novel miRNAs, with UBC samples showing greater miRNA diversity (708 known miRNAs) compared to controls (540). Ten miRNAs were significantly dysregulated in UBC (p < 0.05). Validation revealed fold changes for miR-6724-5p, miR-1273h-5p, miR-7704, miR-200-5p, and miR-10400-5p ranged from [~]46 to [~]2,777 (p < 0.01). Stage-specific analyses highlighted dynamic miRNA expression linked to tumor progression. A three-miRNA panel (miR-6724-5p, miR-10400-5p, miR-7704) demonstrated superior diagnostic performance (AUC > 70%, sensitivity > 90%). ConclusionMature urinary miRNAs exhibit reproducible, stage-specific dysregulation in UBC and a combinatorial panel offers sensitive, specific non-invasive detection. These findings warrant further multicenter validation for clinical application.

cancer biology↗

bioRxiv2025

Behavioral Decoding Reveals Cortical Endocannabinoid Potentiation during Δ9-THC Impairment

How {Delta}9-tetrahydrocannabinol (THC) impairs natural behaviors in mice remains unknown. We developed a video-monitored behavioral platform with machine learning classifiers to unravel discrete changes in natural mouse behaviors. THC infusion into the medial prefrontal cortex (mPFC) disrupted walking kinematic features characteristic of impairment responses. THC predominantly increased mPFC GABAergic activity preceding walk initiation shifting the mPFC excitatory/inhibitory (E/I) balance. Pose-defined closed loop photo-stimulation of mPFC GABAergic neurons demonstrated that THC exacerbates selected parameters of motor impairment. Surprisingly, THC also induced a time locked, movement-induced, transient potentiation of mPFC endocannabinoid (eCB) release and ensuing CB1R-mediated synaptic inhibition. Here we establish that THC-modifies mPFC E/I balance to excitation via dynamic changes in eCB release which acts to induce behavioral impairment.

neuroscience↗

bioRxiv2025

Liver Kinome Profiling Identifies PS1145 as a Potential Therapeutic molecule for amelioration of Systemic Inflammation in Alcohol-related Liver Disease

Background and aimsAlcohol-related liver disease (ALD) has high mortality due to systemic inflammation. We analysed liver and monocyte kinome profiles in a chronic ethanol-fed pre-clinical rat model to identify therapeutic targets that could mitigate inflammation in ALD. MethodKinome profile was performed in liver and circulating monocytes at baseline, and after 8,12,16,20 and 24-weeks of 40% ethanol administration. Pathway-specific inhibitors, including PS1145 (IKK-phosphorylation inhibitor), PH-797804 (MAPK14 inhibitor), resveratrol and prednisolone were tested for their anti-inflammatory effects in ALD-rats, PBMC from patients and NIAAA mouse model. ResultsKinome profiling identified 497 liver and 345 monocyte kinases in ALD rats (FDR<0.01). A time-dependent increase in MAPK14-associated kinases was observed in both tissues (FC>1.5, p<0.05). By 24 weeks, 172 liver and 48 monocyte kinases were significantly upregulated, particularly those linked to MyD88-TLR4, PI3K-Akt, TNF, TGF{beta}, and cellular senescence pathways. Key contributors included TGF{beta}R1, ROS-generating kinases, and IL-1-driven MAPK14 and IKK phosphorylation. Targeting this axis, PS1145 (an IKK inhibitor) suppressed NF{kappa}B activation and inflammation in THP1 and HepG2 cells, as well as PBMCs from healthy and SAH patients, outperforming PH797804, resveratrol, and prednisolone (p<0.05, FC>1.5). PS1145 significantly reduced IL-6, TNF, and NF{kappa}B, while increasing IL-10. In vivo, PS1145 treatment in the NIAAA mouse model markedly reduced hepatic steatosis, cellular stress and inflammatory pathways (cytokine signalling, IL-36 signalling and others) more effectively than standard therapies, Notably, it also downregulated IL36R, impairing TLR receptor dimerization, suggesting a dual mechanism of action (p<0.05) highlighting its therapeutic potential in ameliorating systemic inflammation in ALD. ConclusionOur study highlights the key role of MAPK14 and MYD88-TLR4 pathway kinases in driving systemic inflammation in SAH. The IKK inhibitor PS1145, by blocking both IKK phosphorylation and TLR dimerization, effectively suppresses inflammatory signalling and improves liver pathology, positioning it as a promising targeted therapy for ALD.

pharmacology and toxicology↗

bioRxiv2025

Regulation of the lncRNA malat1/Egr1 Axis by Wnt, Notch and TGF-β signaling: A Key Mechanism in Retina Regeneration

Adult zebrafish retinas rely on the resident Muller glia to maintain homeostasis and enable regeneration. Retina regeneration remains incomplete in mammals despite extensive efforts to emulate zebrafish regenerative conditions. Many studies have examined the reprogramming of zebrafish Muller glia cells, which is necessary for regeneration driven by regeneration-associated gene expression. Here, we show that the lncRNA malat1, crucial for many biological functions, plays essential roles during retina regeneration. We demonstrate that malat1 functions through an Egr-dependent axis, modulated by Wnt, Notch, and TGF-{beta} signaling pathways, and is necessary for effective retina regeneration. Moreover, we uncover that the antisense lncRNA talam1, which regulates malat1 availability, is differentially regulated in zebrafish and mice, highlighting species-specific gene regulatory mechanisms after retinal injury. Cells with active TGF-{beta} signaling stabilize Malat1 in mice while the same signaling destabilizes malat1 in zebrafish. Taken together, our work uncovers a new role for the malat1/Egr1 axis in necessitating retina regeneration, which may have important implications for differential regenerative ability in vertebrates.

cell biology↗

bioRxiv2024

Inclusion of Hsp70 co-chaperone and RNA binding activities facilitates optimal function of spliceosomal disassembly factor Cwf23 in intron-rich Schizosaccharomyces pombe

Cwc23 is an essential J-domain protein that collaborates with the disassembly factor Ntr1 to facilitate spliceosomal disassembly. Although Cwc23 orthologs have been identified in spliceosomal extracts of many eukaryotes, their functionality in intron-rich eukaryotes remains largely unexplored. Here, we investigate the functionality of Cwf23, an ortholog of Cwc23 in Schizosaccharomyces pombe. Our study reveals that while the interaction between Cwf23 and SpNtr1 is conserved, it is not essential in S. pombe. Additionally, the RNA recognition motif (RRM) in Cwf23 is crucial for its function, as mutations in the RRM affect both growth and pre-mRNA splicing. Unlike its budding yeast counterpart, the J-domain of Cwf23 is indispensable, as J-domain mutants cannot support cell viability. These findings suggest a new paradigm in which the presence of a functional RRM and the essential nature of the J-domain underscore the increased requirements for an RNA-binding Hsp70 co-chaperone machinery in spliceosomal remodelling in "intron-rich" eukaryotes.

molecular biology↗

bioRxiv2024

Microarray Integrated Spatial Transcriptomics (MIST) for Affordable, Robust, and Comprehensive Digital Pathology

10X Visium, a popular Spatial transcriptomics (ST) method, faces limited adoption due to its high cost and restricted sample usage per slide. To address these issues, we propose Microarray Integrated Spatial Transcriptomics (MIST), combining conventional tissue microarray (TMA) with Visium, using laser-cutting and 3D printing to enhance slide throughput. Our design facilitates independent replication and customization in individual labs to suit specific experimental needs. We provide a step-by-step guide from designing TMAs to the library preparation step. We demonstrate MISTs cost-effectiveness and technical benefits over Visium and GeoMx Nanostring. We also introduce AnnotateMap, a novel computational tool for efficient analysis of multiple ROIs processed through MIST.

bioinformatics↗

bioRxiv2023

Microevolution of clade II isolates of Candida auris highlights multifaceted intra-clade heterogeneity in acquiring resistance towards amphotericin B

Candida auris exhibits high-level resistance to amphotericin B (AmB). Mechanisms such as ergosterol biosynthesis malfunction, oxidative damage mismanagement, and increased drug efflux contribute to AmB resistance in C. auris. In this study, we experimentally evolved two East Asian drug-susceptible clade II isolates of C. auris (P2428 and CBS10913T) isolated from different geographical locations to develop resistance against AmB. We analysed alterations in karyotype, genome sequence, and gene expression profiles to uncover the mechanisms driving AmB resistance. The independently evolved clade II adaptors displayed up to 4-16-fold higher MIC50, as compared to the parental cells. AOX2 (alternative oxidase) and the cell wall integrity pathway have been identified as critical in the development of AmB resistance. However, we noted certain intra-clade heterogeneity in the associated mechanisms. While in P2428 adaptors (P-lines), the ergosterol and sphingolipid pathways appear to play a crucial role, this was not the case for CBS10913T adaptors (A-lines), which acquired resistance independent of lipid-associated changes. The transcriptomic, WGS, and phenotypic analyses also confirm that the evolved AmB-resistant isolates follow distinct trajectories for adaptation, Furthermore, unlike the fluconazole-resistant isolates, as reported previously, changes in ploidy do not seem to contribute to the differential mechanisms of AmB resistance. Overall, this study not only provides insights into the mechanisms and pathways involved in AmB resistance but also highlights intra-clade-heterogeneity that exists within clade II of C. auris. ImportanceCandida auris demonstrates significant resistance to amphotericin B (AmB) that stems from factors like alteration of ergosterol biosynthesis, perturbation of the oxidative damage response, etc. A comprehensive understanding of underlying mechanisms can be studied in a holistic manner by subjecting resistant as well as susceptible clinical isolates to a comparative genome-level analysis. An alternate and more dynamic approach is to expose susceptible isolates to a certain concentration of drug which is not lethal but can trigger the resistance mechanisms. In the present study, we evolved C. auris towards AmB and observed novel and differential mechanisms of resistance development, in two different isolates despite belonging to the same clade. This study provides insights into the intra-clade heterogeneous behavior of C. auris towards AmB.

microbiology↗

bioRxiv2023

Effect of natural precursors and micro/macroparticles addition on the morphology modulation of Streptomyces toxytricini KD18 stimulates lipstatin productivity

The cellular architecture of filamentous microbes is of great interest because it is frequently associated with secondary metabolite productivity and can be altered by cultivation conditions. Hence, the evaluation of cell morphology is of the utmost significance for better understanding of industrial processes involving filamentous bacteria. In the present study, effect of glass beads and silica particle addition have been seen in the enhancement of lipstatin production along with alterations in the morphology. The addition of glass beads and silica particles directed the morphology of Streptomyces toxytricini KD18 towards the formation of small pellets (0.3 to 0.4mm) with dispersed mycelia as compared to the control conditions (0.04 to 2mm). A four-fold increase in lipstatin production was achieved due to mechanical stress caused by glass beads and silica particles. The addition of natural precursors, i.e., Aloe vera pulp, Hibiscus cannabinus leaves and flowers improved the production of lipstatin from 2.06 mg/ml to 6.76 mg/ml, 11.4 mg/ml and 14.09 mg/ml, respectively along with alteration in the pellet morphology in 500 ml shake flask.

bioengineering↗

bioRxiv2022

Genomic evaluation of BC4, a consortium of four Alkalihalobacillus clausii isolates, confirms its probiotic potential and safety in usage

Four strains of Alkalihalobacillus clausii B603/Nb (resistant to rifampicin), B619/R (resistant to streptomycin), B637/Nm (resistant to tetracyclin) and B106 (resistant to chloramphenicol) were isolated from various sources and used to prepare a consortium designated as BC4. Genomes of the constituent strains of the BC4 consortium were evaluated to investigate their genetic makeup and determine their probiotic potential. Gene prediction and functional annotation were performed using RAST. The data obtained was mined for genes encoding various phenotypic traits. This analysis revealed the presence of several genes encoding probiotic attributes like (i) survivability in the presence of low pH, bile, oxidative stress; (ii) bacterial aggregation and adhesion to gut epithelium, etc.; and (iii) enzymes/ molecules conferring health benefits. Further, the genome analysis also confirmed the genes required for enhancing the nutritional amenability, health-promoting, and disease-preventing traits were present. Several genes encoding multiple antibiotic resistance were detected; however, none of these genes was located on mobile elements such as plasmids, transposons, etc. The absence of genes encoding virulence factors, pathogenic islands, emetic toxins, etc., as well as mobile genetic elements, underscored the safety of BC4 isolates.

microbiology↗

bioRxiv2021

Deltamethrin contact exposure mediated toxicity and histopathological aberrations in tissue systems of cockroach species Periplaneta americana and Blatella germanica

Cockroach species Periplaneta americana and Blatella germanica potentially survive in locations close to human activity. Besides spoiling food material, cockroaches also transfer pathogens of different diseases among human. Since the insecticides have been used extensively to control cockroaches, information on their insecticide susceptibility and toxicity at cellular level may be crucial. In the study, deltamethrin toxicity as well as the deltamethrin-mediated cytomorphological changes in brain, ovary and midgut of the two important cockroach species has been assessed. Different concentrations [0.00025% (0.0025mg/ml), 0.0025% (0.025mg/ml), 0.025 (0.25mg/ml), 0.25% (2.5mg/ml), 0.5% (5mg/ml), 1% (10mg/ml)] of deltamethrin in acetone were used to expose test species in WHO bottle assay. Knockdown was recorded after 5 min interval while delayed mortality was observed after 24 hr. Brain, ovary and gut were dissected post 1 hr exposure and 24 hr holding (for 0.25%, 0.5% and 1% concentration), and tissues were processed for microscopic analysis. Deltamethrin exposed cockroaches and dissected tissues were used to estimate deltamethrin using HPLC. At 0.00025% (lowest concentration), the percentage knock-down observed was 66.7% for P. americana and 80% B. germanica respectively (R2= 0.78; p=0.0001) in 1 hr. KDT50 value was found to be 8.7 min (95% CI: 7.3-10.2), while KDT99 was 20.7 min (95% CI: 16.0-35.7) in P. americana at 1% concentration. Whereas, the KDT50 and KDT99 values for B. germanica were 7.4 min (95% CI: 5.4-9.1) and 27.4 min (95% CI: 18.2-80.0) at similar concentration. LD50 and LD95 values (for 60 min standard exposure) were 0.0006 % (95% CI: 0.00-0.001) and 0.034% (95% CI: 0.013-0.49) respectively for P. americana, while these values were 0.0005 (95% CI: 0.00-0.001) and 0.04 (95% CI: 0.01-0.23) for B. germanica. Exposure to 1% deltamethrin induced considerable toxic effect in the epithelial cells in the midgut. HPLC estimated 0.21{+/-}0.05 mg (95% CI - 0.18-0.25; CoV 23.9%) deltamethrin in P. americana post 1% exposure. Even short term exposure of low concentration of synthetic pyrethroid deltamethrin displayed immediate knockdown and delayed mortality in both the test species. Considerable histological damage was observed in both the insects at 1% exposure. In India, resistance to deltamethrin may have been reported among different insects due its extensive use, however the formulations such as insecticide paints, attractant baits etc. developed using deltamethrin as active ingredient could be useful in cockroach control operations.

zoology↗

bioRxiv2021

Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture proteins, with subsequently washes allowing contaminant removal. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in protein recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief centrifugation--either with or without low-cost inert glass beads--as the means of aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1-5000 {micro}g preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling (n = 9,076) also demonstrated improved recovery by SP4 and a significant enrichment of membrane and low-solubility proteins vs. SP3. The effectiveness of SP4 was verified in three other labs, each confirming equivalent or improved proteome characterisation over SP3. This work suggests that protein precipitation is the primary mechanism of SP3, and reliance on magnetic beads presents protein losses, especially at higher concentrations and amongst hydrophobic proteins. SP4 represents an efficient and effective alternative to SP3, provides the option to omit beads entirely, and offers virtually unlimited scalability of input and volume--all whilst retaining the speed and universality of SP3. BRIEFSolvent precipitation SP3 (SP4) captures aggregated protein for proteomics sample clean-up by omitting magnetic beads, instead employing brief centrifugation--with or without low-cost inert glass beads. SP4 offers improvements to protein yields, higher reproducibility, and greater recovery of membrane proteins, with verifications from three labs. Protein precipitation appears to be the primary mechanism of SP3, with reliance on magnetic beads presenting protein losses, especially at higher concentrations. SP4 presents an effective alternative to SP3 with improved scalability and equal speed and universality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/461247v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ba615corg.highwire.dtl.DTLVardef@14df25dorg.highwire.dtl.DTLVardef@62f0c1org.highwire.dtl.DTLVardef@1ac436a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates(1-3). Sandercyanin binds non-covalently to biliverdin IX (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm(1). Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin ([~]18kDa) binds BLA with similar affinities and show a broad red-shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA-bound lipocalins, known only in phytochromes till date. Significance StatementSandercyanin is a tetrameric red fluorescent protein from a blue variant of walleye (Stizostedion vitreum) that binds to biliverdin IX (BLA). Its biophysical properties and structures have been published earlier(1). A bright and stable monomeric Sandercyanin could be utilized as a fusion protein for fluorescence-based applications. Here we report the first structures and spectral properties of fluorescent monomeric Sandercyanin-BLA complexes and describe the molecular basis of modulated spectral properties due to rotated D-ring pyrrole around C14-C15 bond and re-shuffling of BLA-binding pocket. BLA-bound monomeric Sandercyanin could be engineered into brighter variants for in-vivo applications. Our study also reveals an unfamiliar mechanism in BLA-binding lipocalins that regulates red- and far-red absorbance states.

bioengineering↗

bioRxiv2019

Global kinome silencing combined with 3D invasion screening of the tumor microenvironment identifies fibroblast-expressed PIK3Cδ involvement in triple-negative breast cancer progression

As there is growing evidence for the tumor microenvironments (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3C{delta} (f-PIK3C{delta}) as a key regulator of progression. Although PIK3C{delta} has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3C{delta} was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain- and loss-of functions experiments verified the contribution of f-PIK3C{delta} in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3C{delta} confers its pro-tumorigenic effects. Inhibition of f-PIK3C{delta} promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3C{delta} in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3C{delta} in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3C{delta} inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3C{delta} (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

cancer biology↗