bluuebunny/crossref_metadata_2025 huggingface dataset:
P‐glycoprotein deficiency results in intestinal developmental delay in C. elegans
Authors: Salciccioli Brittany , Braxton Diamond , DeYoung MaKenna , Tanner Scott
Published: May 2022

Inflammatory Bowel Disease (IBD) is a term used to group similar diseases characterized by the chronic inflammation of the digestive tract. IBD is an autoimmune reaction with an unknown cause. There is no known cure, only treatments to relieve symptoms. There are many genes linked to this disease, including MDR1. The MDR1 gene codes for a protein called P‐glycoprotein (Pgp), which is known to be expressed in intestinal cells. The exact relationship between IBD and Pgp is unknown, but it has been observed that some patients with IBD have a mutation that causes a loss of function of Pgp. Our goal is to explore the use of Caenorhabditis elegans as a model for intestinal diseases. Because Pgp deficiency is associated with intestinal damage, we hypothesized to measure changes in the intestine and organism that indicate intestinal damage, particularly changes in tight junctions, which would lead to increased intestinal permeability leading to damage. Tight junctional analysis for using both immunohistochemistry and qRT‐PCR shows a downregulation of the tight junctional protein LET‐413 in Pgp1‐/‐ C. elegans. These data support our hypothesis that Pgp deficiency impacts intestinal tight junctions in C. elegans, potentially leading to intestinal damage. This change in expression is similar to what is see in human inflammatory bowel disease, particularly those individuals who have Pgp polymorphisms. The downregulation of tight junctional genes (let41 and dlg1) was also seen in Pgp3‐/‐ and Pgp1/3‐/‐ C. elegans at the L1 stage. Interestingly, changes in gene expression were only observed in L1 stage C. elegans. Mid to old‐aged larvae (L2‐L4) and adult C. elegans displayed no change in gene expression. Additionally, Pgp deficient C. elegans also developed at a slower rate than the N2 control strain. This suggests that these changes are specific to early development. To explore this, gene expression analysis was also performed on several genes related to the development of the C .elegans intestine and pharynx. Pgp1‐/‐ L1 C. elegans displayed significant increases in several genes (pal1, pha4, elt2, end1,and pie1), with all genes trending upwards. This might indicate that these worms have slower developing intestines, and are still undergoing the developmental process in the L1 stage, where the Pgp3‐/‐ C. elegans has already completed the development of the intestine. Taken with the LET‐413 staining which was also decreased in the Pgp1‐/‐ samples, we conclude that the deficiency of Pgp1 impacts intestinal development, which leads to a change in tight junction expression.
Kinetic networks identify key regulatory nodes and transcription factor functions in early adipogenesis
Authors: Guertin Michael J. , Nguyen Bao , Walavalkar Ninad , Duarte Fabiana , Dutta Arun B.
Published: May 2022

Sequence‐specific transcription factors (TFs) bind DNA, modulate chromatin structure, regulate gene expression, and orchestrate transcription cascades. Activation and repression of TFs drive tightly controlled regulatory programs that lead to cellular processes such as differentiation. We measured chromatin accessibility and nascent transcription at seven time points over the first four hours of induced adipogenesis of preadipocytes to construct dynamic gene regulatory networks. Regulatory networks describe successive waves of TF binding and dissociation followed by direct regulation of proximal genes. We identified 14 families of TFs that coordinate with and antagonize each other to regulate early adipogenesis. We developed a compartment model to quantify individual TF contributions to RNA polymerase initiation and pause release rates. Network analysis showed that the glucocorticoid receptor and AP1 drive immediate gene activation, including induction of Twist2. Twist2 is a highly interconnected node within the network and its expression leads to repression of target genes. Although Twist2's role in adipogenesis has not been previously appreciated, both Twist2 knockout mice and Setleis syndrome (Twist2‐/‐) patients lack subcutaneous and brown adipose tissue. We found that kinetic networks integrating chromatin structure and nascent transcription dynamics identify key genes, TF functions, and coordinate interactions within regulatory cascades.
Therapeutic implication of SOX9 and YAP1 co‐repression in advanced intrahepatic cholangiocarcinoma
Authors: Hu Shikai , Molina Laura , Tao Junyan , Liu Silvia , Hassan Mohammed , Singh Sucha , Poddar Minakshi , Nejak‐Bowen Kari , Oertel Michael , Raeman Reben , Singhi Aatur , Bell Aaron , Monga Satdarshan P. , Ko Sungjin
Published: May 2022

Introduction

Intrahepatic cholangiocarcinoma (iCCA) is a liver tumor of increasing incidence and devastating prognosis. The most critical barrier for developing a globally effective therapy for iCCA is molecular heterogeneity caused by diverse molecular drivers and cellular origins in the liver. Clinically, over 90% of iCCAs show alterations of YAP1 and/or SOX9. Therefore, we investigated the therapeutic potential of co‐repression of YAP1 and SOX9 as a wide range therapeutic option for advanced iCCA.

Methods

By immunohistochemistry (IHC) using 2 tissue microarrays, we examined the localization of SOX9 and YAP in clinical iCCA. We also assessed clinical relevance of AKT‐NICD (AN) /YAP (AY)‐dependent hepatocyte‐driven iCCA by bioinformatic analysis comparing their transcriptome with large human datasets. Lastly, we comprehensively investigated therapeutic effect of simultaneous deletion of Sox9 and Yap1 in advanced murine iCCA using respective floxed mice.

Results

Over 90% of human CCA samples (n=108) exhibited nuclear SOX9 and YAP1. We also identified significant upregulation of p‐AKT, SOX9 and YAP1 in HCs of patients with primary sclerosing cholangitis and nonalcoholic steatotis hepatitis, well‐known risk factors for iCCA. While co‐expression of AN led to hepatocyte‐derived lethal iCCA, conditional deletion of either Yap1 or Sox9 dramatically reduced AN‐ iCCA development. Yap1 deletion impaired HC‐to‐biliary epithelial cell (BEC) fate conversion and tumor cell proliferation, while Sox9 elimination only repressed proliferation. Interestingly, following single deletion of either Yap1 or Sox9 we observed a few iCCA tumors expressing either SOX9 or YAP1 but not both. This is also identified in a small subset of human iCCA; SOX9+/YAP1‐ (4.6%) or SOX9‐/YAP1+ (3.7%), suggesting that single deletion of YAP1 or SOX9 is not sufficient to abrogate clinical iCCA. We also found that simultaneous deletion of Yap1 and Sox9 completely prevents iCCA development. Remarkably, co‐deletion of Yap1 and Sox9profoundly decreases fully developed lethal iCCA burden while extending survival. However, singular deletion of either shows no survival benefit compared to control group harboring advanced iCCA as a therapeutic manner.

Conclusions

We elucidate the critical, but distinct roles of Yap1and Sox9 in iCCA development and demonstrate the therapeutic potential of simultaneous targeting both for wide range of iCCA irrespective of drivers. Ongoing work will comprehensively delineate the molecular mechanism how YAP1 and SOX9 compensate each other when either is removed in iCCA, and will be relevant for studying adaptive resistance against the singular targeting of SOX9 or YAP1 in iCCA.
Scanning‐Free functional Fluorescence Microscopy Imaging Toward Spatial Mapping of Biomolecular Information in Live Cell
Authors: Oasa Sho , Krmpot Aleksandar , Nikolic Stanko , Clayton Andrew , Tsigelny Igor , Changeux Jean‐Pierre , Terenius Lars , Rigler Rudolf , Vukojevic Vladana
Published: May 2022

Live cells form signaling network and control it via molecular interactions and transporting processes. Through molecular interactions and transporting processes, they precisely regulate spatio‐temporally the concentration and mobility of biomolecules. To understand the dynamic integration of molecular interactions and how cells control them spatio‐temporally, quantitative methods with high sensitivity and spatio‐temporal resolution are needed. Fluorescence microscopy‐ and correlation spectroscopy‐based methods have been proven to be indispensable for this purpose, and advanced methods such as Single Plane Illumination Microscopy (SPIM)/Total Internal Reflection Microscopy (TIR)‐based Fluorescence Correlation Spectroscopy (SPIM‐FCS/TIR‐FCS) have been developed. However, both methods also have limitations. In SPIM‐FCS, fluorescence signal is simultaneously read‐out; but light sheet propagation may be affected by obstacles in the sample, giving rise to an uneven fluorescence excitation across the sample. TIR‐FCS is limited to study at the basal plasma membrane due to an evanescence illumination. In this situation, we have developed scanning‐free massively parallel FCS (mpFCS) with high spatio‐temporal resolution (~240 nm and ~10 µs) and single‐molecule sensitivity, which allows us to quantitatively characterize fast dynamic processes and precisely measure the concentration of fluorescently labeled biomolecules in live cells [Krmpot A.J. et.al., Anal. Chem. 2019; Oasa S. et.al., Anal.Chem. 2021]. mpFCS records fluorescence signals from independent 256/1024 excitation spots generated by the diffractive optical elements (DOEs) using single‐photon avalanche diode (SPAD) camera. Time series of fluorescence intensity are transformed to autocorrelation curves to determine the concentration and diffusion time. Our instrument also allows Fluorescence Lifetime Imaging Microscopy (FLIM), to characterize the immediate surroundings via fluorescence lifetime, and FLIM‐based Förster Resonance Energy Transfer (FLIM‐FRET), to assess the biomolecular interaction via donor‐acceptor interaction. We have applied mpFCS/FLIM to spatially map in live cells the local concentration, diffusion and local environment of a transcription factor in the cell nucleus and have characterized the effect of allosteric inhibitor of transcription factor dimerization [Oasa S., et.al., PNAS 2020]. I will also discuss another potential of the instrument in spatial cross‐correlation analysis toward the molecular transporting in live cells.
The new in the enteral dysbiosis correction intensified by heavy metals and mycotoxins
Authors: Ponamarev Vladimir , Popova Olga , Baryshev Viktor
Published: May 2022

The study aimed to research the new treatment and pharmacorrection protocol of toxical body state intensified by heavy metals and mycotoxins in the cattle using native drugs. The studies were carried out on cows aged 3 to 4 years. 2 groups were formed (n=10 by each). The protocol of 1st group cows (n=10) was included the sorbents' complex as a supplementation to the ration (4 % per the daily rate during 14th days) and the hepatoprotective agent “Hepaton‐vet” (the compound: lac Carduus, ursodeoxycholic acid) at a dose of 100 ml per cow during 21 days. The protocol of 2 d group cows (n=10) was also included the sorbents' complex as a supplementation to the ration (4 % per the daily rate during 14th days) and the conventional fluid therapy. We were determined the sorbent net efficiency as a quantity characteristic of loading capacity. The sorption of mycotoxins was indicated by the quantity‐related method under different pH, which was affected the pH replacement of the gastrointestinal tract in animals. The heavy metals in milk and feed samples were analyzed by the periodate method and atomic adsorption spectrometry. The level of mycotoxins was tested by the Agra quant ELISA method. We were used the software program “Statistica 6” with Student's t‐test for dependent adjectives as a statistical analysis of results. The highest level of cadmium was contained in the dry forage samples (0.14±0.05 mg/kg). The difference of their between the dry forage and mixed feed with silage was 27 %. The results of the biochemical analysis were showed hypoproteinemia and hypoalbuminemia (21.23±1.12 g/l). The reason could be the dystrophic change of hepatocytes and the deranged biosynthesis of this fraction with the shift to γ‐globulins increasing. The increase of albumin level was on the 12th day of treatment (29.19±1.32 g/l). The albumin level of cows 1st group was increased by 21.2 % and 17.3 % consequently by the end of the experiment compared with the value of cows control group. Y‐globulins were decreasing by 37.3 % and 40.5 %. The atmospheric methane emissions in the 2d group were decreased by 8 % (84±3.5 mg/m3). So, we can assume that using the complex of sorbents with hepatoprotective agent “Hepaton‐vet” has a positive influence on the liver's metabolic process that has evidenced results of morpho‐biochemical analysis and clinical diagnostic of animals.
Gelatin Methacryloyl (GelMA) as a Suitable Three‐Dimensional Extracellular Environment for the Derivation of hiPSC‐Derived Kidney Organoids
Authors: Clerkin Shane , Wychowaniec Jacek K. , Singh Krutika , Davis Jessica L. , Treacy Niall J. , Krupa Ivan , Brougham Dermot F. , Crean John
Published: May 2022

The generation of human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have facilitated novel insights into renal developmental processes and have the potential to provide personalised treatment strategies for patients with end‐stage renal disease. Traditional protocols for the directed differentiation of hiPSC‐derived kidney organoids have predominantly focused on biochemical cues to specify hiPSCs towards a mesonephric lineage, but have not considered the influence of the surrounding biophysical properties of the extracellular environment on organoid formation. We hypothesised that Gelatin methacryloyl (GelMA), a derivative of collagen with mechanically amendable hydrogel stiffness profiles, could be used to investigate the influence of extracellular matrix stiffness on kidney organoid specification. hiPSC‐derived kidney organoids were differentiated within photo‐crosslinked GelMA hydrogels of defined mechanical strengths. Enrichment of renal cell types in response to the various mechanical microenvironments was subsequently investigated using immunocytochemistry, qRT‐PCR, transmission electron microscopy (TEM) and histological staining methods. Rheological analysis of formulated hydrogels confirmed the generation of matrices with distinct stiffness (Young’s Modulus, G’) profiles. Hydrogels comparable to the stiffness of the gastrulation‐stage embryo (G’ = 400 Pa) and adult human kidney tissue (G’ = 5‐8 kPa) were generated. Importantly, the mechanical properties of the hydrogels showed remarkable stability even with prolonged time in culture. PCNA proliferation and cleaved caspase‐3 apoptotic staining of organoids embedded within scaffolds demonstrated high cell proliferation and viability in the hydrogel conditions by day 24 of differentiation. The formation of glomerular, proximal tubular and distal tubular structures, supported by basement membrane and interstitial cells were confirmed in conditions using immunofluorescent imaging. Interestingly, qRT‐PCR analysis revealed significant upregulation of nephron‐associated genes (including PAX8, NPHS2, NPHS1, SLC3A1 and AQP1) in organoids differentiated within extracellular environments that approximated adult human kidney tissue, when compared to organoids differentiated within the much softer microenvironments. Our results illustrate the influence of the extracellular environment on appropriate cell fate determination and propose GelMA hydrogels as faithful extracellular supports for the specification of hiPSC‐derived kidney organoids.
Discovery of the first tight‐binding reversible antagonists of Hedgehog protein autoprocessing
Authors: Wagner Andrew G. , Stagnitta Robert T. , Xu Zihan , Pezzullo John L. , Kandel Nabin , Giner José‐Luis , Covey Douglas F. , Wang Chunyu , Callahan Brian P.
Published: May 2022

Hedgehog (Hh) signaling ligands (HhN) are expressed as precursors with an adjacent C‐terminal autoprocessing domain, HhC. Autoprocessing activity of HhC releases and cholesterylates the Hh ligand thereby activating the HhN for full biological activity. Inhibitors of HhC activity are of interest as probes of the Hh signaling pathway, and HhC inhibitors may also offer new therapeutic approaches to suppress oncogenic signaling activity of HhN. The intramolecular nature of the HhN/HhC system poses a challenge for reversible inhibitors, and to date most antagonists of HhC bind the active site covalently. Here we report we report the discovery and optimization of the first tight‐binding reversible inhibitor of HhC. Using a light‐to‐dark FRET activity assay for Drosophila melanogaster HhC, we screened a focused library of 1187 sterol‐like analogs in search of HhC inhibitors. Four structurally related hits were identified and further validated by dose‐response assays. The most effective inhibitor from the screen had an IC50 value of 2x10‐6M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT‐HBT, a derivative with a sub‐micromolar IC50of 3x10‐7 M. Inhibition appears to be mediated by a previously unidentified allosteric binding site on HhC. Kinetic analysis, photoaffinity labeling, computational modeling, and point mutagenesis studies suggest that the binding site is nearby, but not overlapping with the cholesterol binding site of HhC.
Analysis of the interactions between the HIV‐1 Spike and the F7‐22 “cooperating” antibody
Authors: Parker Miller Emma , Fera Daniela
Published: May 2022

Broadly neutralizing antibodies (bnAbs) are promising for HIV‐1 vaccine design because they target highly divergent strains of the virus. However, attempts to elicit bnAbs have not been successful because of their long maturation pathways and high mutation frequencies. Thus, the design of an effective vaccine to elicit bnAbs will require an understanding of the co‐evolution process between the virus and antibodies in a host. In one HIV‐1 infected patient called CH848, the elicitation of a bnAb lineage called DH270 was preceded by a cooperating antibody lineage, of which DH272 was a member. This antibody was observed to neutralize, or prevent infectivity, of the initial infecting virus, called the transmitted founder (TF). We characterized an antibody antigen binding fragment (Fab) closely related to DH272, called F7‐22, and its interactions with the TF spike protein, called Env, from patient CH848 using negative stain electron microscopy, molecular modeling, and binding kinetics. Data implicate the Fab CDRH3 and CDRL1 loops and the Env V1/V2 loops as important for complex formation. Further work is needed to develop a more complete model of this interaction and a rationale for the mechanism through which DH272 acts as a cooperating lineage.
In Silico Investigation of Gastroprotective Compounds from n‐Butanol Fraction of Costus igneus on Antiulcer Druggable Targets
Authors: Adetayo Modupe O. , Anyasor God'swill N.
Published: May 2022

Gastric ulcer is a common gastrointestinal tract disorder characterized by discontinuation in the stomach mucosal lining and forming a painful lesion. Costus igneus is an ethnobotanical plant used in the management of gastrointestinal disturbances. This study characterized the gastroprotective bioactive compounds from n‐butanol fraction of C. igneus leaf (CIBF) and investigated their druggability effects on selected antiulcer targets using in silico approach.

CIBF compounds were identified and characterized using gas chromatography‐mass spectrometry (GC‐MS) method. The 3D structures of CIBF compounds were downloaded in SDF format from Pubchem National Library of Medicine compound database and used as ligands to dock H+K+ATPase receptor (PDB ID: 5YLV), muscarinic receptor (PDB ID: 5ZHP), urease (PDB ID: 6ZJA) and histamine receptor (using Beta‐1‐adrenergic receptor [PDB ID: 2YCW] as a template). The 3D structures of the proteins were downloaded from the Protein Data Bank. Omeprazole, cimetidine and vonoprazan were used as reference drugs. The ligands and target proteins were processed and prepared for docking in pdb formats by using Chimera 1.14 and OpenBabel GUI version 2.3.2a, respectively. Multi‐targeted molecular docking was done using PyRx – Python Prescription 0.8. Physicochemical and pharmacokinetic properties were analyzed using Osiris DataWarrior version 5.5.0. Bioavailability and drug‐likeness analyses were done using the SwissADME web tool.

GC‐MS analysis detected 15 suspected bioactive compounds in CIBF. Molecular docking study showed that 10‐11, (6ʹ,7ʹ) naphthoquinono [3.2] paracyclophane (NQP) had the highest ligand binding affinity for H+K+ATPase, muscarinic receptor, urease and histamine receptor when compared with the others 14 compounds, omeprazole, cimetidine and vonoprazan. Physicochemical properties of NQP did not violate the Lipinski, verber and lead likeness rules. Pharmacokinetic predictive analysis indicated that NQP possesses good oral bioavailability, high gastrointestinal absorption, blood‐brain barrier permeability and no inhibitory effect on cytochrome P450s.

Findings from this study showed that NQP exhibited substantial inhibitory effects on proton pump, urease activity, muscarinic and histamine receptors. It also showed good physicochemical and pharmacokinetic properties. It is recommended that n‐butanol fraction of C. igneusleaves should be further explored as a potential source of drug lead candidate for the management of gastric ulcer disease.
A Novel Oral Glutamate Carboxypeptidase II Inhibitor for the Treatment of Inflammatory Bowel Disease
Authors: Pradhan Manisha , Peters Diane E. , Norris Lauren D. , Thomas Ajit G. , Rais Rana , Slusher Barbara S.
Published: May 2022

Inflammatory bowel disease (IBD) affects 1.3% of the US population, and approximately 40% of patients with moderate‐to‐severe disease are non‐responsive to existing therapeutics. Glutamate carboxypeptidase II (GCPII), a membrane‐bound zinc metallopeptidase, is highly upregulated in inflamed biopsies from patients with both Crohn’s disease and ulcerative colitis, as well as in preclinical colitis models. Previously, our lab and others have shown that small molecule GCPII inhibitors are efficacious in mouse colitis models when administered systemically or as enemas. To facilitate the clinical translation of this strategy, we have developed (S)‐IBD3540, a gut‐restricted and orally active small molecule GCPII inhibitor. (S)‐IBD3540 is a potent GCPII inhibitor (IC50=4nM) with a desirable preclinical ADME and safety profile. We have characterized its anti‐colitis activity in both acute and chronic mouse colitis models. When evaluated for efficacy in acute dextran sulfate sodium (DSS)‐induced colitis, (S)‐IBD3540 displayed: i) dose dependent inhibition of colon GCPII activity, ii) robust attenuation of colon inflammation, and iii) superior efficacy relative to clinical agents sulfasalazine and tofacitinib. We further confirmed (S)‐IBD3540’s oral efficacy in spontaneous colitis of interleukin 10 knockout (IL10‐/‐) mice. Here, once daily oral treatment with (S)‐IBD3540 in established colitis improved clinical signs and attenuated colon inflammation as measured longitudinally by ELISA for fecal LCN2, a non‐invasive biomarker that correlates with intestinal inflammation. Overall, the GCPII inhibitor (S)‐IBD3540 has an encouraging preclinical profile with robust efficacy in both acute (DSS) and chronic (IL10‐/‐) mouse models of colitis, and represents a promising new strategy for the treatment of IBD.
Understanding the Tissue‐Specific Transcriptional Effects of Mitochondrial Introgression in Drosophila
Authors: Burgess Alexandra , Santiago John , Sanders Jennifer , Rand David
Published: May 2022

Mitochondrial DNA (mtDNA) mutations are an important cause of inherited disease. According to the United Mitochondrial Disease Foundation, “every 30 minutes a child is born who will develop a mitochondrial disease by age 10”. Mitochondrial Replacement Therapy can be used as a form of in‐vitro fertilization in which mitochondria are moved from a third party donor to a recipient oocyte or embryo. This new therapy enables women with mtDNA mutations to have healthy children. However, little is known about the effects of placing mtDNA into a “foreign” nuclear background. This study uses Drosophila as a model system to investigate how an alternative mitochondrial genotype (Sm21;OreR) effects the nuclear and mitochondrial transcriptome in 3 tissues (head, abdomen, and thorax). The effect of sex on RNA expression in these samples is also investigated. Drosophila melanogaster and Drosophila simulans flies were mated and extensively backcrossed to produce a progeny with isogenic D. melanogaster nuclear genome and D. simulans mtDNA (away team). The transcriptional response of tissues was then examined by performing RNA‐Seq on triplicate biological replicates of male and female Sm21;OreR (away team) and control OreR;OreR (home team) flies. Using bash in Oscar systems RNA‐seq reads from these tissues were aligned to a D. melanogaster genomic map. RStudio was used to perform statistical tests (PCA, Volcano plots, Heatmaps) to measure transcriptional variation within the samples. Our preliminary findings indicate that tissue type and sex are the main drivers of transcriptional difference within these samples.
Methadone disrupts early synaptic development in human cortical organoids
Authors: Dwivedi Ila , Zhou Dan , Caldwell Andrew , Subramaniam Shankar , Haddad Gabriel
Published: May 2022

In the last decade, the national opioid epidemic has yielded a 131% increase in pregnant women diagnosed with opioid use disorder (OUD), which is characterized by opioid abuse and dependence. Pharmacological treatments for OUD commonly involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms. The ability to readily enter fetal circulation, accumulate in neural tissue, and cause long‐term neurocognitive sequelae, has led to concerns regarding the drug’s effect on fetal brain development. Since little is known about the impact of methadone on human fetal brain development, we took advantage of the in vitro induced pluripotent stem cell (iPSC)‐derived human cortical organoid (hCO) technology to probe its effects on the earliest developmental stages. Our lab has demonstrated that 1‐week of chronic exposure to a clinically relevant 1μM dose of methadone leads to the suppression of synaptic transmission in 8‐ to 10‐week‐old hCOs. Therefore, we hypothesized that long term chronic exposure to methadone from the earliest stages of cortical differentiation would alter the molecular mechanisms underlying synaptic development. To test this, we conducted bulk mRNA sequencing of 2‐month‐old hCOs derived from two cell lines that had been chronically treated with 1 μM methadone for 50 days. Differential expression analyses revealed a robust transcriptional response to methadone in these hCOs, with over 2000 genes that were significantly differentially expressed independently of baseline transcriptional differences between the cell lines (|FC| ≥ 1.5, p‐adj < 0.05). Gene set enrichment (GSEA) and gene ontology (GO) analyses revealed alterations in pathways associated with both structural and functional synaptic development, including the downregulation of pathways associated with primary cilia and extracellular matrix (ECM). At a molecular level, preliminary co‐expression analysis (CEMiTool) of 1063 genes associated one or more of these three cellular components yielded a primary co‐expression module of 417 genes associated with synaptic, ciliary, and extracellular matrix function that were also predicted to interact at the protein level. During brain development, primary cilia regulate the integrity and composition of the ECM, influencing synapse formation, differentiation, maturation, and refinement. These results indicate that chronic methadone changes gene expression that fundamentally alter synaptic formation and structure, potentially leading to the observed alterations in synaptic transmission.
Characterization of the Structure, Stability, Bioactivity of Recombinant FGF19
Authors: Ternier Gaëtane , Kumar T.K.S
Published: May 2022

Fibroblast growth factor 19 (FGF19) is a member of the endocrine fibroblast growth factor family, secreted in the small intestine. FGF19 binds to the fibroblast growth factor receptor 4 (FGFR4) in the presence of β klotho to downregulate the secretion of bile acid. All endocrine FGFs, including FGF19 are unstable. In this study, we examine structure, stability, and bioactivity using a wide range of biophysical and biochemical experiments. The protein has been successfully overexpressed in Escherichia coli (E. coli), and optimal conditions for storage have been established. Results of thermal denaturation of recombinant FGF19, using circular‐dichroism and differential scanning calorimetry, suggest that the thermal unfolding of the protein is reversible, and the Tm (melting temperature; temperature at which half of the protein is denatured) was found to be 55 oC. Far UV circular‐dichroism data indicate that the backbone of the protein is predominantly in the beta‐sheet conformation. In addition, cell proliferation and cell metabolism data show that FGF19 exhibits low cell proliferation, but significant metabolic activity. These results will be discussed in details.
Phenotypical resistance profile of Stenotrophomonas maltophilia insolated from farm amimals
Authors: Nikitin Georgy , Makavchik Svetlana , Smirnova Lubov , Sukhinin Alexander , Kuzmin Alexander , Krotova Anastasia
Published: May 2022

Stenotrophomonas maltophilia is a multidrug‐resistant opportunistic pathogen. Infections caused by resistant strains of bacteria in animals are characterized by a more severe manifestation, increased duration of treatment and often leads to recurrence. Isolation, identification and determination of pathogens and interpretation of their antibioticograms are the basis of effective and rational antibiotic therapy. The aim of the study was to analyze the antibiotic resistance of Stenotrophomonas maltophilia cultures isolated in cases of respiratory diseases in livestock. The materials for the study were nose scrapings collected from 50 postpartum period cows and 69 newborn calves, and 30 horses up to 3 years old. Primary cultures of tracheal and nasal swabs from horses, cows, and calves were incubated on meat‐peptone broth with 1% glucose and horse serum at 37℃ for 24 hours. After that the culture was inoculated on nutrient media such as columbia ram blood agar, chocolate agar, Endo medium and then incubated at 37℃ for 24 hours. Identification of the isolated cultures and study of their biochemical properties were performed using Nefermtest 24 (Erba Lachema, Czech Republic) test systems. Antibiotics resistance measurement was carried out by the MIC method using MicroScan WalkAway 40 plus (Siemens, Germany) automatic analyzer and GN3F, RUSTEF, NF Sensititre (Trek Diagnostic Systems, Great Britain) plates. Disk diffusion test with Mueller‐Hinton agar was performed according to the recommendations of the European Committee for the determination of antibiotic susceptibility (EUCAST). Pathogenicity of cultures was studied by in vivo testing on white mice. The total number of multidrug resistant S. maltophilia isolates made up less than 5% of 149 clinical samples. Following groups of antibiotics were selected to test the antibiotic sensitivity of this bacterium based on the study’s results: penicillins combined with β‐lactamase inhibitors (co‐amoxiclav, ampicillin/sulbactam, ticarcillin/clavulanic acid), quinolone antibiotics (levofloxacin, moxifloxacin, ciprofloxacin, enrofloxamethacin (trimethoxoprimolamines), sulfanilamylamines (TMP/SMX), nitrofurans (nitrofurantoin), tetracyclines (doxycycline), and macrolides (erythromycin). S. maltophilia cultures isolated from livestock were sensitive only to levofloxacin and moxifloxacin. Interpretation of antibioticograms is one of the main factors that determine the effectiveness of antibiotic therapy.
The influence of using plant‐mineral bolus in early fresh period on the efficiency of the first inseminations of dairy cows
Authors: Plemyashov Kirill , Korochkina Elena , Nikitin Vladimir , Anistenok Sergey , Nikitin Georgy , Achilov Vadim
Published: May 2022

The early fresh period has a big value in the productive life of animals. The level of management on this time in the farm and the animals’ body state have a correlation with milk production and reproductive efficiency. The purpose of the study was to research the influence of vegeto‐mineral bolus using in early fresh period on the efficiency of the first inseminations of dairy cows. The studies were carried out in one of the breeding farms of the Leningrad region. It was Holstein cows aged from 2 to 7 years. The milk production of cows was 9000 kg. It was two groups (n=80 by each). The cows of the experimental group in the early fresh period received plant‐mineral boluses Metrabol (Techna France Nutrition S.A.S., France). The compound of these boluses includes vitamin E, Mg, Zn, Cu, Se and herbal components (calendula, cinnamun, suisque). It was 2 boluses per animal during the first 12 hours after calving. The protocol of early fresh period of cows (control group) was used the injection of antibacterial, myotonic and multivitamin preparations in the first day of calving. The using of plant‐mineral boluses in the early fresh period had a positive influence on the efficiency of the first inseminations. The high result of first inseminations had cows of experimental group. The difference of values between two groups was 12,8% (first lactation); 1,3% (second lactation); 17,5% (third or more lactation). However, the higher percentage of efficiency after second insemination had cows of control group. The difference of values was 19 % (first lactation); 14,5 % (second lactation) and 15,8% (third or more lactation). More successful third and other inseminations were seen in cows of the experimental group. Thus, the percentage of first lactation’ cows was 6,2%, second lactation – 13,3%, the third and more lactations ‐1,8%. It can be concluded that the using of plant‐mineral boluses during the first 12 hours after calving contributes to a faster recovery of the reproductive system of cows in the postpartum period, which is expressed by a higher efficiency of the first inseminations.
Glycosylated Sulfonylurea (2DGs) Modulates Insulin ‐ dependent and Insulin ‐ independent Signaling Pathways via PI3K and P38 MAPK in L6 Skeletal Muscle Cell Line
Authors: Nalweyiso Jacinta , Okechukwu Patrick N. , Sie Ting Ling N. , Hui Tan Y. , Balachandran Abbirami , Siew Ling Crystale L. , Ghadeer Suaifan , Anisa Fromming Gabriele R. , Johnson Stanlas
Published: May 2022

Background

Metformin and sulfonylureas are the most prescribed oral anti‐diabetic drugs globally. However, side effects like lactic acidosis, cardiovascular risks, hypoglycemia and weight gain limit their use in certain comorbidity cases. Metformin utilizes both insulin‐dependent and insulin‐independent sig naling pathways to modulate blood glucose levels in diabetic patients. Sulfonylureas increase insulin secretion from the pancreas and enhance glucose uptake by skeletal muscles through the PI3K signaling pathway. Glycosylated sulfonylurea (2DGs) is a modified analogue of sulfonylurea developed by integrating an aryl sulfonamide with a glucosamine moiety. Previous research on 2DGs has shown in vitro and in vivo antidiabetic, antioxidant, anti‐glycation activity and alleviation from kidney, liver, and cardiovascular diabetic‐related complications.

Objective

The current study focused on the evaluation of insulin signaling pathways of 2DGs in normal and insulin resistant L6 myotubes.

Method

To evaluate PI3K signaling, insulin resistance was induced in L6 myotubes using high glucose (25 mmol/l) and insulin (100 nmol/l) for 24 hours, followed by low glucose (5mmol/l) and no serum for 5 hours and insulin (100 nmol/l) for 30 mins. Treatment was done for 24 hours with tolbutamide and acetylated and deacetylated 2DGs at 5uM, 10uM. For p38 MAPK signaling, one group of L6 myotubes was incubated with SB203580 (p38 MAPK inhibitor) and second group was without SB203580. Both groups were treated with 2DGs similar as mentioned above. The treatment was left for 1 hour, after which DNA and protein extraction for quantitative PCR, ELISA, and Western blot experiment was done.

Results

There was observed high expression of PIK3CA, SLC2A4 and MAPK14 gene in qPCR and upregulated phosphorylation of PI3K, GLUT 4 and p38 MAPK protein in acetylated and deacetylated treated groups compared to tolbutamide and non‐treated group. A positive Pearson correlation was observed between the PIK3CA, SLC2A4 and MAPK14 gene expressions and PI3K, GLUT 4 and p38 MAPK protein expressions respectively.

Conclusion

Finding indicates that 2DGs insulin signaling pathway seems to be through both insulin dependent via PI3K and insulin ‐ independent via p38 MAPK phosphorylation in insulin resistant and normal L6 myotubes respectively.
Possible cytotoxic effect of allantoin on oral squamous cell carcinoma stemness (in vivo and in vitro model
Authors: Binhudayb Nujud A. , Elsherbiny Nehal , Asseri Saad M. , El‐Sherbiny Mohamed , Aloufi Abdulrahman M. , AlTorkai Roba H. A. , Elsherbini Amira M.
Published: May 2022

Cancer stem cells (CSCs) and associated tumor microenvironment (TM) have been proposed to mediate cancer progression, and therapeutic resistance rendering them valuable therapeutic targets in head and neck squamous cell carcinomas (HNSCC). Oral CSCs drive their tumorigenic effect via stromal fibroblasts. Phytochemicals like allantoin are extensively used in wound healing modulating fibroblast activity. The present study aimed to evaluate whether allantoin can modulate CSCs and/or TM and subsequently contribute to sensitization of cancer cells to chemotherapeutic agents. Hep2, OECs, DPSC cell lines were used to test the cytotoxicity of allantoin on cancer, oral epithelial cells, and normal dental pulp stem cells, respectively. The effect of allantoin and/or 5‐fluorouracil (5FU) on cell cycle, apoptosis and expression of stem cell and apoptotic markers was tested in vitro. Further validation was performed using in vivo 4‐nitroquinoline 1‐oxide model of tongue squamous cell carcinoma model with assessment of cytotoxic effect of allantoin by flow cytometry, immunohistochemical staining, western‐blot and RT‐PCR. Allantoin was safe on both normal oral epithelial cells and dental pulp stem cells. Allantoin showed a statistically significant and dose dependent increase in 5FU apoptotic effect via reduction of cancer stem cell markers CD133 and CD44. Regarding cell cycle arrest, there was no statistically significant difference with addition of allantoin to 5FU. In vivo, allantoin down‐regulated SOX2, NANOG, ALDH and OCT4 gene expression in tongue squamous cell carcinoma. Additionally, allantoin reduced the expression IL‐6, TGF‐Β, PDGF, α‐ SMA and CD163. Our results proposed that high concentration of allantoin drives its antitumorigenic effect. This was mediated by modulating stromal component of TM via down‐regulation of trans‐differentiation of cancer associated fibroblasts and tumor associated macrophages. These findings shed the light on potential use of allantoin as cytotoxic drug either alone or combined with other chemotherapeutic drugs.
Renal gluconeogenesis induction by glucagon and insulin receptor deletion in humans
Authors: Tiwari Swasti , Sharma Rajni , Sahoo Biswajit , Srivastava Aneesh
Published: May 2022

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Recently, we provided evidence to suggest that early insulin resistance may impair fast‐fed regulation of Phosphoenolpyruvate Carboxykinase (PEPCK), rate‐limiting enzyme for gluconeogenesis in humans. To confirm this and to elucidate a link between insulin resistance and fast‐fed regulation of PEPCK in the kidneys, we studied primary proximal tubule cells (hPT) and urinary extracellular vesicles (uEVs) from humans. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate‐2 (IRS2) mRNA levels than fed cells. The fast‐fed regulation was, however, attenuated in insulin receptor knockdown hPTs (IRKO). The insulin receptor knockdown was confirmed by a blunted insulin‐induced suppression in PEPCK and PGC1α mRNA and p‐IR and p‐AKT protein expression in IRKO cells. Exosomes secreted by the wild‐type or IRKO hPT showed similar regulation as observed in the respective hPT. Similarly, in human uE, the relative abundance of IRS‐2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast‐fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK mRNA, IRS2 mRNA, and gluconeogenesis. Insulin receptor deletion further increased their levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation. Funding from ICMR‐CARE and SGPGI intramural to ST and CSIR to RS.
Adjuvants for inactivated vaccine against Avibacterium paragallinarum
Authors: Nikitin Georgy , Pankratov Sergey , Sukhinin Alexander , Prikhod'ko Elena , Rozhdestvenskaya Tatyana , Ruzina Anna
Published: May 2022

Avibacterium paragallinarum causes a severe respiratory disease characterized by catarrhal inflammation of the mucous membranes of the nasal cavity, infraorbital sinuses, and conjunctivitis. The study aims to determine the reactogenicity and protective properties of inactivated vaccines against Av. paragallinarum is made using mineral‐salt adjuvant aluminum hydroxide (AH) and high molecular weight acrylic acid‐carbomer polymer. Vaccine samples were made of inactivated Av. paragallinarum cultures of strains B‐7770 (serotype “A”), “1130917 / AmsB” (serotype “B”), and 150215 / TulaC2 (serotype “C”). A milliliter of each vaccine contained 1.0 billion microbial cells of each Av. paragallinarum serotype. The first sample contained AH adjuvant, and 2nd sample had a carbomer in it. Vaccines were tested on 60 days old layer hens. Ten birds were immunized with each vaccine sample to determine reactogenicity. The vaccine was injected intramuscularly between the radius and ulna in a volume of 2.0 cm3. Ten days after immunization, the birds were euthanized and autopsied to measure the local tissue response at the region of vaccine administration. Three groups of chickens (n=10) were formed to measure the immunogenic activity. Birds of the first and second groups were intramuscularly inoculated with one vaccine sample in a volume of 1.0 cm3. The chickens of the 3rd group were not vaccinated. After 28 days of research, all chickens were infected with a mixture of Av. paragallinarum “A,” “B,” and “C” serotypes. The culture mixture was applied intraocularly at 10 50% infectious doses for each serotype. All birds vaccinated with the AH and carbomer vaccines had swelling of soft tissues and petechial hemorrhagic lesions of the muscles near the inoculation area. Fibrin plates (0.2x1.5 cm) were found in the muscles of the inoculation area in 5 AH cases. No clinical manifestation of the disease was found in both vaccinated groups, which indicates good protective properties of the tested vaccine samples. All 10 chickens of the 3rd group showed symptoms of a respiratory disease after infection with Av. paragallinarum, which reveals a high susceptibility of unvaccinated hens to the bacteria. Inactivated vaccine against Av. paragallinarum with carbomer as an adjuvant is less reactogenic than a similar vaccine made using AH. At the same time, both vaccine samples have good protective properties.
Identification and Characterization of Novel Macromolecular Assemblies Responsible for Cell Adhesion
Authors: Dash Sagarika , Samanta Dibyendu
Published: May 2022

Cadherins and nectins are cell surface glycoproteins that function as cell adhesion molecules (CAMs) to drive the adhesion of cells and thereby organize them into higher‐ordered structures during early development. Homophilic associations among the cadherins and homophilic as well as heterophilic associations among the nectins lead to the formation of adherens junction that drives cell‐to‐cell adhesion. However, several studies suggest for the existence of complex cross‐talk among these CAMs, which remains to be explored in greater detail. Based on SPR‐based protein‐protein interaction studies followed by structure‐guided mutagenesis and complementary biophysical analyses, we observed specific heterophilic associations among cadherins and nectin family members mediated through their membrane distal ectodomains. In particular, N‐cadherin was found to interact with nectin‐2, E‐cadherin to interact with nectin‐4, and E‐cadherin and N‐cadherin were also found to interact with each other. In conclusion to these observations, the biophysical and biochemical studies imply for direct heterophilic recognition and crosstalk among nectins and cadherins and the results obtained elaborate how these novel macromolecular assemblies can also be a part of adherens junction to drive physiologically relevant cell‐cell adhesion.
STAT4 Play A Critical Role in Aging and High Fat Diet‐Associated Decline in Long‐Term, Activity‐Dependent Potentiation of Synaptic Transmission
Authors: Stanton Patric K. , Nadler Jerry L. , Zhang Xiao‐lei , Hollander Callie , Ma Haoqin , Yang Xinyuan , D’silva Melinee , Stahr Natalie , Moriarty Alina , Keeter Coles , Galkina Elena V.
Published: May 2022

Aging and Alzheimer’s disease (AD) both exhibit declines in the magnitude of long‐term, activity‐dependent synaptic potentiation in humans and animals that correlate with declines in cognitive function and long‐term memory in a range of behavioral tasks. Signal Transducer and Activator of Transcription 4 (STAT4) is a transcription factor that is essential for mediating T helper 1 cell differentiation and induction of inflammatory responses to IL‐12. STAT4 plays a vital role in signaling in a range of inflammatory diseases, including cardiovascular inflammation in diabetes, systemic lupus erythematosus and rheumatoid arthritis. It is, however, unknown what role STAT4 plays in central nervous system diseases such as AD, which also have significant inflammatory components to the progression of neurodegeneration. The present study examined the effects of elimination of STAT4 signaling in transgenic mice lacking myeloid cell‐specific STAT4 expression on the expression of long‐term potentiation (LTP) of synaptic transmission, and learning and memory, in mice fed normal versus high‐fat diets. Electrophysiological recordings from Schaffer collateral‐CA1 synapses in hippocampal slices from 3 or 24 week old LysMcreStat4fl/flLdlr‐/‐ mice showed no differences in baseline synaptic transmission, paired‐pulse facilitation (PPF, a measure of presynaptic function), or LTP compared to age‐matched Stat4fl/flLdlr‐/‐ control littermates. Feeding mice a high cholesterol, high carbohydrate diet (DDC) diet from 8‐24 weeks of age resulted in marked, significant impairments in LTP compared to age‐matched mice fed a chow diet, without differences in basal transmission or PPF. In contrast to Stat4fl/flLdlr‐/‐ control mice, LysMcreStat4fl/flLdlr‐/‐ mice receiving the same DDC diet were completely protected from DDC‐associated reductions in LTP. Our current behavioral data support the idea that DDC fed Ldlr‐/‐ mice may experience cognitive and memory declines associated with early AD in comparison with LysMcreStat4fl/flLdlr‐/‐ mice. Taken together, our data support an essential role for the transcription factor STAT4 in the sensitivity of the central nervous system to DDC ‐induced impairments in synaptic plasticity and potentially cognitive function, and suggest that STAT4 pathways are a promising new target for interventions to treat neurodegenerative diseases such as Alzheimer’s, where chronic neuroinflammation is a critical disease mediator.