bluuebunny/crossref_metadata_2025 huggingface dataset:
Reserve‐adaptive potential of sperm plasma in stallions of different ages
Authors: Atroshchenko Mikhail M. , Engalycheva Maria G. , Shitikova Anna M.
Published: May 2022

During the aerobic metabolism of sperm, various reactive oxygen species are produced. Sperm cells are extremely vulnerable to oxidative stress (OS), which causes loss of membrane integrity, resulting in increased cell permeability, enzyme inactivation, structural DNA damage and cellular apoptosis. The aim of this study was to assess the reserve‐adaptive potential (RAP) of sperm plasma in stallions of various ages. RAP is the difference between the level of spontaneous and induced oxidative protein modification (OMP), reflecting the degree of OS. Spontaneous OMP reflects the accumulation of OS products in vivo. To determine the level of spontaneous OMP, a sperctrophotometric analysis of 2,4‐dinitrophenylhydrazones (DNPH) formed during the interaction of carbonyl derivatives of sperm plasma proteins with 2,4‐dinitrophenylhydrazine was carried out (spectrophotometer “SF‐2000”, Russia). R.L. Levine, modified by E.E. Dubinina. Measurements of DNPH derivatives were carried out at 14 wavelengths: in the range 260‐280nm for the registration of aldehyde dinitrophenylhydrazones of a neutral nature (ADNPHn), of a basic nature (ADNPHb) – 258‐264 and 428‐520nm, for the determination of ketone‐dinitrophenylhydrazones of a neutral nature (CDNPHn) 363‐370nm, basic character (CDNFGb) – 430‐434 and 524‐535nm. After that, the total amount of carbonyl derivatives was found. Induced OMP was determined in a similar way, but after adding iron (II) sulfate and hydrogen peroxide solutions to the reaction mixture. This provoked the formation of new carbonyl groups. The more RAP, the higher the ability of the biological material to resist the OS.

Two groups of stallions were formed: the first group consisted of 15 stallions aged 13 to 20 years (average age 15.2±1.8 years), the second group consisted of 15 stallions aged 3 to 6 years (average age 4.0±0.5 years). The spontaneous OMP of sperm plasma in aged stallions was 495.6 Eod/ g protein, the induced one – 573.4 Eod / g protein. In the group of young stallions, spontaneous OMP – 347.2 Eod / g protein, induced – 485.3 Eod / g protein. The OMP level of sperm plasma in older stallions is initially higher than in young stallions (p = 0.04), which indicates the accumulation of carbonyl derivatives in vivo as a result of the prevalence of OS over the antioxidant capabilities of the organism. But when additional oxidizing components are added to the reaction mixture, the OMP of aged stallions does not increase as significantly as in young ones. RAP of sperm plasma of old stallions is 13.56%, of young stallions – 28.75%. The ability to withstand OS, the reserve of antioxidant capabilities of sperm plasma in young stallions is 2.1 times higher than in older stallions.
Shut the F(AAH) Up: Inhibiting Fatty Acid Amide Hydrolase as a Novel Approach to Pain
Authors: Strandberg Stacey , Brzozowski K. , Daily M. , Desjarlais C. , Dougherty M. , Hemsworth K. , Mark K. , Marsho J. , Mora Gallegos L. , Otten E. , Schaefer C. , Scheidt A. , Strandberg S. , Toth H. , Vanderhoef K. , Walesa A. , White T.
Published: May 2022

Promising new therapeutic treatments using the fatty acid amide hydrolase (FAAH) inhibitor can be used to relieve symptoms in a patient suffering from a variety of conditions, including but not limited to insomnia, anxiety, fibromyalgia, migraines, arthritis, and other chronic pain conditions. FAAH is responsible for breaking down anandamide, which is an endogenous agonist of the CB1 cannabinoid receptor and analgesic neurotransmitter. The FAAH protein contains a catalytic triad of Ser 241, Ser 217, Lys 142 embedded in its center. The FAAH monomer assumes a twisted 11 strand β‐sheet in the middle of the monomer and 24 α‐helices surrounding the β‐sheets. The Divine Savior Holy Angels MAPS (Modeling A Protein Story) Team modeled the catalytic domain of FAAH using 3D technology. Inhibitors of the FAAH molecules perform better through noncovalent interactions with the triad active site. The inhibitors stop FAAH activity through hydrophobic interactions that result from the active site changing its shape. Noncovalent and reversible inhibitors bring higher selectivity and less unwanted side effects than already studied covalent inhibitors. Non‐covalent FAAH inhibitors are currently in development as analgesics lacking the adverse effects of opioid‐based analgesics. Further studies are being conducted with selective and potent FAAH inhibitors in order to reduce neurological pain suppression, reduction of cancer cells, and increase feeling of motivation. Currently, the United States is in an opioid crisis that caused 100,000 deaths in the last year, which is why new treatments involving FAAH inhibitors prove promising because they reduce the extreme effects opioid‐based drugs present.
Triggering eye movements in mice to understand stable visual perception
Authors: Robles Maximino , Hunt Joshua , Felsen Gidon , Poleg‐Polsky Alon
Published: May 2022

Our project is motivated by the question of how the brain suppresses perception sometimes. Specifically, during motion signals of motion that we voluntarily generate with our eyes. To have a model for answering this question, we first need to reliably elicit these voluntary eye movements. Presenting a drifting grating, the Felsen and Poleg‐Polsky labs did that in mice except the eye movements were involuntary. Presenting a moving center that radiates dots, other scientists did that in primates and the eye movements were voluntary. We want the same stimulus to work on mice because they’re more ethical subjects. Therefore, we hypothesized that that a mice‐adapted stimulus would work on mice. We discovered that the moving center of our stimulus elicited eye movements. Next, we discovered the moving center of the stimulus elicited involuntary eye movements. Next, we discovered the moving center of the stimulus elicited more eye movements when on the left side of the screen. Since this came from expanding dots, a next step should be to try contracting dots. Ultimately, our data doesn't support the hypothesis that our mice‐adapted optic flow stimulus would elicit voluntary eye movements. We could expect difficulty in eliciting voluntary eye movements in mice because they have such large field of vision and spread out photoreceptors that they may not need voluntary eye movements. Our results were relevant because they say something about the potential of mice as models to understand how we make sense of a moving world when we ourselves move in it.
Characterization of CarR‐CarS Two‐Component System Involved in Polymyxin B Resistance of Vibrio vulnificus
Authors: Choi Garam , Sung Dayoung , Ko Duhyun , Kim Tae Young , Byun Hokyung , Choi Sang Ho
Published: May 2022

Enteropathogenic bacteria inevitably encounter cationic antimicrobial peptides (CAMPs) produced by the host immune defense system. The CAMPs bind to negatively charged lipid A molecules of lipopolysaccharide on the bacterial cell surface through electrostatic interaction, leading to increased bacterial membrane permeability and cell lysis. It is known that a fulminating human pathogen Vibrio vulnificus is resistant to the CAMP polymyxin B (Pm). However, the mechanisms by which V. vulnificusdevelops Pm resistance are still elusive. To identify transcription factors conferring Pm resistance on V. vulnificus, a library of knock‐out mutants lacking putative transcription‐factor genes was constructed and screened for the Pm‐sensitive mutants. From the screening, the VVMO6_RS15995 gene (carRVv) encoding a response regulator homologous to Vibrio cholerae CarR (CarRVc) was discovered to be critical for the Pm resistance of V. vulnificus. CarRVv composes a two‐component system with a histidine kinase CarSVv whose gene VVMO6_RS15990 is located next to carRVv. Of note, homologues of CarRVc‐regulated almEFG that enhance V. cholerae Pm resistance through glycine modification of lipid A were not found in V. vulnificus genome. Instead, qRT‐PCR revealed that CarRVv acts as a positive regulator for the expression of VVMO6_RS15980 encoding a putative phosphoethanolamine transferase EptA as well as VVMO6_RS15985 and VVMO6_RS15975 genes that exist as an operon adjacent to carSVv. Biochemical analyses demonstrated that the products of this eptA operon are required for Pm resistance of V. vulnificus presumably via lipid A modification. Electrophoretic mobility shift assay and DNase I protection assay indicated that CarRVv regulates the expression of the eptA operon by directly binding to its upstream region. In addition, mutational analyses showed that each amino acid residue involved in the phosphorelay signal transduction of the CarR‐CarS two‐component system plays an important role in the activation of the eptA operon and the development of Pm resistance. The combined results suggest that the CarR‐CarS two‐component system of V. vulnificus could contribute to the bacterial survival against host‐derived CAMPs by mechanisms different from those of V. cholerae.
Application of Activity‐Based Probe, MV151, in the Mammalian Nervous System Reveals New Insights into Proteasome Changes in Human Alzheimer's Disease Brain
Authors: Turker Fulya , Margolis Seth S.
Published: May 2022

Proteasome complexes play a critical role in human brain health and disease. Despite enormous effort, proteasome composition, activity, and abundance in the human brain remains poorly understood. In this study, we describe a series of tools that can measure the catalytic activity and levels of individual proteasome β‐subunits and distinct proteasome complexes in mammalian tissue. Adapting these tools to brain tissue from human Alzheimer’s disease (AD) patients, we found that the human brain has a high abundance of catalytically active 20S proteasomes and that these 20S proteasomes exhibit higher total activity in AD when compared to unaffected controls. Additionally, we showed that 20S proteasome abundance is inversely correlated with the severity of the AD case. Taken together, our results indicate that while 20S proteasome abundance is decreasing in AD patient brains, this is counter‐balanced by an increase in proteasome activity. We now propose that homeostasis of 20S proteasome activity is a hallmark of human brain health, and changes in abundance and activity are correlated to the severity of AD disease. This discovery sets the stage for further investigation into 20S proteasome activity in neurodegenerative disease states.
Structural basis for environmental sensing in Pseudomonas fluorescens
Authors: Font Maria E. , Nunez Cinthia , O'Toole George , Sondermann Holger
Published: May 2022

Over the past decades, it has become clear that bacteria often prefer a sessile over a free‐swimming lifestyle. The transition from a mobile to an immobile, social lifestyle, called a biofilm, is induced in response to a variety of environmental stimuli, such as carbon or phosphate sources, linked to signaling via the second messenger cyclic‐di‐GMP (c‐di‐GMP). In gammaproteobacteria, c‐di‐GMP regulates cell adhesion to biotic and abiotic surfaces through the transmembrane Lap system that controls the surface presentation of large adhesin proteins. Signaling specificity is achieved through direct physical interaction between a c‐di‐GMP‐producing diguanylate cyclases (DGCs) and LapD, the c‐di‐GMP receptor central to the Lap system. To date, ten DGCs have been identified as regulators of LapD, affecting biofilm formation. However, how they differ functionally and the stimuli to which they respond are often not well understood. To shed light on the environmental sensing mechanism for DGC activation, we have determined the structure of the periplasmic domain of two DGCs in P. fluorescens, a model organism for biofilm research. These domains, belonging to the CACHE family of protein domains, protrude into the periplasm where they encounter their respective ligands, which correlates with their activation. By comparing the structures of CACHE domains and their ligand binding profiles, we identify molecular signatures that contribute to c‐di‐GMP signaling specificity, environmental sensing, and biofilm formation.
Impact of Gut R. torques on Type 2 Diabetes in Selenium‐deficient Mice
Authors: Huang Ying‐Chen , LeGrand Caleb , Cheng Wen‐Hsing
Published: May 2022

Selenium (Se), an essential trace element, is involved in a variety of biological functions through optimized levels of dietary intake. Obesity and type 2 diabetes have been characterized by altered gut microbiota, inflammation, and gut barrier disruption. We have previously demonstrated that suboptimal Se intake at levels <23% of nutritional needs increases susceptibility to type 2 diabetes in middle‐aged mice. Furthermore, results of 16S rRNA amplicon sequencing analyses show a profound impact of dietary Se deficiency on gut microbiota, inducing an increased abundance of Lachnospiraceae in old mice. Lachnospiraceae, a family of anerobic and spore‐forming bacteria in the phylum Firmicutes, can degrade mucins and are associated with inflammation of the gut and insulin resistance. In particular, many studies link the R. torques species of the Lachnospiraceae family to type 2 diabetes. Thus, we hypothesized that dietary Se deficiency reshaped gut microbiota to modulate type 2 diabetes. The objective of this study was to determine whether and how R. torques and dietary Se interacted to influence type 2 diabetes. Weaning female C57BL/6J mice were fed either a Se‐deficient or a Se‐adequate diet for 27 weeks, and half of them were given an oral gavage of R. torques (2 ×108 CFU in 0.2 mL) daily between weeks 22 and 25. Mice were terminated at week 27. Neither dietary Se deficiency nor R. torquesenrichment influenced body weight or food intake during the time course. Dietary Se deficiency induced (P < 0.05) glucose intolerance (13%) and insulin insensitivity (16%) in the female mice aged 7 months. Although R. torquesenrichment did not aggravate or improve these two type 2 diabetes‐like symptoms, it affected the abundance of Lactobacillus, Faecalibacterium prausnitzii, Roseburia spp./E. rectale, and E. coli in the fecal and/or cecal samples. R. torquesenrichment 1) decreased selenoprotein P and glutathione peroxidase 1 in the liver but not skeletal muscle in Se‐adequate mice; 2) increased liver glutathione peroxidase 1 at the protein level in Se‐deficient mice. Altogether, R. torquesenrichment does not restore dietary Se deficiency‐induced glucose intolerance or insulin resistance in mature female mice despite changed protein expression of two key selenoproteins in Se metabolism.
Comparison of Efficacy and Specificity in Cancer‐Targeting Prodrug Conjugates
Authors: Riederer Lillian R. , Hammers Matthew
Published: May 2022

Current chemotherapy methods are non‐specific, killing cancerous and healthy cells alike. This drug‐related cytotoxicity results in many side effects such as hair loss or lower blood cell count. Prodrug conjugates have been developed with the goal of preferentially targeting diseased cells to minimize interactions with healthy cells, thereby mitigating deleterious off‐target effects. One powerful prodrug strategy exploits higher vitamin receptor expression levels in tumor cells. These abundant receptors drive their rapid, nutrient‐demanding growth rates. As such, a vitamin structural motif may be included in the prodrug conjugate design to target the ever‐growing tumor: a “targeting group” that acts as an appealing disguise for the drug. Biotin, folate, cyclic and acyclic RGD peptides have been found to be effective in targeting tumors over non‐cancerous cells. To systematically compare the selectivity and potency of these targeting groups, we intend to construct fluorogenic prodrug conjugates based on a previously established scaffold. The conjugates include one of the four respective targeting groups, the anticancer drug (camptothecin), a naphthalimide fluorophore component to track drug delivery, and an activatable disulfide linker that cleaves upon entering the reducing cellular environment, activating both the drug and the fluorophore. After these conjugates have been synthesized, comparisons of efficacy and specificity among targeting groups will be made to gain a better understanding of targeted cancer prodrug conjugates. Currently, these conjugates are still in the synthesis stages.
Influence of pole‐like kinase 1 on controlling contractility of the urinary tract and gene activated patterns of human bladder smooth muscle cells
Authors: Wang Xiaolong
Published: May 2022

Background

Pole like kinase (PLK)‐mediated promotion of proliferation and smooth muscle contraction in the lower urinary tract has been recently suggested and may offer putative targets and novel compounds for treatments of lower urinary tract symptoms. However, PLK function for contractility of smooth muscle and proliferation of bladder smooth muscle cells are unknown and the specificity of PLK inhibitors has been questioned.

Methods

Here, human bladder smooth muscle cells (hBSMCs) inhibited with five PLK inhibitors (TAK960, MLN0905, Wortmannin, GW843682X, Centrinone‐B) and hBSMCs cultured in smooth muscle medium were compared. hBSMCs characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Proliferation and viability were assessed by CCK‐8 assay. Contractions of human ureterovesical junction smooth muscle from donation after circulatory death renal transplantation were studied in an organ bath, where they were induced by the α1‐adrenoceptor agonists noradrenaline, phenylephrine, and electric field stimulation. Transcriptome sequencing of hBSMCs were obtained using NEBNext®UltraTM RNA Library Prep Kit for Illumina®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms.

Results

Viability and proliferation of hBSMCs were reduced in a concentration‐ and time‐dependent manner. TAK960 inhibits adrenergic contractions of human ureterovesical junction smooth muscle, and proliferation of hBSMCs. No significant differences in other PLK inhibitors combined with control were found. Compared to hBSMCs in control medium, 323 upregulated genes and 160 downregulated genes were significantly altered by TAK960 24hours 500nM, respectively (FDR=0.05, FC=1.5). Genes most prominently and significantly upregulated by PLK inhibiting condition were IFI30, GBP4, VTN, SCG2, OAS2, RRAD, TNFSF10, KRT18, SFN, meanwhile, downregulated was CCL11 (log2FC ≥1.5 or ≤ ‐1.5). The most significantly relative signaling pathway included cytokine‐cytokine receptor interaction, systemic lupus erythematosus and transcriptional misregulation cancer (enrichment factor ≥0.05). Accession to cite for these sequences read archive data is PRJNA714246.

Conclusion

Our findings point to a certain role of PLK‐1 in of proliferation in hBSMCs and contractility in ureterovesical junction smooth muscle, which may be of relevance in lower urinary tract symptom. PLK‐1 inhibitors block these effects and show shared and divergent effects. PLK‐1 regulation of bladder smooth muscle growth and contraction may include genomic and nongenomic mechanisms.
PIP2 and cholesterol interplay in inflammation and atherosclerosis
Authors: Gulshan Kailash , Iacano Amanda J. , Traughber Cynthia A. , Khan Mariam R. , Opoku Emmanuel , Smith Jonathan D. , Nunn Tina , Sangwan Naseer
Published: May 2022

Introduction

Cholesterol accumulation in arterial macrophages leads to foam cell formation, Nlrp3 inflammasome activation, and IL‐1b release. Phosphatidylinositol 4, 5‐bisphosphate (PIP2) plays a role in the cholesterol efflux pathway and IL‐1b release. The PIP2 phosphatase TMEM55b modulates cholesterol efflux but the mechanism of this effect is not clear. Miltefosine altered PIP2 localization and blunted Nlrp3 inflammasome in macrophages, but the mechanism of its anti‐atherosclerotic properties is not clear.

Objectives

To determine the effect of cellular PIP2 levels on ABCA1 mediated cholesterol efflux. To determine the mechanism by which Miltefosine reduces atherosclerosis in mice.

Methods and results

The correlation between cellular PIP2 levels and ABCA1 mediated cholesterol efflux was determined. PIP2 levels were markedly increased in cholesterol‐loaded cells vs. control macrophages (~ 22 % increase, N=3, mean±SD, *P=0.05). Expression of TMEM55b, a PIP2 phosphatase, in BHK cells resulted in ~ 70 % decrease in ABCA1 mediated cholesterol efflux (N=4, mean ± SD, P<0.0001). Miltefosine treated mice showed blunted in‐vivo Nlrp3 inflammasome assembly and had significantly lowered IL‐1b levels in peritoneal lavage (mean +SD; N=5, **p<0.001). Miltefosine‐fed mice showed significantly higher reverse cholesterol transport to plasma, liver, and feces. The hyperlipidemic apoE‐/‐ KO mice fed with Miltefosine (50 mg/kg/day) gained significantly less weight vs. controls and showed significantly decreased atherosclerotic lesions (≥50% smaller vs. controls; mean + SD, p<0.001 in females with N=10, and p<0.04 in males with N=12). Miltefosine altered gut microbiota in hyperlipidemic apoE‐/‐ KO mice.

Conclusion

PIP2 regulates ABCA1 mediated cellular cholesterol efflux. Miltefosine can dampen in‐vivo Nlrp3 inflammasome assembly, increase reverse cholesterol transport, reduce high fat diet‐induced weight gain, reduce atherosclerotic lesion area and alter gut microbiota.
Worms With Armor: Tegumental Adaptations of Stomach Fluke, Dinurussp. (Trematoda: Hemiuridae)—Ultrastructural Characteristics
Authors: Yesudas Rekha , Ryle Wesley
Published: May 2022

The stomach is a hostile environment rarely utilized as a habitat by parasites, but stomach flukes are unique helminths that have adapted to survive in this acidic environment. Unlike the flatworms, stomach flukes have a cylindrical body like blood flukes. We hypothesized that, in addition to suckers, stomach flukes are equipped with structures to prevent them from being ripped loose by peristaltic movements and that they have secretory glands to resist the pH of the niche. In the present study, we collected Dinurus sp. (Trematoda: Hemiuridae) from dolphinfish Coryphaena hippurus to understand the tegumental structure and host–parasite interactions. We used ultrastructural and histochemical techniques to investigate the functions of the tegument. The prosoma (proper body) was equipped with densely packed frill‐like tegumental plications with rough and smooth lobes, while the ecsoma (tail) was without plications. Smooth‐lobed prosomal plications had pores (5 μm) that were secretory in function. Secretory granules were delivered from the underlying type‐2 tegumental cells through cytoplasmic connections. Electron‐dense granules coalesced within these cells and were secreted by holocrine method. They were also periodic acid‐Schiff (PAS) and Alcian blue positive. Similarly, PAS positive mucus cells were observed in the gastric pits of the host stomach. Rough‐lobed plications of the flukes enable them to cling onto the mucin. In Dinurus, a glycocalyx forms the outer limit of the tegument. Below the glycocalyx is a stream of cytoplasm devoid of mitochondria, suggesting the tegument is for protection and not for absorption. Under similar conditions, tegumental pores of the ecsoma secreted serine proteinase, which hydrolases gelatin but is incapable of hydrolysis in the presence of serine protease inhibitors. Although additional studies are needed to confirm these initial observations, our study demonstrated for the first time that tegumental plications of stomach fluke Dinurus sp. have mechanical and secretory, not absorptive, functions. Related helminths, blood flukes (schistosomes) are medically important digeneans that cause human diseases. Further, tegumental enzymes in the native form are too scarce for analysis in blood flukes. Unlike blood flukes, Dinurus are relatively large (few mm). Based on the year‐round availability, larger size, and nature of the tegument, we propose Dinurus sp. as an excellent study model for tegumental investigations, such as serine proteinase enzyme analysis.
Targeting Pantothenate Kinase as an Effective Strategy for Antifungal Drug Development
Authors: Gihaz Shalev , Gareiss Peter , Choi Jae‐Yeon , Renard Isaline , Chattopadhyay Pal Anasuya , Surovsteva Yulia , Chiu Joy E. , Thekkiniath Jose , Plummer Mark , Hungerford William , Montgomery Micaela L. , Hosford Alanah , Adams Emily M. , Lightfoot Jorge D. , Fox David , Ojo Kayode K. , Staker Bart L. , Fuller Kevin , Ben Mamoun Choukri
Published: May 2022

Fungal infections affect over one billion people worldwide each year and are the leading cause of mortality by eukaryotic pathogens with an estimated 150 million severe life‐threatening cases and 1.7 million deaths reported annually. The rapid increase in the number of clinical cases and the emergence of multidrug resistant pathogens such as Candida auris emphasize the urgent need for new strategies to develop more effective, selective and safe antifungals. Coenzyme A (CoA) plays a central role in key cellular processes including the citric acid cycle, lipid metabolism, and ergosterol biosynthesis. The first step in the CoA biosynthesis pathway is catalyzed by a pantothenate kinase (PanK), which phosphorylates pantothenic acid (PA, or vitamin B5) to form phosphopantothenate. In fungi, this enzyme is encoded by a single copy PanK gene, which plays an essential role in cell viability. Here, we report the first crystal structure of the S. cerevisiae PanK enzyme, Cab1, at 1.8Å resolution. Cab1 was crystallized alone and in the presence of three lead high‐affinity inhibitors identified in a high‐throughput chemical screen of a library of 156,593 small molecules. Structure‐guided mutagenesis together with functional complementation assays in yeast identified key residues in Cab1 that are essential for catalysis, substrate binding and dimerization. Structure activity relationship and biological assays identified a lead inhibitor YU385599, with excellent activity against the human pathogens Candida albicans, C. glabrata, and C. parapsilosis. Together, our data validate PanK activity as a novel drug target and identify a novel class of small molecules for the treatment of fungal diseases.
Downregulation of ANO1 activity in PC‐9 cells modulated by a new novel candidate TMEMinh001
Authors: Woo JooHan , Das Raju
Published: May 2022

Relevant to multiple studies, calcium activated chloride channel called Anoctamin1 (ANO1) is identified as an excellent biomarker for tumor growth reported in different cancer cells such as prostate, breast, and lung cancer. Growing evidence suggests, ANO1 promotes tumor growth via regulating EGFR signaling and modulating MAPK and protein kinase B pathway. In non‐small cell lung cancer (NSCLC), the ANO1 expression level is high, leading to high cancer‐related mortality. To date, several natural and synthetic compounds have shown their modulatory effect on ANO1 activity but are out of the limelight due to selectivity. Our present study is carried out based on finding a selective and potent modulator of ANO1 activity by in silico and in vitro assay. According to the hypothesis, inhibitors of ANO1 are screened out, where an inhibitor reduced ANO1 expression without modulating intracellular Ca+ level and cystic fibrosis transmembrane conductance regulator (CFTR) channel activity. The molecular docking study revealed negative high binding free energy than known inhibitor Ani9 and other candidate compounds. Furthermore, our potential compound suppressed cell viability and migration process in ANO1 expressing PC9 cells, while not in H1975 cells not expressing ANO1. Besides, it initiated the apoptosis process by up‐regulating caspase‐3 activity. Thus, the above results provided significant evidence of the anticancer effect of the final compound against ANO1, reducing its protein level in NSCLC.
Physiological Basis of Adaptation of Yakut Horses to Subarctic Climate
Authors: Plemyashov Kirill , Fedorov Valeriy , Koryakina Lena , Maksimov Vladimir , Grigorieva Natalia , Pavlova Alexandra , Sleptsov Evgeniy
Published: May 2022

The Yakut horse is the northernmost horse on the planet and indeed the most resistant to cold. Local horses are well adapted to extreme environmental conditions, wherein in winter, the air temperature reaches record low values (especially at night) and ranges from ‐59.8 to ‐64.4°C. Despite such extreme conditions, Yakut horses are outdoors all year round, thanks to their compact body structure, thick coat, and the thick layer of subcutaneous fat. In this regard, the study of the mechanisms of survival of local horses to extremely cold environmental conditions is of particular scientific and practical interest. The work aimed to study the mechanisms of physiological adaptation of the Yakut horse to extreme environmental conditions during the cold season. The work was carried out at the Department of Physiology of Farm Animals and Ecology of the Arctic State Agrotechnological University. In Yakut horses in the cold season, the total protein content in the blood is significantly higher by 25.19% than in spring (P<0.001). Reliably high rates are noted for protein fractions: α1‐globulin by 29.95% (P<0.05), β‐globulin by 16.69%, and γ1,2‐globulins by 42.29 and 40.2% (P<0.05 0.001), respectively, compared with those in the spring. At the same time, the activity of enzymes and substrates in the blood serum (AsAT, ALT, and Alk‐Phos) of the studied horses is within the physiological norm. In spring, we note a decrease in the level of total protein in the blood against the background of a decrease in globulins by 29.4%, which is most likely due to the late pregnancy period in mares. The content of albumin in the blood serum in the spring decreases by almost 3%, while the albumin‐globulin coefficient increases by 0.09 units, indicating a more intense course of protein metabolism in animals during this period. Thus, in the cold period, the activity of the immune system in Yakut horses is significantly higher than in spring, which provides high adaptive qualities of animals to shallow air temperatures with poor pasture. In the spring, the Yakut horses have an increase in protein metabolism, which coincides with the physiological state of the mares, caused by an increase in the energy and plastic needs of the fetus.
Role of glucocorticoid‐induced leucine zipper during type II macrophage differentiation induced by dexamethasone and IL‐4
Authors: Lee Choong‐Eun , Jeong Hana , Yoon Hyeyoung
Published: May 2022

Macrophages, central role players in the immune‐inflammatory response, exist in diverse phenotypes in specific tissue environments, ranging from pro‐inflammatory (M1) to anti‐inflammatory (M2) types. Various factors in the microenvironment of infected tissues may lead to M1 vs M2 activation, which include LPS, TNF‐alpha and IFN‐gamma for M1 and glucocorticoid (GC), IL‐4 or IL‐13 for M2, respectively. While GC is considered as a strong inducer of M2 mainly through the inhibition of inflammatory cytokine expression, molecular mechanisms directly responsible for M2 phenotype induction by GC are not well understood. As a GC‐induced signaling mediator, we have investigated the role of glucocorticoid‐induced leucine zipper (GILZ) during dexamethasone (Dex)‐ and IL‐4‐induced M2 differentiation. We have observed that in PMA‐treated THP1 cells representing M0 state, GILZ is induced by Dex or IL‐4 under M2 polarization condition. The induction of GILZ by Dex is ROS‐ and p38‐dependent, which in turn mediates Dex‐induced expression of IL‐10 and other molecular markers of M2 such as Arg1. Importantly GILZ knock‐out resulted in a severe suppression of IL‐10 induced by Dex or IL‐4. On the other hand, STAT6 knock‐out did not negatively affect Dex‐induced M2 response, while substantially reduced IL‐4‐induced IL‐10 levels. Finally, GILZ‐ablated macrophages exhibited a complete inhibition of ROS generation required for M2 polarization induced by Dex. Together these data strongly suggest that GILZ plays a critical role as a common mediator of M2 differentiation induced by GC and IL‐4.
The Gut Microbiome is Significantly Altered in APP/PS1 Mice Following Air Particulate Matter Exposure
Authors: O'Piela Devin R. , Mackos Amy R. , Escobar Yael‐Natalie , Allen Mike , Zhang Yan , Wold Loren E.
Published: May 2022

The detrimental effects of air pollution extend beyond its pathological cardiovascular and pulmonary consequences. Recent research has implicated exposure of air pollution, specifically air particulate matter (PM2.5, <2.5 μm diameter), in Alzheimer’s disease (AD) development, though the pathogenesis remains unknown. Gut dysbiosis (i.e., alterations of gut microbiota resident bacterial populations) can result from dietary changes, stress, medications, particulate matter exposure, and alcohol or chemical consumption. Unbalanced bacterial populations may resolve themselves, though persistent stressors can induce chronic dysbiosis, which is implicated in the development of inflammatory bowel disease, obesity, diabetes, cancer, and AD. Thus, we hypothesized that PM2.5 contributes to gut dysbiosis and detrimental microbial byproducts that may ultimately accelerate AD progression in a transgenic AD mouse model. We tested our hypothesis by subjecting 3‐month‐old AD transgenic (APP/PS1) and non‐carrier wildtype (WT) male mice to filtered air (FA) or PM2.5 exposure for 5 days/week, 6 hours/day for 3 months (n=9‐12). The average daily PM exposure is 90.0 μg/m3, which is a 5‐fold concentration above the ambient mean daily PM2.5 concentration (18.8 ±11.4 μg/m3) at the study site in Columbus, OH. The composition has been previously examined and was found to contain high levels of zinc and iron. Following the 3‐month exposure, fecal samples were collected, and DNA was isolated before 16S Illumina sequencing was performed. Sequencing was implemented to determine gut microbial changes associated with genotype and exposure conditions. This data revealed significant alterations in beta diversity based on genotype and exposure, representing altered bacterial communities associated with experimental conditions (Analysis of Molecular Variance (AMOVA), p<0.001)). Additionally, PM2.5 exposure increased gut microbial alpha diversity for both APP/PS1 and WT mice (Shannon‐Weiner diversity, p<0.05). Taken together, these findings indicate that airborne particulate matter exposure induces gut microbiota changes and contributes to microbial dysfunction in a transgenic mouse model of AD.
Profiling the ubiquitinated proteome in human cells
Authors: Diaz Stephanie S. , Liu Xing
Published: May 2022

Ubiquitination is a post‐translational modification that determines the half‐life of many cellular proteins by conjugating a chain of the small protein ubiquitin to a target protein. The ubiquitin chain is then recognized by the 26S proteasome, and subsequently, the ubiquitinated protein is degraded. This ubiquitin‐proteosome system (UPS) contains various components that function cooperatively to determine the ubiquitination status of cellular proteins that play key roles in biological events. It has become clear that mis‐regulation of protein ubiquitination can lead to diverse human diseases, and multiple UPS components have been identified as promising drug targets. To facilitate studies that define roles of the UPS components in protein ubiquitination, a method that probes the ubiquitination status of the whole proteome in human cells is necessary. Tandem ubiquitin binding entities (TUBEs) is a synthetic protein that binds poly‐ubiquitin chains with a dissociation constant in the nanomolar range. Therefore, TUBEs can recognize the poly‐ubiquitinated protein and enrich them from human cell lysate. Combining the TUBE‐dependent affinity precipitation, stable isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry, we are establishing a method for quantitatively accessing the ubiquitinated proteome in human cells. To preserve the poly‐ubiquitinated protein, HEK293 cells were treated with bortezomib, a proteosome inhibitor, to prevent the ubiquitinated proteins from degradation. To perform the affinity precipitation, the recombinant TUBE was conjugated to biotin and immobilized on resin via the Streptavidin‐biotin interaction. The lysate of the bortezomib‐treated cells was incubated with the immobilized BiotinTUBE to enrich poly‐ubiquitinated proteins, which were then digested by the trypsin protease. Following the trypsin digestion, ubiquitinated proteins generated peptides with the Lys‐ɛ‐Gly‐Gly (K‐ɛ‐GG) remnant, which was further immunoprecipitated by antibodies specifically recognizing the K‐ɛ‐GG remnant and identified by mass spectrometry. To probe proteins with different ubiquitination status under different conditions, SILAC was used to grow one cell sample in normal (light) media and the other cell sample in (heavy) media containing stable isotope labeled amino acids. By comparing the abundance of the light versus heavy peptides from the same ubiquitinated protein, changes in the ubiquitination status of each detected protein can be determined. We anticipate that this method will help profile changes in the ubiquitinated proteome when the UPS components are mutated or interfered by drugs.
Early Life Stress Induced Hypothalamic–Pituitary–Adrenal (HPA) Axis Dysfunction Promotes Chronicity of Experimental Colitis in Mice
Authors: Muir Rachel , Klocke Barbara , Foote Jeremy , Jennings Melissa , Molina Patrick , Kellum Cailin , Pollock Jennifer , Maynard Craig
Published: May 2022

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction that ultimately increases susceptibility for adverse health outcomes later in life. The aim of this study was to determine whether ELS in mice affects susceptibility to, and/or severity of colitis induced subsequent to stress exposure. We hypothesized that ELS would contribute to earlier onset disease and exacerbated colitis. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), where newborn mice are separated from their mothers for 4‐8 hours daily from postnatal day (PD) 2 until early weaning on PD 17. Beginning at PD 28, colitis was induced in MSEW mice and their normally‐reared (NR) counterparts by transient blockade of the interleukin‐10 receptor (IL‐10R). Relative to their NR counterparts, MSEW mice exhibited systemic and colon‐specific deficits of the HPA‐axis endpoint molecule corticosterone, prior to induction of colitis. Following disease onset, NR mice were mostly in remission by 15 days after cessation of IL‐10R blockade, whereas MSEW mice displayed sustained histological pathology with an emphasis on epithelial damage, in addition to consistently elevated colonic tumor necrosis factor (Tnf). Furthermore, pharmacologic induction of HPA‐axis dysfunction via early life exposure (PD 1‐14) to the synthetic glucocorticoid, dexamethasone, mirrors the results we observed in the MSEW model of ELS – both prior to and subsequent to colitis induction. Our results indicate that early life exposure to prolonged stress leads to HPA‐axis dysfunction and colonic corticosterone deficits which may subsequently promote persistent colonic inflammation in susceptible hosts.
Two‐way regulation of protein expression for identification and validation of on‐target inhibitors of Mycobacterium tuberculosis
Authors: Grover Shipra , Engelhart Curtis A. , Perez‐Herran Esther , Tiwari Divya , Li Wei , Abrahams Katherine A. , Papavinasasundaram Kadamba , Bean James M. , Sassetti Christopher M. , Mendoza‐Losana Alfonso , Besra Gurdyal S. , Jackson Mary , Aldrich Courtney , Schnappinger Dirk
Published: May 2022

Whole‐cell screens are a powerful tool to identify small molecules that inhibit bacterial growth by targeting pharmacologically relevant mechanisms. Identification of the target for many of these whole cell hits could often be a complicated and time‐consuming process. To supplement Tuberculosis drug discovery effort in this regard, we developed cell‐based assays that utilizes two‐way regulation of protein expression to readily identify on‐target inhibitors of essential genes in Mycobacterium tuberculosis. We successfully used this approach to identify a novel guanidine‐based inhibitor of MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis from a library of 220 compounds. We next extended this approach to categorize inhibitors that either directly or indirectly impact the activity of Biotin Protein Ligase; BirA. This work highlights the value of target‐based phenotypic screens to identify novel antimycobacterial inhibitors. These assays have significant utility in facilitating prompt identification of new lead molecules and target‐driven optimization of analogues of more advanced scaffolds that inhibit MmpL3 and BirA activity in the cellular context.
Identification of Eral1 Recognition Sequences for Unfolding and Degradation by Mitochondrial ClpXP
Authors: Posner Cori , Kardon Julia
Published: May 2022

Eral1 is a GTPase and ribosomal assembly factor of the mitochondrial small subunit (SSU). Eral1 binds 12SrRNA at a critical juncture, and its association and dissociation are necessary for mitoribosome maturation. Previous work in the literature indicated the proteolytic complex mitochondrial ClpXP (mtClpXP) as a regulator of dissociation: mtClpXP physically associates with Eral1 in vivo and knockout of ClpP results in Eral1 stabilization, accumulation of Eral1‐bound small subunit precursors, and reduced levels of mature mitoribosomes. In order for mtClpXP to both permit Eral1 association and enact its dissociation from the assembling small subunit, it must either recognize a specific Eral1 conformation that indicates completion of its assembly factor function or precisely tune Eral1 levels to achieve a metastable state. We aim to describe the recognition strategy that mtClpXP utilizes to unfold and degrade Eral1 and enable ribosome assembly. We have demonstrated that mtClpXP directly unfolds and degrades Eral1 in vitro. Evolutionary coupling analysis between human Eral1 and mtClpX has pointed to select residues that may mediate the interaction between these proteins. We have shown that mutation of at least one such Eral1 residue blocks its unfolding and degradation. We are utilizing a peptide SPOT array to characterize additional Eral1 motifs that assist in recognition and unfolding by mtClpX and hypothesize that the exposure of these sequences aligns with the Eral1 state that is recognized by mtClpX.
Immune‐stimulating properties of Wild Yam (Dioscorea villosa) root extract in triple‐negative breast cancer cells
Authors: Elhag Rashid , Almalki Abdulaziz , Mazzio Elizabeth , Darling‐Reed Selina , Soliman Karam F. A.
Published: May 2022

Testing the roots of Wild yam (Dioscorea villosa extract (WYE) in vitroconsistently induced cell death at low IC50 concentrations in many types of cancers, including glioblastoma, melanoma, colon cancer, neuroblastoma, and breast cancer. However, reports also demonstrate that WYE contains detergent steroidal saponin surfactants, which act on contact to reduce oil‐water interfacial tensions causing disintegration of the bio‐lipid membranes. In the current investigation we separated cell death from saponins from its biological effects that occur at sub‐lethal doses, where lytic cell death is not an interfering variable in MDA‐MB‐231 triple‐negative breast cancer cells. Moreover, we confirmed the inverse relationship between rising saponin content with destruction to plasma and mitochondrial membranes, concurrent to toxicity in 2D and 3D cell cultures. Once appropriate sub‐lethal concentrations were established, we monitored the changes in whole transcriptomic (WT) mRNA miRNA. Also, the intergenic non‐coding RNAs concurrent to the biological cell proliferation studies, spot test of antibody‐protein arrays, and an ELISA were documented. The results indicate that at sub‐lethal concentrations of WYE, the microarray data show 346 differentially expressed genes (DEGs) of 48,226 transcripts tested; 166 upregulated and 180 downregulated. Upregulated DEGS reflected immune‐stimulating effects on TNF signaling, COX2, cytokine release, and elevated cholesterol/steroid biosynthesis. Downregulated DEGs reveal extensive damage to cell cycle processes, including loss of transcripts for cell division cycle (CDC) genes (25A,45,6,7, A2, A7, A7L,8) cyclins (CCN) (A2, B1, B2, E2, F), cyclin‐dependent kinases (CDKs), centromere proteins (CENP), kinesin family members (KIFs), and polo‐like kinases (PLKs) with exception to a corresponding up‐regulated DEG for p21 gene CDKN1A. The transcriptome data matches cytostatic effects observed in a 6‐day proliferation study. It was concluded that WYE mediates a saponin lytic effect by direct contact and this breast cancer cell model has two different biological consequences at lower concentrations and independent of cell death, including pro‐inflammatory and collapse of the cell cycle effects.
Defining the Hierarchy of Heterogeneity of Digits in the Expression of Fingerprint White Line Count: Forensic Perspective
Authors: Taura Magaji , Adamu Lawan
Published: May 2022

The application of fingerprint white line count (fWLC) in the area of determination of identity is not well investigated in Hausa population of Nigeria. The aim of the present study was to define the hierarchy of heterogeneity of digits in expression of fWLC among Hausa population of Kano state, Nigeria. A total of 285 participants (140 males and 145 females) of Hausa ethnic origin with mean age of 21.56 ± 1.84 (18 – 25) years were involved in the study. A live scan was used to capture the fingerprints from the ten digits for determination of secondary fingerprint associated epidermal creases, fWLC. The most heterogeneous digits in fWLC expression were right index and left ring, followed by left thumb. Left index, right little, right middle and right thumb shared common heterogeneity with significant variation with only two digits. For the left little, left middle, and right ring, the digits were homogenous in expression of fWLC except for at most with one digit. In conclusion, the right index digit was defined as the most heterogeneous digit in fWLC expression among Hausa population of Kano state Nigeria. Hence, attention needs to be given to this digit for different purposes including forensic, diagnosis among others.