bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacological activation of Mac‐1 modulates TLR4 signal transduction pathways
Authors: Stoub Darren , Gupta Vineet 
Published: November 2023

Mac‐1, a β2 integrin, has been implicated in leukocyte‐mediated inflammation and may be involved in the etiology of several diseases, such as serum nephritis and systemic lupus erythematosus. We recently reported the discovery of a novel class of small molecule Mac‐1 agonists, which elicit an anti‐inflammatory effect. Here, we report that LA1, one of our novel agonists, mitigates LPS‐induced inflammation by suppressing TLR4‐dependent pro‐inflammatory signalling in leukocytes. Specifically, LA1 treatment modulates phosphorylation of several relevant signal transduction proteins, as seen by western blot analaysis, and impacts cellular localization of FOX03 and NFkB, as seen through immunofluorescence. We suggest that activation of the alpha subunit of Mac‐1, CD11b, suppresses MyD88 activation, which impacts AKT and IKKα/β phosphorylation, thereby suppressing the production of IRF7, IL‐18 and IFN I. Our results suggest that activation of Mac‐1 may represent a novel mechanism for the treatment of several inflammatory diseases.Support or Funding InformationFunding provided by Rush University Medical Center and Dordt College
Molecular Mechanism Underlying Paradoxical Oncogenic Effect of BRAF Inhibitor Treatment
Authors: Wang Zhihong , Candelora Christine , Cope Nicholas , Wong Kenneth 
Published: November 2023

The serine/threonine kinase, BRAF, is a known driver of melanoma progression and is somatically mutated in 40–60% of melanomas. Inhibitors targeting the ATP binding site of the catalytic domain of BRAF provide significant short‐term clinical benefits in the majority of melanoma patients. Surprisingly, those ATP competitive inhibitors paradoxically activate downstream signaling in melanoma patients bearing wild‐type BRAF. The unexpected consequence of inhibitor‐stimulated growth of certain cancers casts doubt on anti‐BRAF therapy. Thus, alternative BRAF‐targeted therapies are urgently needed and are likely to emerge from a better understanding of the extremely complex regulation mechanisms of the BRAF kinase. In order to identify the regulatory factors that lead to drug paradoxical effects, we characterize the purified full‐length RAF family kinases, in particular BRAF and CRAF. Our preliminary data demonstrate that full‐length BRAF recapitulates the paradoxical effect in vitro. Further biochemical studies were conducted to investigate how phosphorylation and protein‐protein interaction impact BRAF regulation and drug sensitivity.Support or Funding InformationUSciences Startup
Evaluation of Antidermatophytic Activity of Indian Traditional Herb Eclipta prostata in a Rodent Model of Tinea Pedis
Authors: Sivasankaran Ramakrishnan 
Published: November 2023

IntroductionEclipta prostata, known as False daisy is used in preparation of Indian traditional herbal medicine (Ayurveda). The preparation – Neelibringadhi Thailam, a herbal oil is applied locally to treat many dermatological conditions, particularly, dermatophytosis. The properties of this preparation has not been scientifically ascertained, but finds its place in the ancient Indian textbook of formulations called Sahasrayogam.Aim of the studyTo investigate the antifungal activity of the methanolic extract of Eclipta prostata against the dermatophyte – Trichophyton rubrum. Using an animal model, we compared the antifungal properties of the herbal product with a standard antifungal drug alone and in combination with the standard antifungal drug.Materials and methodsTinea pedis was induced on the hind paws of Wistar rats by inoculating conidial suspension of Trichophyton rubrum. The rats were divided into four groups‐‐based on the treatment they received. The groups were treated for 7 days with one of the following‐cream base (control), 1% clotrimazole cream (standard), 10% methanolic extract of Eclipta prostata, or a combination of clotrimazole cream and the extract. At the end of 7 days, the outcome was evaluated by clinical scoring and fungal culture of tissue samples.ResultsComparing the fungal burden scores, it was found that both the herbal extract and the combination of herbal extract with the standard antifungal were significantly superior to the control group (p < 0.05) and the standard group with antifungal therapy alone (p < 0.05). Comparison of the clinical scores showed that both the herbal extract and the combination of herbal extract with the standard antifungal were significantly superior to the control group (p < 0.05). However there was no statistical significance when compared to the standard antifungal therapy group. The therapeutic benefit offered by the test extract was superior to that offered by the standard antifungal clotrimazole in the given period of treatment (7 days). The study thus revealed that the combination of the test extract and the standard drug could reduce the fungal burden to a greater extent than the standard antifungal used alone in the given period of treatment (7 days).ConclusionThe current study has revealed that the methanolic extract of Eclipta prostata is a potent antifungal drug against dermatophytosis. Isolation of biologically active molecule from the methanolic extract could serve as a valuable new antifungal drug. This assumes significance in this era of emerging resistance to antimicrobial agents.
NBCe1‐B/C Knockout Mice Exhibit Signs Associated with Proximal Renal Tubular Acidosis
Authors: Salerno Emily E , Patel Sangita P , Marshall Aniko , Alassane Mballo Cheikh S , Parker Mark D 
Published: November 2023

The SLC4A4 gene encodes three major variants of the electrogenic Na+/HCO3− cotransporter NBCe1: NBCe1‐A, ‐B, and ‐C. NBCe1‐A is expressed in the kidney and contributes to blood pH homeostasis. NBCe1‐B and ‐C are expressed in a diverse array of non‐renal cells and contribute to intracellular pH homeostasis and fluid secretion. Individuals with NBCe1 mutations exhibit proximal renal tubular acidosis (pRTA). Disease signs include low blood pH, diverse ocular abnormalities, and poor dentition. Current treatments for pRTA focus solely on correcting blood pH. Thus it is of interest to determine which signs of pRTA are secondary to the acidosis (due to NBCe1‐A loss) and which are a primary consequence of NBCe1‐B/C loss.There are two previously reported mouse models of the disease: [1] Mice lacking NBCe1 (all variants) that exhibit low blood pH, corneal edema, weak tooth enamel, and impacted intestines; these do not survive beyond 25 days and [2] Mice lacking NBCe1‐A (but expressing NBCe1‐B/C) that exhibit low blood pH and elevated intraocular pressure, but otherwise thrive normally. We have generated a line of mice that, by CRISPR/Cas9 disruption of a common exon, lack NBCe1‐B/C while maintaining expression of NBCe1‐A. These mice have a normal blood pH yet exhibit corneal edema, chalky incisors, and failure to thrive. Thus non‐renal forms of NBCe1 contribute directly to the signs of pRTA and these pathologies, unlikely to be addressed by alkali therapy, require supplemental treatment.Support or Funding InformationStart‐up funds from the Dean of the Jacobs School of Medicine and Biomedical Sciences and The Department of Physiology and Biophysics at the University at Buffalo
Regulation of Aerobic Glycolysis by Na/K‐ATPase
Authors: Cui Xiaoyu , Yu Hui , Xie Joe , Pierre Sandrine V. , Xie Zijian 
Published: November 2023

Mammals express Na/K‐ATPase isoforms in a tissue‐specific manner. Besides the canonical ion‐pumping function of the enzyme complex, we and others have demonstrated the importance of α1 Na/K‐ATPase/Src interaction in cellular signal transduction. Interestingly, α2 Na/K‐ATPase lacks Src interaction. Using cell lines expressing wild‐type or mutant forms of rat α1 and α2 in α1‐knockdown pig kidney cells, the aim of this study was to test whether the difference in Src interaction confers an isoform‐specific phenotype to cellular metabolism.Compared to α1‐expressing cells, α2 cells exhibited a 30% increase in aerobic glycolysis measured by Seahorse Extracellular Flux Analyzer. Consistently, direct measurement of lactic acid content in the medium indicated that both short term (4h) and long term (3 days) release were significantly increased in cell cultures. Consistently, ATP generation of α2‐expressing cells was more dependent on glucose. Indeed, α1‐expressing cells exhibited about 25% drop in cellular ATP content after 45 minutes in 50 mM 2‐DG, while a larger drop of over 50% was observed in α2 cells in the same condition. In α2‐expressing cells, failure to activate glycolysis in response to inhibition of oxidative respiration indicated that the glycolytic reserve was drastically reduced compared to α1‐expressing cells, suggesting that they lacked metabolic plasticity and were more prone to metabolic stress. Taken together, these data suggested that Na/K‐ATPase regulates aerobic glycolysis in a α‐isoform‐specific manner in mammalian cells, with α2 resulting in an increase of glycolysis known as a Warburg Effect. In keeping with well‐known characteristics of cells exhibiting a Warburg Effect, α2 cells were highly dependent on glucose for their growth and survival with significant reduction in number upon glucose‐deprivation that was not observed in α1 cells. Consistent with a critical role of Na/K‐ATPase/Src interaction in this isoform‐specific function, cells expressing mutant forms of α1 which were unable to interact with Src, showed increased glycolysis, decreased metabolic plasticity and high reliance on glucose comparable to those of α2 cells. Conversely, cells expressing mutant form of α2 which was able to interact with Src, showed a α1‐like aerobic glycolysis phenotype. Physiologically, it is known that α2 isoform is highly expressed in tissues/cells such as glycolytic skeletal muscle fibers, astrocytes and adipocytes where glucose is utilized as a major fuel for ATP generation or for providing building materials as well as metabolic intermediates. Thus, our new findings could be highly relevant to Na/K‐ATPase α2‐specific cell physiology. We speculate that co‐expression of α1 in α2‐expressing cells could allow a dynamic regulation of bioenergetics that meet the need of muscle, astrocytes and adipocytes.Support or Funding InformationMarshall Institute for Interdisciplinary Research and NIH Grant HL‐109015
Multiprotein complexes regulating excitatory and inhibitory neurotransmission in the brain
Authors: Mahadevan Vivek , Woodin Melanie 
Published: November 2023

It is now firmly established that imbalances between neuronal excitation (E) and inhibition (I) commonly manifest in neurological and neuropsychiatric disorders1,2. However, the precise molecular machinery that underlies E:I balance regulation in neurons is poorly understood. KCC2 is the only neuron‐specific K+Cl− cotransporter that extrudes Cl‐from mature neurons, enabling them to maintain a low intracellular [Cl−], essential for fast inhibitory neurotransmission3,4. Additionally, KCC2 plays important roles in excitatory neurotransmission by regulating dendritic spine morphogenesis 3. KCC2 dysfunction contributes to human neurological disorders including, epilepsy, neuropathic pain and multiple neuropsychiatric disorders5–10. Since KCC2 is at the crux of both synaptic excitation and inhibition and is implicated in neurological disorders, it is imperative to identify the full‐range of KCC2 binding partners to have a firm grasp of its biology.First, using a candidate approach, we made a surprising discovery that native KCC2 exists in a physical and functional complex with native‐ionotropic glutamate receptor (iGluR) subunit GluK211, and its auxiliary subunit Neto212,13 in hippocampal neurons; and that they regulate KCC2‐mediated neuronal Cl− homeostasis. In the present abstract, we reveal that native KCC2 coexists with several members of recycling machinery at the post synapse that have been established to regulate iGluR recycling and surface expression; and also regulate KCC2 cell biology and function. Overall, we identify the existence of members of synaptic inhibition and synaptic excitation within the same macromolecular complex, creating a framework for (i) understanding the fundamental nexus between cell‐intrinsic synaptic excitation and inhibition, and (ii) provide novel therapeutic strategies to rescue KCC2 deficits that occur in E:I imbalance disorders of the nervous system.Support or Funding InformationCIHR
Biotin/pantothenic acid over‐supplementation to SLC5A6 knockout (SMVT‐cKO) mice leads to normalization of the growth rate, intestinal mucosal morphology, and tight junction integrity of the knockout animals
Authors: Sabui Subrata , Lambrecht Nils W , Kapadia Rubina , Ghosal Abhisek , Said Hamid 
Published: November 2023

We have recently shown that intestinal‐specific(conditional) knockout of mouse SLC5A6gene (which encodes sodium‐dependent multivitamin transporter, SMVT) leads to severe inhibition in intestinal biotin absorption, biotin deficiency and growth retardation. It also leads to the development of spontaneous chronic inflammation (including significant neutrophil infiltration, induction in mucosal level of pro‐inflammatory cytokines like IFN‐γ and TNF‐α), abnormal intestinal morphology, and disruption in tight junction integrity, especially in the cecum (Am J Physiol Gastrointest Liver Physiol.,2013, 304: G64‐71; Am J Physiol Gastrointest Liver Physiol., 2016, 311: G561‐70). In this study, we sought to investigate whether over‐supplementation of the SMVT‐cKO mice with biotin (and pantothenic acid, the other vitamin that is transported by SMVT) could prevent the observed growth and intestinal abnormalities. For this we provided high level of biotin and pantothenic acid (1 mM each in drinking water) to dams during pregnancy and lactation, and to their SMVT‐cKO offspring throughout their life span; same sex wild‐type littermates similarly treated with biotin/pantothenic acid were used as controls. The results showed that biotin/pantothenic acid supplementation of the SMVT‐cKO mice to lead to a complete reversal of all observed abnormal phenotypes including growth retardation, decreased bone density/length, and biotin deficient status; it also prevented early death of the animals. In addition, no histological abnormalities were found in the intestine(cecum) of the supplemented SMVT‐cKO mice. Furthermore, no intestinal inflammation was observed (i. e., no neutrophil infiltration and no changes in level of expression of pro‐inflammatory cytokines, like IFN‐γ and TNF‐α, in the gut mucosa) compared to wild‐type control littermates. Moreover, the supplemented SMVT‐cKO mice showed similar level of expression of tight junction proteins (claudin‐1, claudin‐2 and ZO‐1) and myos in light chain kinase as those of wild‐type control littermates. These findings show that biotin/pantothenic acid play an important role in the maintenance of normal intestinal mucosal health and immunity as well as in tight junction integrity.Support or Funding InformationThis study supported by grants from the DVA and the NIH (DK58057 and DK56061)
Dietary Na+‐depletion stimulates divalent metal transporter 1 (DMT‐1) expression in rat proximal colon
Authors: Minor Emily 
Published: November 2023

Iron deficiency anemia is the most common nutrient deficiency worldwide. The majority of non‐heme iron absorption occurs in the duodenum via divalent metal transporter‐1 (DMT‐1) on the apical membrane and ferroportin (FPN) on the basolateral membrane. People with digestive diseases, such as celiac disease, have impaired iron uptake due to decreased absorptive area in the duodenum. Novel therapies to increase iron absorption are needed because current therapies are ineffective or have serious potential side effects. Sprague dawley rats fed a Na‐depleted diet for 6 days have increased levels of aldosterone, which has been show to upreguate various iron transporters, including DMT‐1 and FPN. RT‐qPCR demonstrates a 4‐fold increase in mRNA abundance of DMT‐1 and FPN in the duodenum, proximal, and distal colon of rats fed a Na‐depleted diet. There was also an increase in protein abundance of DMT‐1 and FPN in rats exposed to the same experimental diet. Aldosterone supplementation and/or dietary Na restriction is thus a potential novel therapy to enable colonic absorption of iron in people with digestive diseases that hinder iron uptake in the duodenum.Support or Funding InformationNIH/NIDDK DK‐018777
To kill a bacterium, you need to think like a bacterium
Authors: Brown Eric 
Published: November 2023

When bacteria are grown in media containing only carbon, nitrogen and essential salts, they shift in their metabolic activities to include the synthesis of essential amino acids, vitamins and other cofactors. Only 303 genes, for example, are essential for growth of E. coli on rich media and some 119 additional genes are required for growth on nutrient‐limited media. Hence compounds that target bacteria under nutrient‐limited conditions could serve as leads for novel antibacterial drugs. In fact, nutrient‐limited media probably provide a better proxy for the host environment. There have been many reports of impaired growth and attenuated virulence in pathogens due to mutations in vitamin, nucleobase and amino acid biosynthetic genes. Nevertheless, systematic searches for antibacterial chemicals have overwhelmingly emphasized rich media conditions. Thus, there is a considerable gap in antibacterial chemical space surveyed to date.In the Brown laboratory, we are trying to understand the potential of nutrient biosynthesis as a new and tractable target in drug‐resistant Gram‐negative pathogens. To this end, we have been screening libraries of structurally diverse synthetic compounds and natural products to find inhibitors of bacterial growth in minimal media. Compounds and extracts active in primary screens are subject to the addition of an array of key metabolites and pools thereof to identify suppressors of growth inhibition and provide hypotheses for physiological, genetic and biochemical experiments to elaborate mechanism of action. More recently, we are developing chemical and genomic platforms to understand the interaction of the nutrient biosynthesis apparatus with all aspects of bacterial physiology using systems approaches. The ultimate goal of these studies is to contribute fresh directions for new antibacterial therapies.Support or Funding InformationI acknowledge operating funding from the Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada as well as a salary support from the Canada Research Chairs Program.
Differential Vascular Contributors to Racial Differences in Aortic Pressure among Children
Authors: Lefferts Wesley K , Augustine Jacqueline A , Atallah‐Yunes Nader , Gump Brooks B , Heffernan Kevin S 
Published: November 2023

African‐Americans (AA) develop elevated blood pressure (BP) earlier in life than white individuals. Traditionally measured brachial BP does not accurately describe the load placed on the central vasculature (i.e. aorta) and heart, thus noninvasively estimated aortic BP may hold greater prognostic value. Racial differences in aortic BP have been documented in young adults and may develop as early as childhood. The aortic BP waveform is comprised of a forward (Pf) and reflected pressure wave (Pb) that are dependent on the geometry and stiffness of the aorta. The purpose of this study was to examine vascular contributors to aortic systolic BP (SBP) in AA and white children. BP and measures of aortic structure/function were assessed in 78 AA (n=38 female, 10.4 ± 0.9 yr, height 146±9 cm, 46.5±15.1 kg) and 68 white children (n=33 female, 10.7±0.8 yr, 145±8 cm, 41.8±11.7 kg) in this cross‐sectional study. Aortic geometry was assessed as aortic root diameter, measured during diastole via echocardiography. Aortic stiffness (carotid‐femoral pulse wave velocity [PWV]) was measured using applanation tonometry. A generalized transfer function was used to estimate aortic pressures based on radial waveforms collected via tonometry. Aortic BP waveforms were separated into Pf and Pb using wave separation analysis and the flow triangulation technique. AA children were significantly younger with greater body mass, and had higher aortic SP (AA 99±9 vs white 95±6 mmHg) and PWV (AA 4.7±0.7 vs white 4.4±0.8 m/s) than white children (p<0.05). Aortic root diameter, Pf, and Pb were not different between AA and white children. After entering age, height, weight, and aortic root diameter, the significant predictors of aortic SP among AA children were PWV, Pf, and Pb (R2=0.59, F(2,70)=14.36, p<0.01). An identical model run for white children revealed that only Pb significantly contributed to aortic SP in the final model (R2=0.35, F(2,60)=4.54, p<0.01). Aortic root diameter was not a significant predictor of aortic SP in either group (p>0.05). Our data suggest that 1) racial differences in aortic BP and PWV are present in children; and 2) there are racial differences in the primary vascular contributors to aortic SP such that forward (Pf) and reflected wave pressure (Pb), and aortic stiffness (PWV) contribute to aortic SP in AA children, while only Pb contributes to aortic SP in white children.Support or Funding InformationFunded by NIH NIEHS R01 ES023252 02 (BBG)
Mechanotransduction in hypertensive pulmonary edema
Authors: Chignalia Andreia Zago , Isbatan Ayman , Patel Milan , Minshall Richard , Dull Randal 
Published: November 2023

Hypertensive pulmonary edema (HPE) occurs acutely, can be life threatening and is typically associated with normal left ventricular performance. Factors other than simple Starling forces may contribute to the molecular mechanisms by which increased blood pressure leads to acute endothelial barrier failure and lung edema. Herein we investigated the contribution of the endothelial glycocalyx and mechanotransduction pathway(s) to HPE.MethodsTo mimic an adrenergically‐mediated hypertensive crisis, we administered norepinephrine (NE) to rats and assessed indices of edema development. Rats were anesthetized with isoflurane and mechanically‐ventilated; animals received an infusion of NE to target a MAP = 150mmHg. Lung wet/dry ratio, DPaO2, plasma hematocrit, plasma volume, lung and BAL MPO activity and lung histological injury were assessed. Albumin transport was assessed in vitro. Inhibition of nitric oxide synthase (NOS) by L‐NAME was used to test the contribution of endothelial mechanotransduction to edema development. Wet/dry ratio was determined in Glypican‐1−/− and Syndecan‐1 −/− isolated perfused lungs exposed to low and high pressure to evidence the role of the glycocalyx in pressure‐induced edema.ResultsNE‐treated rats had a decrease in PaO2 and in plasma volume, an increase in lung wet/dry ratio, plasma hematocrit levels and in lung injury histological score; MPO activity was not altered. L‐NAME significantly attenuated NE‐induced pulmonary edema. Glypican‐1−/− and Syndecan‐1−/− mice were protected from pressure‐induced pulmonary edema. In vitro, albumin uptake and transport was increased by pressure but not by NE; effect inhibited by L‐NAME.ConclusionThese results reveal a determinant role of the glycocalyx in the molecular mechanisms that signal to HPE development.