bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of calcium sulfide in intrinsic and extrinsic apoptotic pathways in non‐small lung cell adenocarcinoma
Authors: Rosado Maria Milagros Figueroa , Forti Kevin Muñoz , Torres Bryan , Bernard Faviola , Arroyo Gerardo , Ruiz Abigail , Santiago Pedro , Perez Jaileene , Gonzalez Jonathan , Castro Miguel , Suarez Edu
Published: November 2023

Apoptosis is the process of programed cell death and it is characterized by metabolic changes and energy‐dependent mechanisms. It occurs normally in the development and regeneration of tissues, to insure proper functionality of systems, and it can also function as part of the cellular response against disease or cytotoxicagents. Still, a variety of internal and external stimuli trigger apoptotic cascades, which are categorized in two signaling pathways, extrinsic and intrinsic; they are based on the origin of the stimuli, and the mediators involved. Previous studies in our laboratory, showed that non‐small lung cell adenocarcinoma cell line (ATCC HCC827) treated with proprietary calcium sulfidenanoclusters (CaS) [3.8μM] showed increased apoptotic activity following 48 hours that become statistically significant at 72 hours (p<0.05) without affecting non‐cancerous cell lines (MCR5) after 24‐hours. The objective of this study was to determine the expression of intrinsic (Bcl‐2 and caspase‐3) and extrinsic (caspase‐8 and Bax) apoptotic factors in HCC827 cells upon addition of a single dose of CaS nanoclusters evaluated at 24 hours intervals up to 72‐hours. We hypothesized that CaS nanoclusters will increase the intrinsic and extrinsic apoptotic signaling pathways. DMSO [1%] was used as a negative control and Etoposide[10μM] as a positive control. The flasks were randomly assigned to one of the treatments in triplicates. We used Western Blot, densitometry, and statistical analyses to evaluate changes in expression of the studied proteins. The preliminary data showed that: (1) at 72 hours, the expression of the pro‐apoptotic Bax increased and the anti‐apoptotic Bcl‐2 decreased; (2) at 24 and 48 hours the expression of full caspase‐8 and their respective cleaved variations increased, as well as the pro‐apoptotic caspase‐3 when HCC827 cells were treated with CaS nanoclusters (p<0.05). These results suggest that CaS nanoclusters have apro‐apoptotic effect, involving both intrinsic and extrinsic pathways during the specified timelines. We are currently studying additional pro apoptotic mediators in the intrinsic (caspase‐9, ‐12) and extrinsic (caspase‐7) to elucidate the level of effect of CaS nanoclusters in these pathways.

Support or Funding Information

This work was supported by: UPRPRISE Program NIH Grant #R25GM096955
TRPC1‐STIM1 Activation Modulates Transforming Growth Factor β‐induced Epithelial‐to‐Mesenchymal Transition
Authors: Schaar Anne , Sukumaran Pramod , Sun Yuyang , Dhasarathy Archana , Singh Brij
Published: November 2023

Objective

Tumor metastasis is the principal cause of cancer‐associated deaths; therefore, identifying the mechanism(s) of Epithelial‐to‐Mesenchymal Transition (EMT) mediated metastasis, including the role of the ubiquitous second messenger Ca2+, is important to limit cancer induced death.

Activation of Epithelial‐to‐Mesenchymal Transition (EMT) is important for tumor metastasis to occur. Although growth factors such as TGFβ and EGF have been shown to induce EMT in breast epithelial cells, the mechanism resulting in migration is not well understood. TGFβ induced EMT down regulates the epithelial factors E‐cadherin and increases expression of mesenchymal neural cadherin (N‐cadherin) through a process known as the ‘cadherin switch’. Cleavage of E‐cadherin in both localized and metastatic tumors is believed to be mediated by the calcium (Ca2+)‐dependent calpain family of cysteine proteases, however the ion channel and the source of Ca2+ entry is not yet identified. Changes in intracellular Ca2+ are the result of either the release of Ca2+ stores from the endoplasmic reticulum (ER) that initiates store‐operated Ca2+ entry (SOCE) mechanism, or direct Ca2+ entry from the extracellular space upon membrane depolarization. During initiation of SOCE (when ER Ca2+ stores are depleted) Stromal Interaction Molecule 1 (STIM1) is shown to aggregate at the ER‐PM junctions, where it interacts with proteins such as the Transient Receptor Potential Canonical 1 (TRPC1) channel causing Ca2+ influx. Herein, we provide evidence that Ca2+ entry into the cell, especially upon store‐depletion, plays an important role in TGFβ‐induced EMT by promoting cellular migration and potentially leading to metastasis. The increased migration by TGFβ in non‐cancerous cells was due to the loss of E‐cadherin along with a subsequent increase in N‐cadherin levels. Importantly, TGFβ‐treatment increases store‐mediated Ca2+ entry, which was essential for the activation of calpain cleavage of E‐cadherin. Inhibition of Ca2+ entry by using non‐specific Ca2+ channel blocker SKF‐96365, significantly decreased Ca2+ entry, decreased TGFβ‐induced calpain activation, and suppressed the loss of E‐cadherin along with inhibiting cell migration. Furthermore, TRPC1 function as an endogenous Ca2+ entry channel and silencing of either TRPC1 or STIM1 significantly decreased TGFβ induced Ca2+ entry as well as inhibited TGFβ‐mediated calpain activation and cell migration. Together these results suggest that disrupting Ca2+ influx via TRPC1/STIM1 mechanism reduces calpain activity, which could restore intercellular junction proteins thereby inhibiting EMT induced motility.

Support or Funding Information

Financial support was received from the National Institutes of Health (DE017102) awarded to B.B.S. and 5P20GM104360 awarded to AD.
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 Information

Funding 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 Information

USciences Startup
Evaluation of Antidermatophytic Activity of Indian Traditional Herb Eclipta prostata in a Rodent Model of Tinea Pedis
Authors: Sivasankaran Ramakrishnan
Published: November 2023

Introduction

Eclipta 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 study

To 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 methods

Tinea 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.

Results

Comparing 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).

Conclusion

The 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.
Effects of TCDD on Wnt Signaling in AhR Congenic Mice
Authors: Huot Joshua R. , Landon Chelsea D. , Munger Michael , Brown Kyle , Arthur Susan T. , Huet Yvette M. , Steuerwald Nury , Leamy Larry J.
Published: November 2023

The Aryl Hydrocarbon Receptor (AhR) is essential for cell‐cell communication as well as cell‐matrix communication. The halogenated aromatic hydrocarbon 2,3,7,8–tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental contaminant known to elicit a wide range of toxic effects and developmental perturbations, primarily through the AhR signaling pathway. Wnt signaling plays a pivotal role in the regulation of development, growth, regeneration, activation of adult stem cells, and cadherin‐based adherence. However, little is known if TCDD‐induced AhR signaling has any impact on Wnt signaling. The objective of this project was to test if TCDD‐induced AhR signaling affects Wnt signaling in liver. Congenic mice (Ahrb1/Ahrb1 and Ahrd/Ahrd) were developed through 20 generations of back crossing, in which pregnant females were dosed on gestation day 13 with TCDD (0, 0.01, 0.1, or 1 μg/kg body weight). In 10 week old congenic mice, markers of Wnt signaling (Activated β‐catenin (ABC), GSK3βP‐y216, and Lef1) were measured. Western blot analysis of liver tissue revealed that ABC (P = 0.047) and Lef1 (P = 0.001) were significantly increased in Ahrd/Ahrd mice, whereas GSK3 βP‐y216 (P < 0.0001) was significantly lower in Ahrd/Ahrd mice. Additionally, none of the Wnt markers revealed a difference between sex. Our results indicate that the AhR locus may play an important role on mediating effects of TCDD‐induced AhR signaling on Wnt signaling.
Tannic Acid is a Tissue‐Specific Dual Modulator of Angiotensin Type 1 Receptor and Low‐Density Lipoprotein Receptor in Rat Aortic Vascular Smooth Muscle and Liver Epithelial Cells: An Insight into the “French Paradox”
Authors: Iyer Laxmi , Zhang Yong , Thekkumkara Thomas
Published: November 2023

“French Paradox,” suggests the protective effect of various polyphenols present in red wine against cardiovascular diseases. Although tannic acid (TA), one such hydrolyzable polyphenol, has diverse pharmacological effects, it's cardioprotective mechanism is not well understood. In this study, we have investigated the effects of tannic acid on angiotensin type 1 receptor (AT1R) and low‐density lipoprotein receptor (LDLR), two major players associated with cardiovascular disorders such as hypertension and hyperlipidemia. Under normal culture conditions, exposure of the primary rat aortic vascular smooth muscle cells (RASMCs) and rat liver epithelial (WB) cells to TA inhibited cell‐surface expression of AT1R an effect that is time and dose dependent without any significant change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA was reversible after providing a TA‐free media for 24 hours. The inhibition of AT1R was further correlated with a significant decrease in the AngII‐mediated release in intracellular calcium. The down‐regulation of AT1R protein is associated with an inhibition of AT1R mRNA expression. In these cells, TA phosphorylated p42/44 MAP Kinases. Nevertheless, pretreatment with a MEK‐specific inhibitor PD98059 prevented TA mediated down‐regulation of the AT1R suggesting a role for MAP Kinase. Moreover, AG1478, an EGFR inhibitor blocked TA mediated MAP kinase activation and down‐regulation of AT1R. Under similar conditions, we observed a significant increase in TA mediated LDLR expression in WB cells, which was not observed in RASMCs. Furthermore, proprotein convertase subtilisin/kexin type 9 (PCSK9), a crucial enzyme responsible for LDLR turn over, was decreased upon TA exposure in WB cells whereas increased in RASMCs. The inverse correlation between LDLR and PCSK9 expression suggests that TA induced LDLR expression is at least in part through preventing PCSK9‐mediated degradation of LDLR protein in these cells. Taken together, the present study show for the first time that TA mediated activation of MAP Kinase through EGFR is critical for the regulation of AT1R and LDLR in RASMCs and WB cells, which can simultaneously prevent hypertension and hyperlipidemia through distinctly targeting AT1R and LDLR, therefore, providing the mechanistic insight into the “French Paradox.”
Deep mapping of the podocyte proteome unravels altered protein dynamics during differentiation
Authors: Schroeter Christina , Benzing Thomas , Brinkkötter Paul , Rinschen Markus
Published: November 2023

Podocytes are epithelial postmitotic cells which maintain the renal filtration barrier. Immortalized mouse and human podocyte cell lines‐both in the differentiated and undifferentiated state‐are widely utilized tools to estimate podocyte injury and cytoskeletal rearrangement processes in vitro. Here, we mapped the cultured podocyte proteome of both mouse and human origin at a depth of more than 8800 proteins in the differentiated and undifferentiated state, respectively. We performed comparative analyses of both “deep” proteomic profiles. Cultured podocytes express abundant copy numbers of endogenous receptors, such as tyrosine kinase membrane receptors, the G protein‐coupled receptor (GPCR), NPR3 (ANP receptor), and several poorly characterized GPCRs. Some protein classes associated with podocyte disease, such as slit‐diaphragm associated proteins and ion channels, were hardly detected. Differentiation‐induced changes were comparable in mouse and human podocytes, but slight differences, e.g. in proteins associated with lipid metabolism were detected. Notably, this data set detected general perturbations in proteostatic mechanisms as a dominant alteration during podocyte differentiation irrespectively of the species: Proteasome activity and kinetics were high in the undifferentiated state and lysosomal activity was high in the differentiated state. Proteome‐wide stability measurements in differentiated and undifferentiated podocytes demonstrated preferential degradation mechanisms in each state, which may partially explain the cellular morphology of each state. In conclusion, podocyte differentiation in vitro is largely associated with a proteostatic shift, and the deep proteomic mapping approach utilized here may demonstrate the limitations, but also the potential of podocyte cell culture.
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 Information

Start‐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
DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells
Authors: Ochi Rikuo , Chettimada Sukrutha , Gupte Sachin A
Published: November 2023

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.
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 Information

Marshall 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 Information

CIHR
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 Information

This 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 Information

NIH/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 Information

I 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 Information

Funded 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.

Methods

To 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.

Results

NE‐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.

Conclusion

These results reveal a determinant role of the glycocalyx in the molecular mechanisms that signal to HPE development.
Augmented Muscle Mass as a Novel Buffer against Obesity‐Derived Hypertension
Authors: Butcher Joshua T , Larion Sebastian , Mintz James , Fulton David J , Stepp David W
Published: November 2023

Obesity is well known to significantly compromise cardiovascular and metabolic function and a front line intervention for the management of obesity is exercise, which reduces adverse cardiovascular related outcomes by increasing weight loss and augmenting muscle mass. Myostatin is a potent negative regulator of skeletal muscle shown to be upregulated in human and animal models of obesity, thus making myostatin an intriguing target for therapeutic intervention, particularly in patients who cannot undergo extensive lifestyle modifications due to various comorbidities.

The model used was the db/db mouse, a well characterized mimic of the human condition of obesity, in combination with myostatin deletion. The study used four groups of mice, lean control, lean myostatin KO, obese control, obese myostatin KO.

The current study found that augmented muscle mass, through myostatin deletion, prevents hypertension in the db/db mouse. Blood pressure (measured using radiotelemetry in conscious freely moving animals) between the lean and lean KO mice was similar (108 ± 1.5 vs. 105 ± 2.1 mmHg) but obese mice have significantly elevated blood pressure compared to obese KO (117 ± 1.6 vs. 108 ± 2.1 mmHg, p < 0.05). The obese groups have similar mass, fat profiles (NMR), activity (CLAMS) and heart rate, thus the improved cardiovascular outcomes occur despite full presentation of the classic db/db phenotype, indicating that the positive cardiovascular benefits conferred from augmented muscle mass are independent of weight loss. Further studies in lean mice have shown that myostatin deletion significantly delays the onset of hypertension with Angiotensin 2 infusion (800 ug/kg/min). This study also presents the novel finding that deletion of myostatin is accompanied by significant improvement in renal fluid dynamics, both in the lean and obese groups. The lean KO mice have significantly increased urine sodium concentration compared to lean control (197.9 ± 16.4 vs. 155 ± 13.7 mM, p < 0.05) and the obese KO also is significantly increased compared to the obese control (130 ± 7.9 vs. 62 ± 28.58 mM, p < 0.05), Importantly urine micro‐albuminuria, a clinically relevant measure of kidney function assessed by the albumin to creatinine ratio, remains similar in the lean and lean KO (328 ± 85.9 vs. 126 ± 29.3 ug/mg/day) but is rescued in the obese KO compared to obese control (315 ± 77.6 vs. 1037 ± 219.1 ug/mg/day, p < 0.05). Together, these experimental observations suggest that myostatin inhibition could serve as an effective target for conferring the protective cardiovascular benefits of augmented muscle mass (independent of exercise) and prevent/reverse obesity‐derived changes to metabolic, renal and cardiovascular function.

Support or Funding Information

Stepp and Fulton NHLBI RO1 HL124773
Blood Pressure Responsiveness to Muscle Metaboreflex Activation in Older Adults Following Dietary Nitrate Supplementation
Authors: Schneider Aaron C , Hughes William E , Kruse Nicholas T , Ueda Kenichi , Casey Darren P
Published: November 2023

There is an increased risk for adverse cardiac events in older individuals that exhibit exaggerated blood pressure (BP) responses to exercise. Evidence suggests that the exaggerated BP responses to exercise in older individuals are due in part to greater muscle metaboreflex activation. The vasoactive molecule nitric oxide (NO) contributes to the regulation of BP and may play a role in the metaboreflex. With aging, NO bioavailability is often reduced but can be improved through nitrate (NO3) supplementation via the nitrate‐nitrite‐NO pathway. Therefore, we sought to test the hypothesis that dietary NO3 supplementation would reduce the BP responses to muscle metaboreflex activation in healthy older adults. To test this hypothesis we utilized a randomized, double‐blind, placebo controlled crossover study design in 8 healthy older adults (67 ± 2 years; 5M/3F). Each subject was studied before and after 4 weeks of NO3 supplementation (beetroot powder) and placebo (beetroot powder devoid of NO3) for a total of 4 study days. The muscle metaboreflex test consisted of 2 min of isometric handgrip (IHG) at 30% of maximal voluntary contraction followed by 2 minutes and 15 seconds of post exercise forearm ischemia (PEI) to isolate muscle metaboreflex activation. PEI was achieved by inflation of a blood pressure cuff around the upper arm to 240 mmHg 3 seconds before the end of handgrip exercise. Beat‐to‐beat peripheral BP was measured via finger plethysmography) and heart rate via ECG throughout the muscle metaboreflex test. BP responses were analyzed as the change (Δ) from baseline to the end of the IHG and PEI periods. Both IHG and PEI increased systolic (SBP) and mean arterial pressure (MAP) relative to resting (baseline) measurements (P < 0.05 for both). No significant interaction (P = 0.39) was found for changes in ΔSBP when comparing 4‐weeks of NO3 supplementation (14 ± 4 vs. 11 ± 3 mmHg) vs. 4‐weeks of the placebo condition (12 ± 4 vs. 15 ± 3 mmHg). Likewise, the change in ΔMAP after 4 weeks did not differ between NO3 supplementation (10 ± 2 vs. 8 ± 2 mmHg) and the placebo condition (8 ± 1 vs. 8 ± 2 mmHg) during IHG (interaction, P = 0.29). Conversely, the ΔSBP (19 ± 3 vs. 13 ± 4 mmHg, P = 0.03) and ΔMAP (11 ± 2 vs. 7 ± 2 mmHg, P = 0.03) were reduced during PEI following 4‐weeks of NO3 supplementation, whereas placebo had no effect on ΔSBP (13 ± 2 vs. 15 ± 2, P = 0.31) or ΔMAP (7 ± 1 vs. 9 ± 1, P = 0.22) during PEI. The reduced BP during PEI suggests that dietary nitrate supplementation is capable of attenuating the muscle metaboreflex during exercise in older adults. Since the metabolic component of the exercise pressor reflex is augmented in older adults with hypertension, it may be possible that nitrate supplementation could have an even greater impact in this patient population.

Support or Funding Information

Research supported by Neogenis Inc.
Advancing Age Is Associated with Augmented Venoarteriolar Response to Leg Dependency
Authors: Yoo Jeungki , Parker Rosemary , Sun Dan‐Dan , Urey Tony , Roberts‐Reeves Monique , Lawlery Justin , Romero Steven , Crandall Craig , Sarma Satyam , Levine Benjamin , Vongpatanasin Wanpen , Fu Qi
Published: November 2023

Background

The venoarteriolar response (VAR) is a non‐baroreflex mediated regional vasoconstriction mechanism which has been proposed to contribute to about 45% of the increase in total peripheral resistance during orthostasis. Advancing age is related to elevated total peripheral resistance and it is involved in the pathogenesis of hypertension. However, whether advancing age is associated with an enhanced VAR is not known.

Objective

To test the hypothesis that there are sex and age differences in the VAR among healthy individuals.

Method

18 (10 women) young (mean±SD; 28±4 years) and 22 (11 women) older (72±4 years) healthy normotensive adults participated in the study. Brachial and femoral artery diameter and blood flow velocity were measured using doppler ultrasound. Forearm and calf cutaneous blood flow were measured using laser‐doppler flowmetry. Assessments were performed while the arm and leg were in the horizontal position and repeated following limb dependency (35–40 cm below the heart level) for 5 min. Blood pressure (BP, SunTech) and heart rate (HR, electrocardiogram) were also measured. The percent reduction in vascular conductance (=blood flow/mean BP) during limb dependency was used to assess the VAR.

Results

The VAR was not different between dominant and non‐dominant limbs. The VAR was more pronounced in the leg compared with the arm (24+/−7% vs. 18+/−5%; P=0.04) while it was not different between men and women. Older subjects had a greater VAR in the leg compared with their younger counterparts (29+/−8% vs. 21+/5%; P=0.04), but the VAR in the arm did not differ between the groups (17+/−6 vs. 19+/−4%, young vs. old; P=0.80). Cutaneous VAR showed similar results as those measured by doppler ultrasound. BP and HR remained unchanged prior to and during limb dependency.

Conclusion

Aging is associated with augmented venoarteriolar response in the leg but not in the arm. The enhanced venoarteriolar response induced vasoconstriction may contribute importantly to hypertension in the older population.
The Influence of Acute Hypernatremia on Pressor and Sympathetic Responses during Exercise Pressor Reflex Activation
Authors: Brian Michael S , Matthews Evan L , Wenner Megan M , Rose William R , Stocker Sean D , Farquhar William B
Published: November 2023

Acute hypernatremia has been shown to increase sympathetic nerve activity in animals and humans, however the impact on exercise blood pressure (BP) responses is unknown. The purpose of this study was to test the hypothesis that acute hypernatremia would increase BP and muscle sympathetic nerve activity (MSNA) responses to exercise pressor reflex activation (EPR). EPR activation was achieved during 2 minutes of handgrip (HG) exercise followed by post exercise ischemia (PEI). BP and MSNA responses were measured pre‐ and post‐a 23 min intravenous infusion of 3% hypertonic saline (HSI) to acutely increase serum sodium (SNa+) and plasma osmolality (pOsm). Eighteen participants (age 23±1 yrs; BMI 24±1 kg/m2) completed the protocol; SNa+ and pOsm significantly increased from pre‐ to post‐HSI (138.2±0.3 to 141.3±0.4 mmol/L; 285±1 to 291±1 mOsm/kg H2O; p<0.05 for both). Resting MSNA increased pre‐ to post‐HSI (n=13, 11±2 to 15±2 bursts·min−1; p<0.05), while resting BP remained stable (p=0.98). HG exercise and PEI led to robust increases in MSNA and BP pre‐ and post‐infusion. MSNA responses during HG were greater following HSI (Δ9±2 vs. Δ14±3 bursts·min−1, p<0.05), but not during PEI (Δ8±2 vs. Δ9±3 bursts·min−1, p>0.05). Mean BP responses were similar pre‐ to post‐HSI during HG and PEI (HG: Δ16±2 vs. Δ18±2 mmHg; PEI: Δ13±2 vs. Δ15±3 mmHg, p>0.05). A second set of participants (n=7, age 23±1 yrs; BMI 25±1 kg/m2) completed a time control protocol, consisting of quiet rest instead of an infusion. The time control condition yielded no change in resting SNa+, pOsm, mean BP, or MSNA (all p>0.05); HG exercise and PEI led to robust increases in BP and MSNA (p<0.05) but responses were not different pre‐ to post‐quiet rest. In summary, acute hypernatremia increases resting MSNA and MSNA response during HG exercise.

Support or Funding Information

Supported by R01 HL128388‐01A1