bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spectroscopic Investigation of Novel New Delhi Metallo‐β‐Lactamase‐1 Inhibitors
Authors: Bergstrom Alex , Cheng Zishou , Miller Callie , Chen Allie , Thomas Pei , Bonomo Robert , Fast Walter , Cohen Seth M , Page Richard C , Tierney David L , Crowder Michael W 
Published: November 2023

The prevalence of antimicrobial‐resistant pathogens is an ever‐growing threat to world health, and the pipeline supplying new classes of antibiotics has begun to run dry. The β‐lactams are a class of antibiotics that revolutionized healthcare and have been a mainstay in medicine for decades. However, their efficacy towards pathogenic bacteria is threatened by the transmission and evolution of β‐lactamases, enzymes capable of inactivating this important class of antibiotics. The metallo‐β‐lactamases (MBLs) are a class of β‐lactamases of which there exist no clinically known inhibitors. Yet, they are capable of hydrolyzing almost all β‐lactams. These enzymes rely on either one or two Zn(II) ions within their active site which are necessary for catalysis. New Delhi MBL‐1 (NDM‐1) is of particular concern due to its global spread and rapid transmission between pathogens. Recently, 2,6‐dipicolinic acid (DPA), as well as several derivatives, were identified as potent inhibitors of the MBLs. Here we report the spectroscopic techniques used to elucidate the mode of inhibition, which aided in the discovery process.This investigation aimed to fit the mode of inhibition of NDM‐1 into one of two models: through metal scavenging, or through competitive inhibition. EDTA, a well‐known metal sequestering agent, was selected as a standard for interpreting spectroscopic data of an inhibitor fitting the first model. Alternatively, L‐captopril, a known competitive inhibitor of the MBLs, was selected for the second model. Using 1H NMR, CW‐EPR, and equilibrium dialysis coupled ICP‐AES on either the di‐Zn(II) or di‐Co(II) metalloforms of NDM‐1, it was discovered that DPA best fit a model of non‐selective metal scavenging. However, the data suggest that several of its derivatives form a stable ternary complex between NDM‐1:active site ions:inhibitor in a concentration dependent manner. At lower concentrations, closer to that which would be found in a cell, there is little evidence of metal scavenging effects. However, at high concentrations they better fit a metal‐sequestering model, though with a preference for targeting the Zn2 site before the Zn1 site. These spectroscopic techniques allowed for the minimization of non‐selective metal scavenging effects during the drug discovery process and may be useful for guiding future investigations into other Zn(II) metalloproteins.Support or Funding InformationThis work was supported in part by the National Institutes of Health (GM111926) and by the Robert A. Welch Foundation (F‐1572 to WF).
Spinal Cord Injury and the Gut Microbiome: Mechanisms and Therapies for Bowel Dysfunction
Authors: Jeffrey Elisabeth , O'Connor Greg , Deo Sapna , Dietrich Dalton , Daunert Sylvia 
Published: November 2023

Both experimental and clinical studies have shown that Spinal Cord Injury (SCI) has detrimental effects on remote organs and systems, yet there have been limited studies focusing on these more widespread effects. Despite attention to the pathophysiology of SCI with a goal of protection and repair, there is very little known about these gastrointestinal side‐effects or their effect on the gut microbiome. We sought to characterize long term effects of SCI on the gut by identifying changes in bacterial communication, microbiome composition, and inflammation. Bacterial quorum sensing molecules (QSMs) regulate various communication processes including growth, biofilm formation, and virulence factor production, thus providing information on the state of gut microbiota. Despite a repeatedly observed link between the intestinal microbiome and health, there is very limited data characterizing the gut microbiome in adult SCI patients with bowel dysfunction. We have employed a SCI rat model to investigate changes in the intestinal microbiome and gastrointestinal tract which may potentially contribute to or cause the GI dysfunction experienced by patients with SCI. Specifically, we determined the changes in the intestinal microbiota post‐SCI, most notably there was a significant increase in the relative abundance of Bifidobacteriaceae and Clostridiaceae post‐SCI, and investigated the potential underlying mechanisms via levels of pro‐inflammatory cytokines (IL1‐β, IL‐12, MIP‐2, and TNF‐α) and QSMs. This study continues to provide new information regarding the injury‐dependent changes on the gut microbiome and revealing microbiome‐host interactions that may contribute to the long term effects of SCI on the GI tract function. Identifying mechanisms behind these changes will aid in the development of new therapeutic strategies such as dietary modulation to improve quality of life for people living with SCI.Support or Funding InformationUniversity of Miami Miller School of Medicine, Miami Project to Cure Paralysis, Department of Biochemistry and Molecular Biology
SP‐A2 contributes to miRNA‐mediated sex differences in response to ozone‐induced oxidative stress: pro‐inflammatory, anti‐apoptotic, and anti‐oxidant pathways are involved
Authors: Thorenoor Nithyananda , Noutsios George T , Zhang Xuesheng , Phelps David S. , Umstead Todd M. , Durrani Faryal , Floros Joanna 
Published: November 2023

Human host defense molecules, surfactant protein‐A1 (SP‐A1) and SP‐A2 differentially affect alveolar macrophage (AM) function and their proteome. We hypothesized, that SP‐A genes differentially regulate the AM miRNome. SP‐A knockout (KO), humanized transgenic mice expressing SP‐A1 and SP‐A2 and SP‐A2 gonadectomized mice were exposed to O3 or filtered air (FA) and AM isolated. Of the miRNAs (n=372) studied, 11 miRNAs were differentially expressed under O3‐induced oxidative stress (OxS) in SP‐A2 males. Their gene targets included IL‐6, STAT3, BCL2, NF‐kB, and FOXO1. a) We observed increase in STAT3 mRNA (p < 0.05) in SP‐A2 after 4hr of O3 exposure (4hrO3). To account for half‐life differences, we measured mRNA after 18hrO3. BCL2 increased (p < 0.05) in SP‐A2 AM, whereas, the other genes significantly decreased compared to FA. TUNEL assay revealed significant decrease and increase in apoptotic cells under OxS in SP‐A2, and KO, respectively, indicating a role for SP‐A2 in apoptosis. b) The IKKβ mRNA level after 18hrO3 decreased significantly in SP‐A2, but not in KO; a number of SP‐A2‐downregulated miRNAs correlated with the upregulation of target genes, TNF‐α, IL‐1β, and IL‐6, indicating a role in pro‐inflammatory gene expression. c) Although FOXO1 did not change, SOD2 and CAT decreased and increased significantly in SP‐A2 and KO, respectively, after 18hrO3, indicating that SP‐A2 contributes to ROS homeostasis by decreasing the expression of anti‐oxidant enzymes. d) Sex hormones contribute to differential miRNA expression, as demonstrated by gonadectomy. Thus, SP‐A2 affects miRNA‐mediated sex‐specific differences in response to OxS by involving inflammatory, anti‐apoptotic, and anti‐oxidant pathways.Support or Funding InformationCHILD fund, Dept of Pediatrics, Penn State University, College of Medicine
Tales from the Crypt: Malic Enzyme 1 (ME1) Links Metabolism to Intestinal Cancer
Authors: Fernandes Lorenzo , Al‐Dwairi Ahmed , Marji Meera , Brown Dustin , Simmen Rosalia C.M , Simmen Frank A. 
Published: November 2023

Colorectal cancer (CRC) is the third leading cause of cancer‐associated deaths in the USA. CRC has significant metabolic underpinnings but the connections between metabolic dysregulation and CRC remain elusive. Our laboratory studies the metabolic pathways involving Malic Enzyme 1 (ME1), the cytosolic enzyme that provides NADPH for fatty acid and cholesterol biosynthesis. Up‐regulation of ME1 has been implicated in oncogenesis, however, its role in CRC remains unknown. Here, we established a new CRC mouse model by crossing mice overexpressing ME1 in the intestinal mucosa (ME1‐Tg) with Apcmin/+ mice that bear a mutation in the Adenomatous Polyposis Coli gene (Apc). Both male and female Apcmin/+/ME1‐Tg offspring exhibited an increase (P≤0.05) in the numbers of adenomas in their duodenum, jejunum and ileum compared to Apcmin/+nontransgenic mice. Immunohistochemical analysis revealed increases in the sizes and numbers (P≤0.05) of β‐catenin‐stained adenomas in ilea of Apcmin/+/ME1‐Tg compared to Apcmin/+ mice. BrdU staining showed no significant differences in proliferation between Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. However, TUNEL staining demonstrated a tendency for a decrease (P=0.08) in apoptotic cell death in the Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. Immunostaining for ME1 revealed a greater (P≤0.05) percentage of positive staining in cells of the villi and adenomas of ilea from Apcmin/+/ME1‐Tg vs. Apcmin/+ mice, while crypt cells exhibited a tendency (P=0.06) to have higher percentage of positive ME1 staining. Fatty Acid Synthase (FASN), a cognate lipogenic pathway partner of ME1, was abundantly expressed in ilea of both Apcmin/+/ME1‐Tg and Apcmin/+ mice, but without significant differences between the groups. To determine the mechanism(s) responsible for higher tumor burden, we performed targeted gene expression analysis of intestines from the two groups. Transcription factor Sp5 mRNA was significantly up‐regulated (by 2.5 fold; P≤0.05) in the jejunums of Apcmin/+/ME1‐Tg vs. Apcmin/+ mice. Immunostaining for Sp5 corroborated our gene expression results revealing a greater percentage of Sp5 staining (P≤0.05) in the cells of the villi of ilea from Apcmin/+/ME1‐Tg compared to Apcmin/+ mice. Our findings are consistent with previous reports of increased Sp5 expression in CRC. To determine a direct role of ME1 in CRC proliferation, we performed in‐vitro assays on HCT116 CRC cell lines using siRNA Knockdown. A significant reduction in HCT116 cell proliferation (P≤0.05) was observed with siRNA‐mediated ME1 knockdown relative to scrambled siRNA treatment. Our collective data establish a link between ME1 up‐regulation and higher tumor burden in the duodenum, jejunum and ileum in a mouse model and suggest the Wnt target and stem cell modulator Sp5 as a potential mediator in these events.Support or Funding InformationGrant Support: NIH R01CA136493, NIH UL1TR000039
Reliability and Validity of Accelerometry Methods Used to Assess Facet Joint Crepitus and Cavitation
Authors: Cramer Gregory D , Budavich Matthew , Bora Preetam , Koo Terry , Madigan Dana , Braden Amanda , Ross Kim 
Published: November 2023

ObjectiveNovel accelerometry methods have been developed to assess and localize vibrations from zygapophyseal (Z) joint audible sounds (crepitus and cavitations) during normal lumbar motion and side‐posture spinal manipulation. This study assessed the reliability and validity of the methods.MethodsA lumbar spine was embedded in silicone in the prone position. Before pouring the silicone, tunnels were created from the posterior aspect of the Z joints to the surface. Once the spine phantom was completed, a specialized mechanical device was lowered into each tunnel and was used to strike the Z joints with a force of approximately 7 N to simulate joint crepitus, and 67 N to simulate cavitation (2 experiments). Ten accelerometers applied in a previously developed pattern were used to localize the specific origin of the joint vibrations. For each experiment (crepitus and cavitation), each joint was struck on three different passes (n=30) while recordings from the accelerometers were made. The order of joint strikes was randomized twice for use in two observation sessions. Two observers blinded to the joint strikes, results of one another, and to their previous results, analyzed the oscilloscope recordings of the accelerometers on two separate occasions, separated by a minimum of 2 hours, to identify the joint from which the vibrations originated. Intra‐ and inter‐observer reliability and validity (actual joint struck vs. observer determination from recordings) were calculated.ResultsCrepitus, Observer 1: validity Test (Run) 1 = 0.93; validity Test 2 = 0.96; intra‐observer reliability (Test 1 vs. Test 2) = 0.96; Crepitus, Observer 2: validity Test 1 = 0.89; validity Test 2 = 0.96; intra‐observer reliability = 0.93; Crepitus, interobserver reliability (Observer 1 vs. Observer 2) = 0.94. Cavitation, Observer 1: validity Test 1 = 1.00; validity Test 2 = 1.00; intra‐observer reliability (Test 1 vs. Test 2) = 1.00; Cavitation, Observer 2: validity Test 1 = 1.00; validity Test 2 = 1.00; intra‐observer reliability (Test 1 vs. Test 2) = 1.00; Cavitation, interobserver reliability (Observer 1 vs. Observer 2) = 1.00.ConclusionsThe methods as tested in a highly controlled environment were in almost perfect agreement for both simulated crepitus and simulated cavitation. The methods should be further developed and refined for use in human subjects.Support or Funding InformationNIH/NCCIH Grant # 3R01AT000123‐06S2
P311, an Intrinsically Unstructured Protein, in Adipogenesis
Authors: Badri Kameswara Rao , Muppuru Kesavulu Muni , Samuel Raymond E 
Published: November 2023

Obesity is becoming a global pandemic with about one third of obese adults reported in United States. This debilitating condition decreases the quality of life, which can ultimately influence the lifestyle leading to various disorders including insulin resistance, diabetes mellitus, dyslipidemia and cardiovascular diseases. There are very limited drugs and invasive procedures available to treat obesity. Hence, there is every necessity to explore and understand novel molecular mechanisms of adipogenesis to control this global pandemic. Recent developments identified white adipose tissue as endocrine organ and brown adipose tissue in thermogenesis recognizing potential complex regulation of adipose tissue in health and disease. P311 is a small 8kDa protein that is abundant in the brain, smooth muscle, skeletal muscle and lungs. We recently showed the role of P311 in the regulation of blood pressure. Here we studied the role of P311 in adipogenesis and elucidated the structure of P311. For the current research, we used 3T3‐L1 in‐vitro cell culture model to study the role of P311 in adipogenesis. We treated 3T3‐L1 cells with adipogenic cocktail (1μM dexamethasone, 10 μg/mL insulin and 0.5mM 3‐isobutyl‐1‐ methylxanthine), let them differentiate into adipocytes for different days (0 to 13 days) and determined the levels of P311 and key adipogenic marker, PPARγ2. We observed significant increase in the expression of P311 as fibroblasts differentiated into adipocytes. In addition, P311 expression is positively correlated with the expression of key adipogenic transcriptional regulator, PPARγ2. In dual luciferase promoter reporter assays, P311 activated the PPARγ2 promoter mediated luciferase expression indicating the importance of P311 in PPARγ2 transcription. Our studies identify P311 as an intrinsically disordered protein and a key molecule that mediates adipogenesis.Support or Funding InformationU54MD008621 (HU‐160026) to Hampton Univeristy
Yeast Pah1 Phosphatidate Phosphatase Regulates the Expression of Phosphatidylserine Synthase for Membrane Phospholipid Synthesis
Authors: Han Gil‐Soo , Carman George M. 
Published: November 2023

Pah1 phosphatidate phosphatase (PAP), which catalyzes a committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of PA required for de novo phospholipid synthesis. In this study, we examined the Pah1 PAP‐mediated regulation of phosphatidylserine (PS) synthase, which catalyzes a committed step for the synthesis of major membrane phospholipids. PS synthase activity was highly increased by the lack of Pah1 PAP with a growth‐dependent upregulation from the exponential to the stationary phase. Immunoblot analysis showed that the elevation of PS synthase activity results from an increase in the level of the enzyme encoded by CHO1. By promoter truncation analysis and site‐directed mutagenesis, the induced expression of CHO1 in the pah1Δ mutant was shown to occur through the inositol‐sensitive upstream activation sequence (UASINO, −163 ~ −154), a cis‐acting element for the PA‐controlled Ino2‐Ino4/Opi1 regulatory circuit. The abrogation of CHO1 induction by the UASINO mutation suppressed the pah1Δ phenotypes related to the increase of PA levels (e.g., increased phospholipid synthesis, nuclear/ER membrane expansion, and reduced lipid droplet formation) with its effect stronger in the exponential phase than in the stationary phase. These results demonstrate that Pah1 PAP regulates the expression of PS synthase through its control on the PA level.Support or Funding InformationSupported by NIH grant GM028140
Evidence for Muscle Pump Enhancement of Capillary Filtration as a Redundant Mechanism for Bone Fluid Flow
Authors: Winet Howard 
Published: November 2023

The current popular paradigm for generating bone fluid flow (BFF) sufficient to stimulate osteocytes combines gravity, poroelastic bone bending and glycocalyx receptors to amplify fluid shear stress to values sufficient for stimulation of bone growth[1]. Given the well‐established influence of muscle pumps on circulation we tested the hypothesis that they can modulate trans‐luminal pressures that drive capillary filtration in cortical bone, thereby serving as a redundant mechanism for BFF.Optical bone chambers were implanted in tibias of 21 female NZW rabbits divided into control and stimulated cohorts, and allowed to fill with bone slivers. Weekly intravital microscope observations were video‐recorded and images processed as usual[2]. During the last observation, 2.54cm TENs electrodes were attached directly to the exposed and released gastrocnemius/soleus muscle of the implanted leg. It was stimulated with a 0–4V biphasic waveform at 4Hz. The one hour stimulation was stopped at intervals after injection of fluorescent dyes to record capillary filtration of the dyes. Dye concentrations as a function of fluorescence intensity were calibrated by the method of Wu et al.[3]. Bending strain on the tibia during stimulation was measured and compared to that of the controls to monitor gravity effects. Measurements were evaluated with Students t‐test.Capillary filtration in stimulated rabbits in the absence of bone bending was significantly greater than in controls. There was no significant gravity‐generated bone strain in either group.We conclude that tibial muscle pump‐generated pressure waves in cortical bone significantly enhance capillary filtration independently of poroelastic effects of bone bending by muscle or gravity. Since fluid flow through bone interstitium or in canaliculi was not directly measured here, no assessment could be made of shear stress on osteocytes or osteoblasts.Published poroelastic models are optimization—for engineers—or adaptationist—for natural selection biologists—in nature. As such they do not allow for redundant mechanisms that can assist or substitute if the original is disabled. As Gould and Lewontin have shown[4], adaptation‐limited models ignore three of the four mechanisms of Neo‐Darwinian evolution paradigms. Evolution includes redundancy and pleiotropy. One consequence of this oversimplification is the absence of a BFF mechanism to explain bone healing in a nailed or casted long bone.Support or Funding InformationSupported by UCLA Dept. Bioengineering
Regulatory Mechanisms of Sec1/Munc18 Proteins in Intracellular Vesicle Fusion
Authors: Yu Haijia , Liu Yinghui , Shen Jingshi 
Published: November 2023

Intracellular vesicle fusion is mediated by a conserved core machinery composed of SNAREs (soluble N‐ethylmaleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins. The core domains of the vesicle‐rooted SNARE (v‐SNARE) and the target membrane associated SNAREs (t‐SNAREs) zipper into a four helix coiled‐coil bundle between two apposed bilayers. The SNARE bundle assembles toward the membranes and provides the energy to bring the two membranes into close apposition to fuse. SM proteins are soluble factors that promote membrane fusion through binding to their cognate trans‐SNARE complexes. It is well known that SM proteins interact with multiple domains on their cognate SNAREs and contribute to the compartmental specificity of intracellular vesicle fusion. However, the molecular mechanism of how SM proteins regulate the vesicle fusion has not been fully elucidated. In a reconstituted fusion system, we observed that the SNAREs themselves were inefficient in driving membrane fusion. The fusion rate was strongly increased by either the cognate SM protein or a Vc peptide corresponding to the C‐terminal half of the v‐SNARE. Further analysis showed that SM proteins promote membrane fusion in a similar manner as the Vc peptide, through restructuring the C‐terminal part of the t‐SNAREs.Support or Funding InformationNational Institutes of Health grants GM102217
Peroxynitrite Produced in Isolated Guinea Pig Cardiac Mitochondria: Role of Mitochondrial NOS
Authors: Gerdes Harrison John , Heisner James S , Yang MeiYing , Camara Amadou KS , Stowe David F. 
Published: November 2023

The presence of an endogenous nitric oxide synthase (NOS) isoform in cardiac mitochondria remains controversial. NOS catalyzes production of the gaseous signaling molecule nitric oxide (NO•) from arginine. During cardiac ischemia/reperfusion (IR) injury, NO• can react with superoxide (O2•−), derived from a free electron and O2, to form the strong oxidant peroxynitrite (ONOO−), which initiates deleterious post‐translational modifications of cellular proteins and can cause tissue damage. We hypothesized that isolated mitochondria contain endogenous NOS. Our aim was to assess production of ONOO− in isolated cardiac mitochondria under conditions of oxidative/nitrosative stress, and to determine if ONOO− production is dependent on the presence of endogenous mitochondrial NOS. To investigate first if isolated guinea pig cardiac mitochondria contain NOS, Western blot analyses were performed using whole‐heart tissue and percoll‐purified cardiac mitochondria samples with anti‐eNOS and anti‐nNOS antibodies. The bands showed that the reacted eNOS antibody (140 kDa), but not the nNOS, antibody, was detected in both the whole‐heart and isolated mitochondria samples, where band density was much higher. Additionally, mitochondria were suspended in respiratory buffer (pH 7.15), and treated with succinate, a complex II substrate, and CaCl2 followed by menadione, a redox cycler that generates large amounts of O2•−. In physiological buffer, added L‐tyrosine is oxidized by ONOO− to form the fluorescent dimer dityrosine (diTyr). We found that exposing isolated mitochondria energized with succinate to CaCl2 and menadione resulted in an increase in the diTyr fluorescence signal, indicating that the simulated IR injury conditions enhanced mitochondrial ONOO− production. To verify that mitochondrial ONOO− production was dependent on NOS, mitochondria were incubated with the NOS inhibitors L‐NAME/L‐NNA, PTIO, a potent global NO• scavenger, and TEMPOL, a superoxide dismutase (SOD) mimetic. L‐NAME, L‐NNA and PTIO significantly decreased succinate, Ca2+ and menadione‐mediated ONOO− production. TEMPOL also decreased ONOO− levels by converting O2•− to H2O2, as assessed by the HRP/amplex red assay. Some reports furnish evidence that NOS migrates from the cytosol to locate in mitochondrial outer membrane. These results suggest that mitochondrial NOS is responsible for the production of ONOO− during simulated oxidative/ nitrosative stress.Support or Funding InformationNational Heart, Lung, and Blood Institute Training Grant T35 HL072483