bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti‐oxidants reverse DNA damage caused by fluconazole in the pathogenic fungus Cryptococcus neoformans
Authors: Hill Amari , Buccilli Roberta , Bodner Nicole , Chandrasekaran Srikripa
Published: May 2022

Understanding development of resistance to anti‐fungal drugs in the pathogenic fungus, Cryptococcus neoformans, is crucial for improving anticryptococcal therapy. We have previously determined that presence of anti‐oxidants in growth medium can reverse the fungal inhibition caused by the azole drug, fluconazole. However, the host/pathogen response to co‐treatment with fluconazole and anti‐oxidants remains poorly characterized. In our study, we have used growth assays, infection assays using a heterologous host model, Minimum Inhibitory Concentration (MIC) assays, and flow cytometry to determine how anti‐oxidants influence susceptibility and resistance of C. neoformans to fluconazole. The larval host, Galleria mellonella, was susceptible to C. neoformans and showed higher survival rate when co‐treated with fluconazole and the pathogen. Co‐treatment with select anti‐oxidants, including Pyrrolidine Dithio Carbamate (PDTC), reversed the therapeutic effect of fluconazole. Fluconazole alone causes an increase in DNA content in C. neoformans, detected as additional DNA peaks using flow cytometry, while co‐treatment with fluconazole and select anti‐oxidants reversed the presence of extra stained DNA. Finally, preliminary evidence suggested that C. neoformans lacking the cell cycle regulator, a homologue of Wee1, exhibit higher resistance to fluconazole. Defects in cell division partly account for inhibition of growth of C. neoformans by fluconazole and the ability of anti‐oxidants to reverse an increase in DNA content caused by fluconazole. Our studies suggest that the cell cycle regulator, Wee1 may be important for C. neoformans susceptibility to fluconazole.
Isc10 is a Dual Mechanism Inhibitor of CMGC Kinases
Authors: Rimal Abhimannyu , Swayne Thomas M. , Kamdar Zeal P. , Tewey Madison A. , Winter Edward
Published: May 2022

Autophosphorylation of the activation loop is a common step that converts many protein kinases from an inactive to an active state. Despite the physiological importance of these reactions, the mechanisms that control activation loop autophosphorylation are poorly understood. Smk1 is a meiosis‐specific MAPK in yeast that autophosphorylates its activation loop on tyrosine (Y), thereby upregulating its own catalytic activity. Previously, we identified Isc10 as an inhibitor of Smk1. Here, we show that Isc10 inhibits Smk1 by two mechanisms. First, the C‐terminal segment of Isc10 binds to Smk1 and specifically inhibits the cis‐autophosphorylation of Y209 in its activation‐loop. Second, prior to Smk1 autophosphorylation, the low activity state Smk1 that is bound to Isc10 phosphorylates it on serine 97 (S97). The phosphorylated S97, in turn, becomes trapped in the Smk1 active site. This prevents phosphorylation of downstream targets before activating signals promote Isc10 degradation and the release of active Smk1. Further, Isc10 inhibits autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. Taken together, these findings define a novel class of developmentally‐regulated molecules that inhibit CMGC‐group kinases.
Structural Investigation of AlsA, a Radical S‐adenosylmethionine Enzyme Involved in the Biosynthesis of the Oxetane‐containing Herbicide Albucidin
Authors: Avalos Dante , Fan Po‐Hsun , Geng Yujie , Liu Hung‐wen , Drennan Catherine L.
Published: May 2022

Herbicides are a class of molecules commonly employed to maintain high crop yields by targeting weeds, a particularly damaging class of pests in agriculture. Albucidin is a novel herbicide found in Streptomyces albus subsp. Chlorinus NRRL B‐24108 that shows a suspected new mode of action. Albucidin is a nucleoside analogue containing an oxetane moiety similar to the antiviral compound oxetanocin‐A (OXT‐A). OXT‐A is the only other known naturally occurring oxetane‐containing nucleoside analogue and has a biosynthetic pathway with two key enzymes: OxsA and OxsB. OsxB is a B12‐dependent radical S‐adenosylmethionine (RS) enzyme and catalyzes formation of the oxetane ring via an oxidative ring contraction. A similar reaction has been proposed to be catalyzed by the B12‐dependent RS enzyme AlsB in the biosynthetic pathway of albucidin; however, the two biosynthetic pathways appear to diverge at this point as well. While OXT‐A is formed upon subsequent reduction and OxsA catalyzed dephosphorylation of the OxsB reaction product, the chemistry leading to albucidin is much less clear. In particular, one carbon must be eliminated, and AlsA, which is a biotin synthase‐like RS enzyme also encoded in the albucidin biosynthetic cluster, has been proposed to participate in this reaction. AlsA is most closely related to BioB‐like enzymes and demonstrates high sequence similarity with the canonical TIM barrel domain; however, AlsA also contains an N‐terminal domain of about 100 amino acids that shares minimal sequence homology to other RS enzymes with known structures. Herein, recent developments in the structural analysis of AlsA using X‐ray crystallography are reported.
Efficacy of post‐injury anti‐miR‐181a and anti‐miR‐200c intravenous treatment in protection against experimental stroke in aged mice
Authors: Griffiths Brian , Xu Lijun , Stary Creed
Published: May 2022

Introduction

Stroke remains the second leading cause of death worldwide and the primary cause of long‐term disability in the US. To date, the only pharmacological treatment for stroke remains tissue plasminogen activator. A major factor for translational failure is likely overwhelming use of young adult rodent models despite the fact that stroke occurs predominantly in aged populations. MicroRNAs (miRs) are non‐coding RNAs that regulate protein translation, and we have previously demonstrated in young adult mice neuroprotection against experimental stroke with anti‐miR‐181a and anti‐miR‐200c. In the present study, to extend translational impact, we tested efficacy of post‐injury intravenous anti‐miR‐181a and anti‐miR‐200c in clinically relevant aged mice.

Methods

All studies were conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, with protocols approved by the Stanford Animal Care and Use Committee. Animal treatment groups were randomized by coin flip, and all analyses were performed by an observer blinded to conditions. Transient focal cerebral ischemia was induced by suture occlusion of the middle cerebral artery (MCAO) for 1h in 20 month‐old male C57BL/6 mice (n=8 per cohort and treatment group). Sham surgery consisted of anesthesia and internal carotid exposure without suture occlusion (n=4 per group). Intravenous infusion: animals were re‐anesthetized and 30 pmol/g anti‐miR‐181a, anti‐miR‐200c or control (MM) in sterile saline (100 μl) was infused over 1 minute into the internal jugular vein at 2h reperfusion. Neurological status was assessed by 4‐point neurologic deficit score at 48h after MCAO. Infarct volume was assessed after sacrifice by anesthesia overdose at 48h after MCAO with triphenyl tetrazolium chloride staining. Infarct volume was calculated as percentage of hemisphere corrected for edema.

Results

MCAO in all groups resulted in significant decreases in neurological score and increased infarct volume relative to sham treated animals. Both anti‐miR‐181a and anti‐miR‐200c treatment resulted in significantly (p<0.05) higher neurological scores and significantly lower infarct volumes relative to MM‐control treated animals. No differences in neurological score or in infarct volume were observed between anti‐miR‐181a and anti‐miR‐200c treatments.

Conclusions

Both anti‐miR‐181a and anti‐miR‐200c are effective non‐invasive post‐stroke interventions to reduce injury in clinically‐relevant aged mice. Future investigations should be extended to testing in aged female cohorts and identifying relevant downstream targets of miR‐181a and miR‐200c.
Tattoos Effect Local Sweat Excretion Rate of Epidermal Growth Factor During Outdoor Group Fitness Exercise
Authors: Brown Shyretha , King Michelle , Keyes David , Engel Megan , Ciciora‐Gold Matthew , De Chavez Peter , Baker Lindsay
Published: May 2022

There is interest in the potential of sweat cytokines to serve as non‐invasive biomarkers to monitor immune system function, but many practical questions remain. For example, sweat constituents collected from tattooed (TAT) skin regions may differ from skin regions without permanent tattooing. The purpose of this investigation was to determine if tattooed skin (TAT) differed from contralateral non‐tattooed skin (NT) for sweat cytokines including: EGF, IL‐10, IL‐1α, IL‐1β, IL‐6, IL‐8, TNF‐α, and IL‐31 concentrations and excretion rates during a group fitness exercise session. Moderately trained individuals (males n=8, females n=8, 34±8 y) with at least one permanent tattoo on the upper body (wrist, forearm, tricep, bicep, scapula, shoulder, or torso) participated in a ~60 minute outdoor (~27±3°C, 63±4% RH) group fitness exercise session. Absorbent patches (3M Tegaderm™ + Pad) were used to collect sweat on both TAT and NT skin. Patches were monitored during the exercise session and removed upon moderate saturation. Sweat cytokine concentrations were measured using Multiplex (EMD Millipore, MagPix) customized kits. Local sweat rate (LSR) was calculated from sweat mass over patch surface area (11.9 cm2) and duration of exercise (56±2 min). Excretion rates were calculated as the product of LSR and cytokine concentration. To evaluate differences between TAT and NT skin, paired t‐tests or Wilcoxon signed rank tests were used. Significance level was set at p<0.05. Data are presented as mean±SD for normally and non‐normally distributed data. There were no differences in LSR for TAT vs. NT skin regions (1.20±0.62 vs 1.24±0.53 mg/cm2/min; p=0.70). There were no differences between TAT and NT skin for concentrations of EGF (45±29 vs 53±31 pg/mL; p=0.10), IL‐10 (0.03±0.04 vs 0.07±0.13 pg/mL; p=0.21), IL‐1β (1.66±1.12 vs 1.86±1.07 pg/mL; p=0.55), IL‐8 (0.60±0.50 vs 0.56±0.64 pg/mL; p=0.27), IL‐1α (618±550 vs 807±913 pg/mL; p=0.3) or TNF‐α (0.40±0.05 vs 0.40±0.04 pg/mL; p=0.85). There was below minimum or no level of detection for TAT and NT skin for IL‐6 and IL‐31. Excretion rates were significantly different between TAT and NT skin for EGF (0.05±0.04 vs 0.06±0.03 pg/cm2/min; p=0.04). In summary, EGF excretion rate was lower in TAT skin than NT skin during exercise, but TAT did not affect the concentrations or excretion rates of any other cytokines measured.
Antibiotics and High‐Fat‐Diet Induced Gut Inflammation and Impaired Gut Permeability are Improved by Dietary Blueberries Possibly Through NFκB Signaling
Authors: Mattos Della Lucia Ceres , Petersen Chrissa , Adhini Kuppuswamy Satheesh Babu , Pon Velayutham Anandh Babu
Published: May 2022

Background

Evidence indicates that a high fat diet can promote gut inflammation and increase gut permeability. The use of antibiotics further exacerbates these complications and can lead to the development of inflammatory bowel disease. Blueberries are rich in bioactive components such as anthocyanins. We recently reported the vascular and prebiotic effects of dietary blueberries. However, the effects of blueberries on gut inflammation and permeability are unknown. In the present study, we tested the hypothesis that blueberries improve antibiotics and high‐fat‐diet (HFD) induced gut inflammation and impaired gut barrier function in C57BL/6J mice.

Methods

7‐week‐old male mice (Jackson Lab) consumed standard diet (10% kcal fat) (CON) or HFD (60% kcal fat) with antibiotic cocktail (ampicillin, vancomycin, neomycin sulfate and metronidazole) in drinking water (HFA) or 3.7% freeze‐dried blueberry powder supplemented HFD with antibiotic cocktail in drinking water (HFA+BB) for 12 weeks. Body weight and body composition were assessed at the end of the experiment. Inflammation and permeability of the colon were assessed by the mRNA expression of inflammatory markers (IL‐1β, IL‐6, iNOS, MCP‐1); gut barrier markers (MUC‐2, cldn‐1 and TJP‐10); and markers of NFκB signaling (p65, IKK‐ β and IκBα) by qPCR using specific primers and SYBR green.

Results

HFA mice had an increased body weight, reduced lean body mass, and increased body fat compared to CON mice. Blueberry supplementation did not alter these metabolic parameters in HFA+BB vs HFA mice. mRNA expression of inflammatory markers (IL‐1β, iNOS, MCP‐1); gut barrier marker MUC‐2; and markers of NFκB signaling (p65 and IκBα) were increased in HFA vs CON mice. However, blueberry supplementation improved inflammation and permeability of the colon as shown by a reduced colonic expression of IL‐1β, iNOS, MCP‐1 and MUC‐2 in HFA+BB vs HFA mice. Further, a reduced mRNA expression of p65 and IκBα in HFA+BB vs HFA mice indicates that the protective effect of blueberry on the colon could be mediated through NFκB signaling.

Conclusion

Blueberry supplementation suppresses colon inflammation and ameliorates colon permeability possibly through NFκB Signaling. Consumption of blueberry is a potential dietary approach to improve gut health.
Effect of Acute Isometric Handgrip Exercise on Vascular Function in Children with Congenital Heart Disease
Authors: Khan Rafique , Boyes Natasha G. , Luchkanych Adam M. S. , Erlandson Marta , Wright Kristi D. , Pockett Charissa , Olver T. Dylan , Tomczak Corey R.
Published: May 2022

Objective

The reactivity of conduit vessels such as the brachial artery is important in influencing arterial diameter, and subsequently, blood flow. Flow‐mediated dilation (FMD) measures vessel reactivity. The increase in shear stimuli from transient arterial occlusion enhances arterial diameter in an endothelial‐dependent manner, and this is thought to be impaired in individuals with congenital heart disease (CHD). Isometric handgrip (IHG) exercise is a proposed intervention to improve vessel reactivity by optimizing the shear stimuli to provoke changes in arterial diameter. Indeed, IHG training has been found to improve FMD in healthy adults and children. Whether IHG training improves endothelial dysfunction in CHD remains unknown. Improving vessel reactivity in the young becomes increasingly important in optimizing vascular health into adulthood. The effects of acute IHG exercise on the brachial artery were examined in children with CHD.

Hypothesis

We tested the hypothesis that acute IHG exercise would increase FMD in children with CHD.

Methods

Four subjects with CHD (mean, 13 ± 1 year, 3 female) completed a single session of 4 × 2 mins of acute IHG (30% maximum voluntary contraction using the right hand) with 1‐min rest intervals between bouts. Before and after IHG exercise, the right brachial artery was imaged with Duplex ultrasound using a 1‐min baseline, 5 mins of forearm circulatory occlusion, followed by 3 mins of cuff deflation. Blood pressure and heart rate were measured continuously.

Results

Pre‐FMD (baseline) mean arterial pressure (pre: 102 ± 17 mmHg vs. post: 110 ± 16 mmHg, p = 0.496) and heart rate (pre: 75 ± 13 bpm vs. post: 66 ± 5 bpm, p = 0.253) were not different. Baseline brachial artery diameter increased following acute IHG in 3 of 4 subjects (pre: 3.11 ± 0.45 mm vs. post: 3.23 ± 0.39 mm, Cohen’s d = 0.3). Peak shear rate was greater post‐IHG in 3 of 4 subjects (pre: 995 ± 346 sec‐1vs. post: 1158 ± 501 sec‐1, Cohen’s d = 0.4). FMD% (4.7% vs. 6.3% and 8.2% vs. 10.5%) and FMD% normalized to shear rate area under the curve (AUC) ratio (2.2E‐5 vs. 7.4E‐5 a.u. and 3.1E‐5 vs. 4.3E‐5 a.u., Cohen’s d = 2.0) increased post‐IHG in 2 of 4 subjects. When brachial artery diameter increased, peak shear rate, FMD%, and FMD% normalized to shear rate AUC improved post‐IHG in 2/3 subjects whereas subject four showed a decrease in brachial artery diameter, FMD% and FMD% normalized to shear rate AUC, with the exception of peak shear rate post‐IHG.

Conclusion

Favorable changes in brachial artery diameter, peak shear rate, FMD% and FMD% normalized to shear rate AUC following acute IHG may be a promising intervention for improving vascular tone and endothelial function in children with CHD.
Connexin43 Overexpression Exacerbates Myocardial Ischemic Reperfusion Injury in Diabetes via Modulating Cardiac Autophagy
Authors: Liu Danyong , He Jianfeng , Zhou Dongcheng , Chen Jiajia , Zhang Liangqing , Xu Aimin , Xia Zhengyuan
Published: May 2022

Background

Patients with diabetes are more vulnerable to myocardial ischemia/reperfusion injury (MIRI). Abnormal increases of cardiac Connexin43 (Cx43) were shown to be associated with a variety of pathological conditions including myocardial ischemia and diabetic cardiomyopathy, concomitant with various degrees of changes in autophagy. However, the role of Cx43 in MIRI in diabetic conditions and in particular its potential interplay with cardiac autophagy in this pathology is unknown. Thus, the present study was aimed to explore the role and mechanism of Cx43 in diabetic MIRI.

Methods and Results

Streptozotocin (STZ)‐induced diabetic and age‐matched control rats were subjected to MIRI by occluding the left coronary artery for 30 minutes followed by 2 hours of reperfusion at 5 weeks of diabetes induction. In vitro, rat origin H9C2 cardiomyocytes (H9C2) were exposed to 35mmol/L high glucose(HG)for 48 hours(h) in the absence or presence of Cx43 gene knockdown with siRNA, followed by hypoxia for 6h and reoxygenation for 12h. Myocardial infarct size was assessed by TTC staining. Cell viability was detected by the CCK‐8 assay, apoptosis by TUNEL assay. Cardiac Cx43 protein, apoptosis‐related proteins and autophagy‐related LC3, p62 and mTOR proteins were assessed by Western blotting. Post‐ischemic myocardial infarct size and cardiac CX43 protein expression were significantly increased in diabetic rats as compared with that in the control (p<0.05), concomitant with increases in the ratio of LC3 II/I and in the protein expression of P62 and mTOR (all p<0.05). In vitro, exposure of H9C2 cells to HG significantly increased cell injury evidenced by increased LDH release and reduced cell viability, and significantly enhanced cell apoptosis and reactive oxygen species(ROS) production that was accompanied by increased cardiomyocyte CX43 overexpression and enhanced autophagy but paradoxically also enhanced mTOR activation, similar to that seen in vivo. All these changes were further exacerbated following H/R under HG. Cx43 Knockdown in H9C2 results in decreased ROS production and decreased apoptosis after H/R under HG leading to increased cell viability (all p<0.05), while Cx43 knockdown attenuated autophagy. Application of the selective mTOR inhibitor rapamycin abolished the abovementioned beneficial effects of Cx43 Knockdown.

Conclusion

Overexpressed CX43 in the diabetic heart exacerbates MIRI in diabetes through enhanced cardiac autophagy under hyperglycemic condition.
Primary cilia control kidney inflammation and scarring in response to obstruction
Authors: Aka Amandine , Viau Amandine , Quatredeniers Marceau , Terzi Fabiola , Bienaimé Frank
Published: May 2022

Primary cilium is a unique antenna‐like structure that protrudes from the surface of most mammalian cells. Primary cilia are located at the apical surface of renal tubular epithelial cells, where they are bathed in the forming urine. Overall, primary cilia are signaling platforms that integrate mechanical and chemical cues. Yet, the functions of tubular cells cilia in kidney physiology remain unclear. Mutations in genes encoding ciliary proteins frequently lead to the development of chronic kidney diseases such as Autosomal Dominant Polycystic Kidney Disease or Nephronophthisis. A common denominator of these “renal ciliopathies” is the infiltration of the kidney by immune cells and myofibroblasts that prompt renal scarring. In the absence of mutations, such events can notably be triggered by tubular obstruction, which abolished urinary flux and therefore may affect cilia signaling. Yet the contribution of primary cilia in kidney response to obstruction is unknown.

Here, we combined comparative analysis of kidney gene expression datasets, models of mechanical tubular obstruction and genetic ablation of primary cilia to explore this question. Our results indicate that the renal cytokine landscape of ciliopathies massively overlaps with the one induced by ureteral obstruction. Applying this latter model to mice with tubule specific cilia ablation, we demonstrated that cilia are instrumental in the induction of most of these cytokines. Subsequently cilia ablation mitigated macrophage and neutrophil recruitment to the kidney and reduced fibrosis burden.

All together, these results show that primary cilia evoke a coordinated response to tubular obstruction reminiscent of genetic renal ciliopathies. Elucidating the mechanisms underlying this function of primary cilia may illuminate the pathophysiology of both genetic and non‐genetic chronic kidney diseases.

AA* and AV* these authors equally contributed to this work
Multiplex Mass Spectrometry for Enzymatic Activities Screening of Lysosomal Storage Diseases
Authors: Jayaweera Taniya , Darie Ion Laura , Petre Brindusa‐Alina , Darie Costel
Published: May 2022

Lysosomal storage diseases (LSDs) are a group of rare and inherited disorders of lysosome function and they are classified into more than 50 types (e.g., mucopolysaccharidoses, neuronal ceroid lipofuscinoses, Fabry’s Disease; Pompe Disease, etc.). The majority of LSDs result from defective lysosomal enzyme activity leading to accumulation of endogenous macromolecules in the lysosome causing severe disease symptoms and finally death. For some LSDs, treatment is available in the form of enzyme replacement therapy with good results if the therapy is started as soon as possible, making the diagnosis of crucial importance. State of art now in LSDs diagnostic employs in first stage, enzymatic determinations by fluorimetry or by mass spectrometry (in few cases).

In this report, we describe a highly specific and sensitive diagnostics on dry blood spots (DBSs) for (i) molecular determinations of LSDs, particularly mucopolysaccharidoses (MPSs) and neuronal ceroid lipofuscinoses (NCLs), by simultaneous fluorimetric and mass spectrometric analysis using new types of substrate derivatives based on different alkyl‐umbelliferone; (ii) clinical diagnostics of LSDs by multiplex‐MS‐MRM analysis using new type of substrate derivatives and (iii) clinical validation of the new developed methods using DBS samples for patients with established LSDs. We have developed a high‐throughput single and multiplexing assay for rare diseases (e.g. MPS 1, MPS 2, MPS 6, NCL 2, NCL 10, Fabry Disease, etc.) in DBSs. A common substrate for the fluorimetric measurement of a lysosomal enzyme activity is a molecule with 2 different parts which are bound covalently: a group recognized by the target enzyme (e.g. sugar, lipid) and 4‐umbelliferone and the product resulted after the enzymatic reaction is always 4‐methyl umbelliferone. In our tests, the enzymatic activity levels in DBSs were determinate by fluorimetry or multiple reactions monitoring mass spectrometry in the presence of an internal standard showing a good statistical correlation in singles assays. Moreover, we developed duplex and triplex assays for the diagnosis of different LSDs from the muccopolysacharidoses family using modified substrates based on different coumarin derivates obtained through Pechman condensation. The new umbelliferone derivatives were synthesize by substituting groups in position 4 and 7 of the alpha‐ coumarin molecule with others e.g. 4‐ ethyl‐ umbelliferone, 4‐propyl‐umbelliferone, etc.

In conclusion, by quantification of umbelliferone derivatives as products of enzymatic reactions either by fluorimetry or mass spectrometry, the newly proposed substrates can be easily use for clinical diagnosis of several LSDs.
Effect of Fluid Shear Stress on Human Breast Cancer Cells
Authors: Mallick Ditipriya , Jana Siddhartha S.
Published: May 2022

The importance of Hemodynamic forces such as interstitial and vascular Fluid Shear Stress (FSS), which are exposed to cancer cells, is not well understood. Here, we report that human breast cancer cells, MDA‐MB‐231, show increased cytosolic and nuclear area, nuclear flattening, focal adhesion and stress fiber formation in the presence of 6 dyn/cm2 orbital shear stress. Withdrawal of FSS restores the morphology within 12 hours. Time lapse video microscopy reveals that FSS pre‐exposed cells migrate more slowly compared with the control cells which are not pre‐exposed with FSS. Interestingly, when we seed these cells on soft agar plate, cells are able to grow, and the size of the colonies are significantly larger compared with the control cells. The transcriptional co‐activator, responsive to biomechanical cues, YAP‐1 translocates into nucleus in the presence of FSS. Altogether, these data suggest that FSS can change homeostasis of cancer cells to facilitate their survival.
Progesterone prolongs time to delivery and attenuates blood pressure possibly by improving inflammation and endothelial function in response to preeclampsia
Authors: Amaral Lorena , McCray Morgan , Evans Kymberlee , Deer Evangeliine , Herrock Owen , Ingram Nicole , Wallace kedra , Turner Ty , Campbell Nathan , Hogg James , Solise Dylan , Reeve kristin , LaMarca Babbette
Published: May 2022

Preeclampsia (PE), new onset hypertension during pregnancy, affects 5‐7% of all pregnancies in the U.S. and is associated with reduced fetal weight, increased inflammation, vascular endothelial dysfunction (increased endothelin‐1 (ET‐1) and decreased nitric oxide (NO)) and hypertension. To date the best treatment remains early delivery of the feto‐placental unit. Activated lymphocytes during normal pregnancy (NP) express progesterone receptors, which stimulate a protein called Progesterone Induced Blocking Factor (PIBF) that is reduces during hypertensive pregnancy disorders. Therefore, this study was designed to test the hypothesis that progesterone, in the form of 17‐hydroxyprogesterone caproate (17‐OHPC), reduces inflammation, markers of endothelial dysfunction while reducing blood pressure and prolong time to delivery in PE women. In our ongoing clinical trial, PE participants received 17‐OHPC (250 mg, I.M.) with blood draws before and after injection. Placentas were collected at delivery. PIBF was 18.6 +/‐1.0 pg/mL in NT (n=4), 14.53 +/‐ 1.0 pg/mL in PE (n=10, p<0.05), and 15.78 +/‐0.85 in PE+17‐OHPC (n=6). Placental CD4+ T cells were 6.5+/‐ 2.7 in PE (n=3), 4.6+/‐ 2.0 in PE +17‐OHPC (n=4). Circulating CD4+ T cells were 18.23 +/‐4.8 in PE (n=4), 14.4+/‐1.2 % gate in PE+17‐OHPC (n=5). Placental TH2 cells were 81.7+/‐10.6% gate in PE and 84.7+/‐11.0 in PE+17‐OHPC. Placental and circulating NK cells were 20.84 +/‐ 6.8 % gate, 7.2 +/‐ 2.0 in PE which reduced to 4.7+/‐1.03, 5.3 +/‐ 1.5 % gate in PE+17‐OHPC. Circulating TNF‐alpha was 32.0 +/‐ 3.4 pg/mL in PE (n=8), which decreased to 21.1+/‐5.5 in PE+17OHPC (n=4). Circulating ET‐1 was 2.53+/‐ 0.4 pg/mL in healthy normal pregnant (NP, n=5), 6.7 +/‐ 1.4 in PE (n=18, p<0.05) and 4.9 +/‐ 1.3 in PE+17‐OHPC. Placenta preproendothelin ‐1 (PPET‐1) increased 1.5 fold change in PE (n=4) compared to NP, which was reduced to 0.96 in PE+17‐OHPC (n=4). Importantly, endotheling‐1 measured in HUVECS media treated with PE sera was 68 +/‐ 22 pg/mg of protein in PE which reduced to 57 +/‐19 in PE+17OHPC (n=6). Moreover, circulation nitrate‐nitrite was 50 +/‐ 9 uM in PE and significantly increased to 94 +/‐14 in PE+17‐OHPC. 17‐OHPC prolonged time of delivery beyond 72h on average and average systolic blood pressure was 151 +/‐ 5 mmHg in PE (n=18) and 137+/‐4 in PE+17‐OHPC (n=13). Our results suggest that 17‐OHPC reduced inflammation, markers of endothelial dysfunction, lowered blood pressure and prolonged time do delivery in PE women.
Development of a Dietary and Life‐style algorithm for Prophylactic‐Remission of IBS: A Case Study
Authors: Shujaat Tayyaba , Malik Samina
Published: May 2022

Inflammatory Bowel Disease is considered incurable in allopathy. Medical professionals hardly advise nutritive‐therapy or life‐style management. This adds to the prevalence of chronic illness and may end into carcinoma. This case study is a success‐story of a young student at 24 years of age who was diagnosed with IBS on colonoscopy. The only medicine advised was hydrocortisone‐enema which did not benefit her. Stress related to father’s demise and unhealthy‐lifestyle and diet were the identified precipitating factors. History included off and on constipation, bloody‐motion with mucus discharge, pain‐free defecation, gaseous‐abdomen with bloating and dull‐pain. Life‐style behavior included poor physical activity, disturbed sleep‐wake cycle, compromised hostel diet and academic‐deadlines related stress.

The impact of Dietary variation was monitored in terms of variation in clinical features. Flare was observed with red‐beans, chick‐peas and black‐lentil. Gluten and Lactose intolerance were ruled out by diet‐variation. Probiotic‐treatment and low FODMAP diet helped in remission. The specific algorithm that facilitated in recovery included figs, strawberries, cranberries, pomegranate, Salmon fish, Greek‐yogurt, olive‐oil, oatmeal, walnuts, dates and green‐leafy‐vegetables. Life‐style modification included mindfulness meditation, walking/jogging, soul‐cycling and counselling. Outcome was measured and compared. Remission was achieved within 4‐5 months and the patient is recovered since last 1.5 years as evidenced by laboratory data. It includes CRP reduction within normal range, hemoglobin improvement (10.2 to 12.2 g/dl), MCV improvement (73 to 86.9 fL), MCH improvement (21.2 to 27.5 pg), MCHC improvement (29.1 to 31.7 g/dl), HCT improvement from 35% to 38.5%, Fecal Calprotectin reduction from 712.9 to 415.3 µg/g and total protein reduction from 8.39 to 7.48 g/dl. Hopefully continuous alternate treatment may normalize the calprotectin marker as well.
Outcome of short‐term physiotherapy intervention program on early stage of multiple sclerosis ‐ A case report
Authors: Nandennagari Sailaja , Preethi Annam , Bethala Krupavarm
Published: May 2022

INTRODUCTION

Multiple sclerosis is a chronic autoimmune progressive disease of demyelination of central nervous system with subsequent axonal damage. The symptoms are due to central nervous system lesions resulting from loss of oligodendrocytes, which myelinates CNS leading to tingling and numbness of limbs and reactive gliosis with no cure for the disease. But CNS diseased effects are often controlled through rehabilitation.

OBJECTIVE OF THE STUDY

The aim of this study is to observe the importance of early physiotherapy rehabilitation along with immunosuppressants to suppress and reduce the symptoms and slows down the disease process beginning from the initial day of hospitalization to six weeks which causes progression in the patient reducing the incapacity and improves functional measures and quality of life in multiple sclerosis patients. Muscle weakness, and spasticity, balance and coordination can be improved through stretching and strengthening exercises.

METHOD

Patient strictly followed intervention for a period of six weeks under the supervision of Physiotherapist. Intervention includes strengthening, aerobic conditioning, flexibility exercises, coordination exercise, balance exercises, gait training using assistive devices, sensory, functional, bowel and bladder control training. Interventions were gradually progressed up to the patient every week according to their symptoms. Patient was assessed in 1st and 7th week using the outcome measures namely Functional independence measure (FIM) and Short Form survey‐12 (SF‐12) assessing the impact of health on patient’s everyday quality of life.

RESULTS

After the 7th week re‐assessment, it has been shown there is a difference in the value of outcome measures. FIM level has improved from 3‐moderate assistance with helper to 6‐modified independence without helper. SF‐12 physical score showed marked improvement from 48.020307 to 56.57706 while SF‐12 mental score change showed very minor change.

CONCLUSION

Multiple sclerosis disease impairment is primarily a consequence of disease progress, but it can be aggravated by reduced physical activity leading to muscle weakness, decreased ROM, spasticity. Exercise and training have been shown to improve the above said deteriorations in multiple sclerosis patients and certainly can be alleviated.

Score through FIM and Health survey scale are tabulated as follows

Outcome measure
Preintervention
Postintervention

FIM, Functional Independent Measure (in scales)
3‐‐ Moderate Assistance(>= 50%): Helper‐Modified Dependance
6‐‐ Modified, Independence (Device): No Helper

SF‐12, Quality of life measure
PCS‐12 (Physical Score): 48.020307 MCS‐12 (Mental Score): 59.04606
PCS‐12 (Physical Score): 56.57706 MCS‐12 (Mental Score): 60.75781
Congested Heart Failure in Small Breeds Dogs
Authors: Morris Tori , Chambers Kyara
Published: May 2022

Mitral valve disease, commonly known as MVD, is the most common heart disease in dogs, accounting for nearly 75% of all dogs affected. The characteristic of this predominant disease includes collagen degradation, elastic fiber fragmentation and proteoglycan accumulation, leading to deterioration of the mitral valve and ultimately, congestive heart failure (CHF). Treatment to reverse or even delay the damaging effects of MVD has not yet been discovered due to a lack of awareness of the underlying processes. Furthermore, this disease in dogs has many similarities with the human MVD and may potentially serve as a translational model for studying MVD further. Therefore, it is imperative to identify the causes and underlying mechanisms associated with canine MVD in order to treat these patients more efficiently at the earliest detectable stage. MicroRNAs (miRNAs) are small, 21‐25 nucleotides in length, non‐coding RNAs that can down‐regulate the expression of protein coding genes by binding to the 3’UTR sites in their messenger RNAs (mRNA), causing either degradation or gene silencing. Prior studies have shown that one miRNA may regulate multiple mRNAs, subsequently effecting the downstream levels of gene expression. Furthermore, if miRNAs prove to be a useful biomarker of heart disease, not only may they serve as detectors of early disease onset, but they may also be useful as therapeutic agents. Given this recent data, we propose to test the hypothesis that circulating miRNAs may be useful biomarkers for detection of CHF, secondary to MVD. Liquid biopsies (blood samples) were collected in EDTA tubes from 7 healthy dogs with no prior history of heart disease and 7 dogs being treated for CHF secondary to MVD. Total serum RNA was isolated using miRNeasy Serum/Plasma Kit (Qiagen). Isolated RNA was then reverse transcribed using miScript II RT Kit (Qiagen) and lastly, miRNA was quantified using miScript SYBR Green PCR Kit, along with miScript miRNA PCR Arrays (Qiagen).
A suicide diiron oxygenase in p‐aminobenzoate biosynthesis in Chlamydia trachomatis
Authors: Pedraza Andrew V. , Wooldridge Rowan , Allen Kylie
Published: May 2022

Folate is an essential one‐carbon carrier cofactor required for essential biological reactions such as amino acid and DNA biosynthesis. Folates are comprised of three chemical moieties: a pteridine ring, p‐aminobenzoate (pABA), and glutamate residues. Chlamydia trachomatis has been shown to synthesize folate de novo; however, it is missing several genes involved in the canonical folate biosynthesis pathway. Previous work has demonstrated that a single gene, ct610, functionally replaces the enzymes responsible for pABA production in the canonical biosynthesis pathway. However, CT610 does not use chorismate as the precursor to pABA as is used in the canonical pathway. Further experiments showed that isotopically labeled tyrosine was incorporated into the pABA molecule when synthesized by CT610 in vivo. However, in vitro experiments demonstrated that CT610 produces pABA in the presence of oxygen and a reducing agent without any added substrates, including tyrosine. This led to the hypothesis that CT610 is a self‐sacrificing “suicide” enzyme that donates an active site tyrosine residue in synthesizing pABA. CT610 has low sequence similarity to non‐heme diiron monooxygenases such as methane monooxygenase and class Ia ribonucleotide reductase, and the previously solved crystal structure of CT610 revealed a diiron active site. Here we describe our recent progress towards understanding this unique suicide mechanism for pABA biosynthesis. To determine the tyrosine residue sacrificed in the reaction, we generated five active site tyrosine to phenylalanine mutants and found that two of the variants completely abolished pABA production, suggesting that one of these residues is the precursor to pABA. Further, a conserved lysine residue is also essential for the reaction and may be a sacrificial amino group donor. We have confirmed the oxygenase activity of wild‐type CT610, which requires only a reducing agent and is not stimulated by the presence of tyrosine or other possible substrates. Further, we developed an Fe(II) reconstitution procedure, where the reconstituted enzyme exhibits a drastic increase in oxygenase activity. However, surprisingly, the reconstituted enzyme does not exhibit increased pABA synthase activity, although EDTA treatment of the as‐purified enzyme almost completely inhibits pABA production. This suggests that a metal other than iron is required for the reaction and thus current work is focused on testing other potential metal cofactors that may play a role in the novel suicide reaction.
In Vivo Effects of AICAR and Prodrug 39 (P39) on Anabolic Pathways Following Refeeding in Skeletal Muscle
Authors: Pace Tyson , Marchant Erik , Wilkinson Cameron , Ashby Jared S. , Galvan David A. , Rojas Joseph , McClune Derek , Saunders Oliver , Hancock Chad R. , Thomson David M.
Published: May 2022

Exercise training is known to elicit many beneficial adaptations. However, the mechanisms by which this happens are not entirely understood. During muscle contraction, increased intracellular AMP levels mediate many exercise‐induced adaptations by activating AMP‐activated protein kinase (AMPK), among other targets. AMP mimicry may, therefore, be an attractive pharmacological approach to stimulate some of exercise’s benefits. The prodrug AICAR is a well‐established AMPK activator when phosphorylated in the cell to form ZMP, an AMP mimetic. However, it has limited pharmacological potential due to poor pharmacokinetics. Prodrug 39 (P39) uses an alternative approach to deliver ZMP to the cell. The purpose of this study was to compare AMPK and P39 effects on blood glucose levels, AMPK activation, and anabolic signaling in the fasted and refed states. C57BL/6 mice were subjected to a 12 hour fast, followed by 1 hour of refeeding. Following the 12‐hour fast, blood glucose was measured, and mice were injected with saline, AICAR or P39 (400 mg/kg BW). Mice were immediately transferred to cages with food, except for a fasted saline group. After 1 hour of refeeding, food was removed from cages. Blood glucose was measured 25 minutes later, and mice were sacrificed. Gastrocnemius‐plantaris‐soleus complex (GPS) muscles were quick‐frozen for protein analysis. Fasting blood glucose levels were 80 ± 4 mg/dL prior to refeeding and were elevated to 144 ± 8 mg/dL (p<0.0001) after food reintroduction. Both AICAR and P39 caused a significant reduction in blood glucose compared to refed controls (p<0.0001, 54 ± 9, and 56 ± 10 mg/dL, respectively). In GPS muscle, AMPK phosphorylation status was unaffected by any treatment. However, ACC phosphorylation, a downstream target of AMPK, was ~3‐fold higher in AICAR‐treated animals compared to refed controls (p<0.01), with no significant effect of P39. Refeeding induced an increase in anabolic signaling, as indicated by an increase in S6 phosphorylation (~4‐fold, p<0.01) and a trend for increased eIF4E‐binding protein (4E‐BP1) phosphorylation (p = 0.1). This increase was entirely prevented in AICAR and P39‐treated animals. Overall, these results suggest that AICAR and P39 are similarly effective in lowering blood glucose and dampening anabolic signaling following refeeding. However, AICAR was more effective in activating AMPK signaling in skeletal muscle at this dose.
Pharmacokinetics of a 40mg Sublingual Dose of Delta‐Tocotrienol Powder
Authors: Hanway Tim R. , Wallace William A. , Gillespie Jonathan C. , Hodges Falyn L. , McCoun Julie A. , Raub Betsy J. , Serrano Eric R. , Moon Jordan R.
Published: May 2022

Tocotrienols and tocopherols are lipid‐soluble chemicals that make up vitamin E. Vitamin E can be subcategorized as (α‐tocopherol, β‐tocopherol, γ‐tocopherol, and δ‐tocopherol) and four primary tocotrienols (α‐tocotrienol, β‐tocotrienol, γ‐tocotrienol, δ‐tocotrienol). Tocotrienols receive less recognition for their effects on health compared to their counterparts, tocopherols. The purpose of this study was to investigate the pharmacokinetics and bioavailability of a powdered, sublingual form of DT3 in healthy men and women. We hypothesized that there would be an increase in plasma DT3 followed by a decrease after reaching peak concentration over an 8‐hour period. Volunteers (m=1, f=4; age: 36.6 +/‐ 10 years, 169 +/‐ 6 cm, 77 +/‐ 14.6 kg) were administered a 40mg of DT3 powder after fasting for 8 hours and given a meal consisting of carbohydrates, proteins, and fats after 4 hours. Volunteers were asked to keep the powder under their tongues for 30 seconds or until the powder was completely dissolved. Blood samples were collected at 0, 30, 45, 60, 90, 120, 180, 240, 360, and 480 minutes. Blood samples collected were placed in the centrifuge to separate plasma from blood. The plasma collected was stored at ‐80°F in a cryogenic freezer. DT3 concentrations were measured from plasma using protein precipitation followed by liquid‐liquid extraction. Analytes were separated by HPLC with the extracts assayed against a calibration curve. DT3 bioavailability was assessed using the parameters of peak plasma concentration (Cmax), time to reach peak plasma concentration (Tmax) and total area under the plasma concentration‐time curve (AUC). Plasma concentration of DT3 reached 47.1 ng/ml (Cmax) 360 minutes after administration (Tmax). DT3 concentration decreased between 360 and 480 minutes. The 0‐8 h AUC reached 10,093 ng/ml*min. Ingesting a 40mg dose of DT3 dissolved orally and then swallowed appears to produce a slow progressive upward absorption trend over the time span of 6 hours after. Following the 6‐hour increase in DT3 concentration a subsequent decrease in plasma DT3 was observed. Optimal dosing and timing of DT3 when dissolved in the mouth may enhance the efficacy of δ‐tocotrienols on health.
Impact of Obesity on Exercise‐Induced Skeletal Muscle Exosomes
Authors: Wen Yuan , Valentino Taylor , Depa Lauren , Goh Jensen , Alimov Alexander , Vechetti Ivan , Peterson Charlotte A. , McCarthy John J.
Published: May 2022

Exosomes are small extracellular vesicles (EVs) that can function as effective delivery vehicles through systemic circulation due to the protective effects of the phospholipid bilayer. Our previous work in mice indicated that resistance exercise induces the release of skeletal muscle EVs to promote adipose lipolysis through the action of microRNA‐1. To better understand the effect of resistance exercise on exosomes in humans, we collected blood samples as well as skeletal muscle and adipose tissue biopsies before and after a single bout of resistance exercise. Using the ExoView platform, we were able to analyze subpopulations of exosomes based on their surface tetraspanin markers (CD63, CD81, and CD9). Our preliminary findings show that serum CD81/CD9 exosome abundance is significantly lower in high BMI (>30) subjects, and serum CD63/CD81 exosome abundance may be differentially affected by exercise in subjects with high BMI. Further studies are necessary to define the nucleic acid and protein contents of the exercise‐induced exosomes. To this end, we have generated a transgenic mouse with an inducible skeletal muscle specific exosome surface protein reporter, which will allow definitive tracking of exosomes released from skeletal muscle that are delivered to other organ systems.
Aging Alters Integrin‐mediated Vascular Smooth Muscle Function in Soleus Feed Arteries
Authors: Shin Song Yi , Ojha Krishna R. , Padgham Samuel , Trache Andreea , Woodman Christopher
Published: May 2022

Introduction

Previous data have shown that aging impairs vasomotor function and alters vascular smooth muscle cell (VSMC) properties. Integrins play an important role in regulating many cellular functions including arterial contractility. However, how aging affects integrin function, and their contribution to impaired constriction in aged arteries has not yet been elucidated. Thus, the purpose of this study was to investigate the age‐induced alteration of integrin function in isolated VSMC and in intact soleus muscle resistance arteries.

Methods

Soleus muscle feed arteries (SFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats, cannulated with glass micropipettes and pressurized at 90cm H2O. Endothelial cells were removed (denuded) by passing 5 ml of air through the artery lumen. Vasoconstrictor responses were assessed by adding of norepinephrine (NE; 10‐9‐10‐4M), angiotensin II (Ang II; 10‐11‐10‐7 M), or phenylephrine (PE; 10‐9‐10‐4 M) in the presence or absence of RGD, an integrin inhibitory peptide. RGE, a non‐inhibitory peptide was used as control. To investigate changes in integrin expression and recruitment at focal adhesions with age, RT‐qPCR, cell adhesion assays, and total internal reflection fluorescence (TIRF) imaging were performed using VSMC isolated from young and old SFA.

Results

Old denuded SFA had reduced vasoconstrictor responses to NE, PE, and Ang II. In the presence of the RGD‐inhibitory peptide, constrictor responses to Ang II were significantly inhibited in denuded old vs. young SFA. Constrictor responses to NE and PE were not significantly altered by RGD. Constrictor responses to NE, PE, and Ang II were also not affected by RGE. Gene expression of specific integrins expressed by VSMC were downregulated in old VSMC compared to young. In addition, RGD‐treated VSMC showed decreased adhesion to fibronectin in both young and old VSMC compared to RGE non‐inhibitory peptide. Quantitative analysis of TIRF images showed that recruitment of integrin α5 and β3 was diminished in old VSMC while β1 was not altered with age.

Conclusions

Taken together, these data suggest that aging leads to integrin dysfunction, which contributes to decreased contractile properties of vascular smooth muscle in SFA.