bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Player in Platelet Function
Authors: Vemana Hari Priya , Karim Zubair , Conlon Christine , Khasawneh Fadi
Published: June 2021

While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor‐operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel‐6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation, secretion (dense and alpha granules), integrin IIb‐IIIa, Akt and ERK phosphorylation in a thromboxane receptor (TPR)‐selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR‐specific. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR‐dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.
Persistent Phenotypic Shift in Cardiac Fibroblasts Following Transient ACE Inhibition
Authors: Madhavpeddi Lakshmi , D'Souza Karen , Biwer Lauren , Hale Taben
Published: June 2021

Cardio‐protection afforded by ACE inhibition may be related in part to fibroblast‐specific changes. We have previously shown that transient ACE inhibitor treatment produces long‐term protection against cardiac fibrosis and inflammation in response to myocardial injury induced by L‐NAME. The present study investigates whether transient ACE inhibition produces a persistent phenotypic shift in fibroblasts that may mediate these protective effects. Adult SHR were divided into 3 groups: Control, Control+ L‐NAME (C+L), enalapril (14‐day treatment + 14‐day washout) + L‐NAME (E+L). Cardiac fibroblasts were isolated following in vivo treatment and cultured to passage 1 for measures of macrophage‐recruiting chemokine release, cellular proliferation, and collagen gene expression. In contrast to fibroblasts isolated from C+L, those from E+L rats did not show any increase in Collagen I gene expression, proliferation rate, or release of macrophage‐recruiting chemokines (MCP‐1, GM‐CSF). Our findings demonstrate that prior ACE inhibition resulted in a phenotypic change in fibroblasts to one that resisted L‐NAME‐induced activation. Understanding the mechanism by which ACE inhibition alters fibroblast response to myocardial injury may reveal a novel treatment strategy to prevent pathological cardiac remodeling and ultimate progression to heart failure. Support: Sarver Heart Center
Purification, Characterization and Anti‐osteocarcinoma Study of a Novel Hemagglutinin from the Northeast China Black Beans
Authors: Dan Xiuli , Wong Jack Ho , Fang Evandro Fei , Ng Tzi Bun
Published: June 2021

In the present study we isolated a novel hemagglutinin from an edible legume and explored the potential of this protein in the treatment of osteocarcinoma and liver cancer. The protein was purified by liquid chromatography techniques which entailed affinity chromatographyon Affi‐gel blue gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion‐dependent and stable over a wide range of temperature and pH values. Anti‐proliferative activity was observed in the tumor cell lines MG‐63 and HepG2, but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization and apoptosis. Besides, this hemagglutin was found to possess cytokin‐simulative effect on splenocyte in vitro. The mRNA expression level of interleukin‐2 (IL‐2), interleukin‐6 (IL‐6), interleukine‐1 beta (IL‐1β), interferon‐ gamma (IFN‐γ), and tumor necrosis factor alpha (TNF‐α) were found to be up‐regulated to different extents after the treatment of this hemagglutinin. The secretion of IL‐1β, TNF‐α and IL‐2 also increased with the concentration of this hemagglutinin.
Targeting IP3‐dependent Calcium Signaling Enhances Sorafenib Lethality for Hepatoma via ER Stress Related Apoptosis
Authors: Yin Xiu‐Xiu , Ye Hui‐Lan , Zhang Guo , Chen Xing‐Zhou , Chen Li , He Li‐Jun , Zhang Ji‐Wang , Zhang Fa‐Can
Published: June 2021

IRE1, hallmark of endoplasmic reticular (ER) stress, has been previously shown as a prosurvival molecule in cancer therapy by multikinase inhibitor sorafenib. However, given that the detail mechanism by which trigger IRE1 to impair the sorafenib lethality remains unclear, the present study was designed to clarify whether calcium signaling was involved in this pharmacologic process in HCC. Firstly, under confocal microscopy, sorafenib (100μM) was found to drive a dramatic intracellular calcium mobilization in HepG2 cells. Secondly, western blotting shew that IRE1 was significantly increased in cells either in the presence of sorafenib (20μM) or directly addition of low dose of CaCl2(0.4mM) for 24h. Interestingly, while using 2‐Aminoethyl diphenylborinate (2‐APB) (75μM), the inhibitor of IP3 receptor, to blunt the sorafenib induced‐calcium release in HepG2 cells, the increment of IRE1 and its activity evoked by sorafenib were substantially attenuated in these cells. Meanwhile, flow cytometry shew that 2‐APB and sorafenib(20μM) amazingly caused a synergistic effect versus sorafenib alone with about 50‐60% augmentation of apoptosis. Taken together, IP3‐elicited Ca2+ releaselikely plays a critical role in the sorafenib up‐regulating IRE1 activity and that is emerging as a novel potential target for anti‐HCC therapeutic strategy in the future.(Supported by NSFC Grants 881172260, 81360077 to G.Z. and 81102495 to L.C.)
Mitochondrial Buffering of cAMP in Adult Cardiac Myocytes
Authors: Agarwal Shailesh , Clancy Colleen , Harvey Robert
Published: June 2021

Compartmentation of the diffusible second messenger cAMP plays a key role in producing unique responses to different G‐protein coupled receptors. It has been proposed that this can only happen if the movement of cAMP within the cell occurs at rates well below those of free diffusion. To directly test this hypothesis we used Raster Image Correlation Spectroscopy (RICS) to measure the diffusion coefficient of fluorescently labeled cAMP, 8‐[Pharos‐450]‐cAMP (P450‐cAMP) in live cells. Confocal imaging showed that incubation with the membrane permeable P450‐cAMP led to a distinct longitudinal pattern of fluorescence accumulation in adult rat ventricular myocytes. This was in contrast to the uniform distribution of cAMP‐free dye (P450), suggesting that P450‐cAMP binds to immobilized target. Interestingly, expression of PKA type II regulatory subunit labeled with CFP (RII‐CFP) exhibited a pattern similar to P450‐cAMP, which corresponded with the extra‐myofibrillar space populated by mitochondria, as verified by labeling with MitoTracker Red. RICS analysis found the diffusion coefficient (μm2/s) of P450‐cAMP (9.7±1.6, n=37) to be significantly slower than that of P450 (17.6 ± 1.73, n=14), but similar to that of RII‐CFP (12.1±1.85, n=11). Furthermore, Ht31, a peptide that disrupts interactions between RII and A kinase anchoring proteins (AKAPs), caused the distribution pattern of both P450‐cAMP and RII‐CFP to become more diffuse. Correspondingly, the diffusion coefficients for P450‐cAMP (34.4±4.37, n=15) and RII‐CFP (32.0±4.53, n=8) increased significantly, while that of P450 did not. These data suggest that the movement of cAMP in adult cardiac myocytes is limited by interactions with PKA RII subunits associated specifically with mitochondria.
Silencing of striatin alters the association of caveolin‐3 with the L‐type calcium channel in cardiomyocytes.
Authors: Nader Moni , Khalil Bariaa , Bakheet Dana , Dzimiri Nuduna , Kvietys Peter
Published: June 2021

Striatin (STRN) is a dynamic protein containing binding domains for caveolin (Cav), protein phosphatases 2A (PP2A), and calmodulin (CaM). Although these proteins regulate the kinetics of the L‐type calcium channel (LCC), the role of STRN in this context remains unknown. We have previously reported that STRN can modulate the intracellular calcium and the contraction rate of cultured cardiomyocytes (CMs). Here we show that STRN is highly expressed in adult rat ventricles as compared to atria. Biochemical analysis revealed that neither the knockdown of STRN (shRNA) nor its overexpression in CMs modulated the expression level of Cav‐3, PP2A, or LCC. Interestingly, silencing of STRN enhanced the association of Cav‐3 with CaM but not PP2A. In contrast, the overexpression of STRN significantly reduced the interaction of Cav‐3 with CaM and PP2A. In both cases, the amount of LCC interacting with STRN/Cav‐3/CaM/PP2A complex was unchanged. Taken together, our data suggest that the regulation of intracellular calcium by STRN may be due to alterations in Caveolin‐3 binding to CaM/PP2A/LCC complex thus regulating the kinetics of LCC in cardiomyocytes.
Effects of total knee arthroplasty on skeletal muscle morphology and gene expression 2 hours after surgery
Authors: Mangum Joshua , Hocker Austin , Jewett Brian , Lantz Brick , Dreyer Hans
Published: June 2021

Osteoarthritis (OA) is the leading cause of hospitalization for older adults and total knee arthroplasty (TKA) is one of the most effective surgical remediations of long standing OA. During TKA, a tourniquet is used to maintain a clear operating field and reduce intra‐operative blood flow. As a result, ischemia‐reperfusion (IR) occurs after tourniquet pressure is released, which may negatively impact muscle metabolism. Our prior reports on the acute effects of IR during TKA suggest decreased protein synthesis, increased protein degradation and activation of the endoplasmic reticulum stress response in skeletal muscle. The purpose of this experiment was to determine if skeletal muscle morphology and skeletal muscle gene expression were altered two hours after TKA. Muscle biopsies were obtained immediately before tourniquet was applied, at maximal ischemia, and two hours post‐surgery (reperfusion). There were 12 subjects (n= 8 female, 4 male; age = 68±5.8) and tourniquet time was 43±10 minutes and post‐surgery biopsy time was 138±32 minutes. Histological analysis of skeletal muscle cross sections was performed to examine cross sectional area (CSA) in all fiber types. There was a 20% increase in Type I fiber CSA, 18% in IIa and 22% in IIx 2 hours post‐surgery (p<0.05). These preliminary results suggest that IR during TKA induces changes in muscle morphology and gene expression that persist two hours post‐surgery. Clinically, these data may support our hypothesis that anoxia occurring during tourniquet application followed by reperfusion may negatively impact muscle cell metabolism and potentially contribute to muscle atrophy and reduced functional mobility in these older adults
ANO1 and TRPC6 Channels are Functionally Coupled in Cerebral Artery Smooth Muscle Cells
Authors: Wang Qian , Narayanan Damodran , Neeb Zachary , Jaggar Jonathan
Published: June 2021

Activation of anoctamin 1 (ANO1 or TMEM16A), a calcium (Ca2+)‐activated chloride channel, in myocytes contributes to pressure‐induced vasoconstriction (the myogenic response) in small arteries. ANO1 is activated due to Ca2+ influx through a stretch‐activated non‐selective cation channel that has not been identified. We used biochemical, fluorescence and patch‐clamp techniques to identify the cation channel associated with ANO1 in cerebral artery myocytes. ImmunoFRET experiments indicated that transient receptor potential channel 6 (TRPC6) and ANO1 were located in close spatial proximity in arterial myocytes. ANO1 antibodies co‐immunoprecipitated both ANO1 and TPRC6 protein from arterial lysate. A TRPC6 antibody that is a selective TRPC6 channel inhibitor blocked swelling‐activated ANO1 currents. HYP9, a TPRC6‐specific channel activator, activated Cl‐ currents that were blocked by ANO1‐inh, a ANO1‐specific inhibitor. Substitution of EGTA with BAPTA, a fast Ca2+ chelator in the patch pipette solution inhibited HYP9‐induced ANO1 current activation. ANO1 knockdown using siRNA, reduced both ANO1 protein and the amplitude of HYP9‐induced ANO1 currents. These data indicate that TRPC6 and ANO1 channels are spatially localized and that Ca2+ influx via TRPC6 channels activates ANO1 currents in arterial myocytes.
Effects of Acute Dietary Nitrate Supplementation on Aortic Blood Pressure and Wave Reflection in Young and Older Adults
Authors: Treichler David , Ueda Kenichi , Ganger Charles , Schneider Aaron , Hughes William , Casey Daren
Published: June 2021

Inorganic nitrate supplementation reduces peripheral blood pressure (BP) in several human populations. We examined whether acute nitrate supplementation decreases central aortic pressures and indices of wave reflection in young and older adults. In 13 young (25±1 yr, 10M/3F) and 12 older (64 ± 1yr, 9M/3F) normotensive adults, noninvasive aortic pressure waveforms were synthesized from high‐fidelity radial pressure waveforms via applanation tonometry before and following (60, 90, 120, 150, and 180 min) consumption of a nitrate rich beetroot juice (BR). Aortic BP and indices of aortic wave reflection (Augmentation Index; AIx and AIx normalized for heart rate; AIx@75bpm) were calculated from the generated aortic pressure waveform. Plasma nitrite concentration increased in both groups 60‐180 min following BR (P<0.001). Peripheral (Systolic: ~5mmHg; Diastolic: ~4mmHg) and aortic BP (Systolic: ~6mmHg; Diastolic: ~4mmHg) were reduced 90‐180min post BR in older (P<0.05), but not young adults. Conversely, AIx (3±3 vs ‐5±3%) and AIx@75bpm (‐6±3 vs ‐15±3%) were reduced in young adults 180 min post BR (P<0.05 for both). Whereas, AIx (28±3 vs 28±2%, P=1.0) and AIx@75bpm (21±3 vs 20±3%, P=0.99) did not change in the older adults. Our data suggest that acute dietary nitrate supplementation reduces central aortic BP in older adults, despite not altering indices of aortic wave reflection.

Supported by ACSM Foundation
The Effects of Relative Exercise Intensity vs Individual Metabolism on LV Twist and Untwisting Rate
Authors: Stöhr Eric , Samuel Jake , Yarlett Andrew , Armstrong Craig
Published: June 2021

Left ventricular twist and untwisting rate ('LV twist mechanics') are fundamental to cardiac function during exercise. However, studies have reported large inter‐individual variability in LV twist mechanics which complicates the interpretation of patient responses. To determine the source of variability in LV twist mechanics during exercise, this study aimed to examine whether LV twist mechanics respond more consistently to the same relative exercise intensity compared with exercise performed at individual anaerobic threshold (IAT). 16 healthy non‐endurance trained males (Age: 22±4 years, O2peak: 45.5±6.9 ml/min/kg, range of IAT: 32–69% of O2peak) were enrolled. Following assessment at rest, each volunteer performed short‐duration exercise at: i) the same relative exercise intensity, 40%peak, ii) at 2% above their IAT and iii) at 40%peak during hypoxia (FiO2=12%). No differences in LV volumes were observed during exercise conditions (p>0.05). However, LV twist mechanics responded more consistently during 40%peak compared with exercise at IAT (F‐ratio 2.080). When exercise at 40%peak was performed in hypoxia, LV twist mechanics increased (P<0.05) but the variability of responses remained similar to 40%peak in normoxia. Overall, LV twist mechanics were related to myocardial work. In conclusion, LV twist mechanics respond more consistently during exercise at the same relative intensity than IAT and diagnostic exercise tests in the clinical and research setting should be standardized accordingly. Importantly, the response during hypoxia suggests that the interpretation of LV twist mechanics must take into account the prevailing myocardial work, which may further improve the diagnostic accuracy.

No external funding was required for this study.
Effect of Remote Ischemic Perconditioning on Renal Water and Salt Handling in Rats Subjected to Renal Ischemia/Reperfusion
Authors: Kristensen Marie Louise , Lassen Casper , Birn Henrik , Nørregaard Rikke
Published: June 2021

Renal ischemia/reperfusion (I/R) can lead to acute kidney injury, characterized by increased urine output and fractional excretion of sodium (FE(Na)). Previous studies have shown an improvement of these parameters when applying local ischemic preconditioning (IPC) to ischemic kidneys. Here we investigate the effect of remote ischemic perconditioning (rIPeC) on the renal water and salt handling in ischemic rat kidneys.

Male Wistar rats were subjected to 37 min of unilateral renal ischemia and three days reperfusion. rIPeC was applied to the abdominal aorta. Blood and urine was collected on day 2 and 3 post‐operatively for clearance studies. The expression and localization of renal aquaporins (AQP1, AQP2, phosphorylated S256‐AQP2 and AQP3) and the sodium transports Na‐K‐ATPase and NKCC2 was analyzed using immunoblotting and immunohistochemistry (IHC). Finally, the renal injury was evaluated by morphological examination and detection of renal injury markers.

As previously shown, renal I/R resulted in an increase in urine output and FE(Na) and a decrease in urine osmolality compared to Sham group. rIPeC attenuated all these parameters. The protein level of AQPs, Na‐K‐ATPase and NKCC2 was down regulated in the I/R group compared to Sham, and this reduction was attenuated in the I/R+rIPeC group. IHC showed weak labeling of AQP2 and pAQP2 in collecting ducts in I/R kidneys compared to sham kidneys. rIPeC increased the labeling and showed more apical localization of AQP2 and pAQP2.

In conclusion, data suggests that rIPeC prevents dysregulation of renal water and salt handling by attenuating down regulation of aquaporins and sodium transporters in ischemic rat kidneys.
High Glucose Induces Aerobic Glycolysis and Vascular Endothelial Growth Factor‐C through LPA Signals in Human Prostate Cancer PC‐3 Cells
Authors: Yang Ya‐Jan , Lin Chu‐Cheng , Lu Kuan‐Ying , Chen Wei‐min , Lin Yueh‐Chien , Chang Cheng‐Chi , Lee Hsinyu
Published: June 2021

Prostate cancer is one of the most commonly diagnosed cancers in males and frequently metastasizes to lymph nodes. Clinical evidences suggested that type II diabetes mellitus were closely associated with the prognosis of prostate cancer. Furthermore, hyperglycemia increases aerobic glycolysis and promote cancer growth. However, the effects of hyperglycemia in prostate cancer remain elusive. Lysophosphatidic acid (LPA) is a small lipid growth factor with multiple functions by activating G‐protein coupled LPA receptors. We demonstrated that LPA enhance VEGF‐C expression through activating LPA receptor 1 and 3 in prostate cancer cells. Furthermore, high glucose treatment has also been shown to enhance VEGF‐C expression in human synovial fibroblasts. In this study, we used PC‐3 cell to investigate whether high glucose induces VEGF‐C expression and aerobic glycolysis through LPA signals to enhance cancer progression. Our preliminary results demonstrated that the mRNA expression level of autotaxin (ATX), an enzyme responsible for LPA synthesis, was increased in PC‐3 cell by 0.02 M glucose treatments. Protein levels of VEGF‐C were also increased after high glucose treatments. Furthermore, the enhancement of VEGF‐C was mediated through ROS‐ and LEDGF‐dependent pathways. On the other hand, high glucose treatments also enhanced aerobic glycolysis rate and affected mRNA expression levels of glucose transporter and hexokinase. Taken together, these results suggested that unregulated glucose metabolism might lead to LPA synthesis and therefore subsequent pathological conditions of prostate cancer.
Seasonally Induced Hepatotranscriptomic Changes in the Freeze Tolerant North American Wood Frog Rana sylvatica
Authors: Kiss Andor , Posner Mason , Mock Thomas
Published: June 2021

The North American wood frog Rana sylvatica uses an overwintering strategy that includes metabolic depression and freeze tolerance. Summer‐acclimated wood frogs will not survive freeze events, suggesting that there is a seasonally induced acclimation. Previous hepatoproteome analysis indicated a shift in metabolism in summer vs winter acclimated animals. The current study evaluated the differential expression between seasonally acclimated wood frogs. Using frogs just thawed and four weeks post‐thaw, we isolated hepatic total RNA, verifying integrity by RIN蠅8. Total RNA (3 frogs, each in duplicate) were used to construct Illumina TruSeq RNA v2 libraries. All twelve samples were sequenced (100 bp PE) on an Illumina HiSeq2000. FASTQ files were processed by SICKLE and AlienTrimmer. De novo transcriptome assembly was performed in CLC Genomics Workbench 7.5. The assembled wood frog transcriptome generated 44,023 contigs, which were annotated (BlastX, GO, IPS, EC, KEGG) via Blast2GO. The annotated transcriptome served as the reference for RNA‐Seq based differential gene expression (DGE) using bROC. DGE analysis identified 1090 candidate cDNAs exhibiting significant (CONF >0.95) expression differences between just thawed and four week post‐thawed frogs. We are currently sequencing the wood frog genome using the 'moleculo' approach (Illumina) to aid in identification of novel DGE cDNAs. Pathways analysis, in addition to comparisons to other freeze‐tolerant animals, are being analysed for similarity of differential gene expression during seasonal acclimation.
Role of the locus coeruleus in susceptibility to social stress‐induced cardiovascular dysfunction
Authors: Lombard Calliandra , Sanner Casey , Wilson L Britt , Wood Susan
Published: June 2021

Psychosocial stress precipitates psychiatric disorders and cardiovascular disease in vulnerable individuals. The locus coeruleus (LC), a major source of norepinephrine in the brain is implicated in depressive disorders and indirectly increases sympathetic activity. We previously identified two distinct phenotypic responses to social defeat (SD) in rats, characterized by either passive coping (PC, submissive postures) or active coping (AC, upright postures, resistance to defeat). PC was associated with endocrine and behavioral adaptations resembling depression and enhanced inflammation and decreased heart rate variability indicating greater cardiovascular disease risk. Using cardiac telemetry the present study investigated the impact of DSP4 (400ug/rat, icv), a noradrenergic neurotoxin that selectively lesions the LC, compared with vehicle on the cardiovascular response to SD or control manipulations (30 min/day, 7 days) and persistent changes in resting cardiovascular parameters in PC and AC rats. Blood pressure and heart rate significantly increased in AC and PC rats during SD vs controls (day 1). This cardiovascular response to SD was reduced by day 7 in the AC rats, but not PC rats. Interestingly, DSP4 treatment decreased the SD‐induced pressor response in the PC rats. Furthermore, 24 hr telemetry indicated a significant pressor response in PC vs AC rats that persisted for at least 5 days after the 7th and final defeat. Chronic elevations in blood pressure evident in PC rats were also blocked by DSP4 treatment. These data implicate a role for the LC in the enhanced cardiovascular susceptibility evident following social stress in passive coping individuals. Supported by 13BGIA14370026
Sweat‐Induced Skin Wetness Perception Can Be Significantly Manipulated Independently of the Level of Physical Skin Wetness
Authors: Filingeri Davide , Fournet Damien , Hodder Simon , Havenith George
Published: June 2021

Skin wetness perception is driven by inputs from cold and mechano sensitive skin afferents. We hypothesized that in the absence of skin cooling, sweat induced wetness perception can be manipulated by altering the mechanical interaction between skin, sweat and clothing. Ten males (22±2years) performed an incremental walking protocol (5Km/h; gradient: 2 to 16%) during two trials designed to produce the same level of physical skin wetness but to induce lower (TIGHT‐FIT) and higher (LOOSE‐FIT) wetness perception. In the TIGHT‐FIT, a tight fitting clothing ensemble was worn to reduce the mechanical interaction between skin, sweat and clothing. In the LOOSE‐FIT, a loose fitting ensemble augmented this interaction. To limit sweat evaporation and skin cooling, a vapour impermeable ensemble was also worn during the trials. Heart rate, rectal temperature, mean skin temperature, skin conductance (SC), whole body skin wetness (wbody) and wetness perception were recorded. Exercise induced sweat production and physical skin wetness increased significantly (SC: 3.1±0.3 to 18.8±1.3µS, p<0.01; wbody: 0.26±0.01 to 0.95±0.2nd, p<0.01) with no differences between TIGHT‐FIT and LOOSE‐FIT (p>0.05). However, the reduced mechanical interaction generated by the TIGHT‐FIT ensemble lowered significantly wetness perception (p<0.01). This reduction was more pronounced when wbody ranged between 0.4‐0.8nd. We conclude that in the absence of skin cooling, sweat induced wetness perception is primarily driven by the mechanical interaction between skin, sweat and clothing. Manipulating this interaction can alter skin wetness perception, independently of the level of physical wetness.
Genetic Analysis of Replication Protein A Large Subunit Family in Arabidopsis Reveals its Role in the DNA Damage Response Pathway
Authors: Fernandes Nancy , Aklilu Behailu , Culligan Kevin
Published: June 2021

Replication Protein A (RPA) is a heterotrimeric single stranded binding protein required for eukaryotic DNA replication, repair and recombination. The primary biochemical function of RPA is to protect and preserve single stranded DNA from nucleolytic degradation and hairpin formation. Downstream cellular responses to detect DNA damage include regulation of cell‐cycle checkpoints, induction of DNA repair and programmed cell death. These responses are mediated through two closely related protein kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia Telangiectasia‐mutated and Rad3‐Related). ATM is activated by double strand breaks while ATR is activated by a variety of lesions resulting in stalled replication forks. Genetic analysis of RPA1, by employing T‐DNA insertion mutants of the five Arabidopsis RPA1 subunit genes, revealed divergence of the RPA1 gene family into three functionally distinct groups. RPA1B and RPA1D are required for normal DNA replication, RPA1A and RPA1C play a role in meiosis, and RPA1C and RPA1E are primarily involved in DNA repair. Our analysis shows that within the repair group individual RPA1 subunits display unique functions in response to DNA damage. Analysis of hypersensitivity responses to replication blocking agents and DNA damaging agents suggests that RPA subunits RPA1C and RPA1E play leading roles in DNA damage repair. Genetic interactions of RPA1C, RPA1E and ATR revealed that a combination of rpa1c, rpa1e and atr mutants create additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways.
The Effect of High Hydrostatic Pressure on α‐Synuclein Fibrils Probed by NMR
Authors: Silva Jerson , Freitas Monica , Foguel Debora , Cruzeiro‐Silva Carolina
Published: June 2021

The human α‐synuclein (α‐syn) is a natively unfolded protein composed of 140 amino acids. Its fibrilization is a hallmark of Parkinson's disease and other α‐synucleinopathies and it is associated to cell toxicity and tissue degeneration. The lack of structural information concerning fibril formation makes it difficult to find new treatments for the associated diseases. We had previously found that a‐syn fibrils are dissociated by pressures (Foguel et al. (2003) Proc Natl Acad Sci U S A. 100:9831‐6). Pressure‐dissociated a‐syn undergoes fibrillogenesis with typical slow kinetics, demonstrating the reversible character of the pressure effects. We have undertaken a new approach based on high hydrostatic pressure NMR to study the disassembly of fibrils into oligomeric and monomeric species. NMR spectra were acquired on 800 MHz 1H frequency (Bruker Avance III) at 15oC and different pressures.At 1 bar no signal was observed as expected for a sample composed of fibrils. However, it was possible to observe some signals coming up at 500 bar. At pressure of 2250 bar, the spectrum was very similar to that obtained for intrinsically disordered monomers of α‐synuclein. We also characterized the species that appeared on decompression. The intermediate species were structurally characterized. Supported by FAPERJ, CNPq, and CAPES.
n‐6 Fatty Acids Instead of n‐3 and Saturated Fatty Acids Correlate with Body Fat Distribution From Subcutaneous Adipose Tissue to Visceral Adipose Tissue in Healthy and Overweight Subjects
Authors: Jiao Jingjing , Zhu Shankuan
Published: June 2021

Objective

Our aim was to investigate the association between the plasma phospholipid (PPL) fatty acid profile and fat distribution in both healthy and overweight subjects.

Methods

Totally 108 of 1066 Chinese adults stratified by gender and body mass index (BMI) were enrolled. The subjects received lifestyle questionnaire survey, physical examination and magnetic resonance imaging (MRI) scan. The PPL fatty acid profile was determined by gas chromatography. The fat distribution including subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), intramuscular adipose tissue (IMAT) and skeletal muscle (SM) was measured by MRI.

Results

PPL n‐3 PUFA levels were inversely associated with SAT in healthy but not overweight subjects. However, no correlation between PPL n‐3 PUFA levels and VAT was observed. PPL n‐6 PUFA levels were directly correlated with SAT and VAT in healthy subjects, while PPL SFA levels were positively associated with SAT and VAT in overweight subjects. Multiple regression analysis revealed PPL n‐6 PUFA were positively correlated with SAT instead of VAT in healthy subjects, but positively associated with VAT instead of SAT in overweight subjects after adjustment for age, sex, BMI, smoking, drinking and education level. PPL n‐3 PUFA did not significantly correlate with both SAT and VAT after adjustments. Logistic regression results showed that PPL n‐6 PUFA levels could be used to estimate the obese state.

Conclusions

PPL phospholipid n‐6 PUFA instead of n‐3 PUFA and SFA as important predictors positively correlate with fat distribution from SAT to VAT. Consequently, PPL n‐6 PUFA levels could be used to estimate the obese state.
Providing Insight Into Complex Disease: Metabolomics Links Genetic Loci To Phenotype
Authors: Mohney Robert , Milburn Michael
Published: June 2021

Metabolomics has emerged as a powerful technology for providing biological insight into disease processes and for identifying biomarkers to diagnose, monitor, and treat disease, in part, because metabolomics provides a highly‐sensitive and real‐time assessment of health status. The utility of metabolomics is further expanded when combined with other 'omics‐based approaches. Integration of metabolomics and GWAS approaches has provided unprecedented insight into how genetic variation influences human metabolism, complex disease, and drug response. This combined GWAS/metabolomics approach has resulted in the generation of a network atlas or metabolic network, including a large number of novel associations, which provides a link between genes and disease phenotype. These results illustrate how metabolomics provides a powerful tool for understanding the molecular events that contribute to disease and help provide the groundwork for personalized medicine approaches that will enable a better understanding of the health status of the individual and their risk for disease.
Discovery of novel Pneumococcal surface adhesin A (PsaA) inhibitors using fragment‐based drug design
Authors: Bajaj Megha , Mamidyala Sreeman , Zuegg Johannes , Begg Stephanie , Ween Miranda , Huang Johnny , Kobe Bostjan , Paton James , McDevitt Christopher , Cooper Matthew
Published: June 2021

Streptococcus pneumoniae is a leading cause of pneumococcal infections, especially in young children in developing countries. Treatment relies heavily on β‐lactam antibiotics, however, rapid emergence of multidrug‐resistant strains of S. pneumoniae over the past two decades has shifted the focus towards identification of novel drug targets. Pneumococcal surface adhesin A (PsaA) is one such drug target, found on the cell surface of S. pneumoniae. It functions as surface adhesin, as well as substrate binding protein, facilitating acquisition of Mn2+, an essential cation that plays an important role in protecting S. pneumoniae from reactive oxygen species produced during oxidative stress. PsaA is essential for bacterial survival and is an important virulence factor, rendering it a promising target for antibiotic development.

To design novel PsaA inhibitors, we used a combination of de novo fragment‐based drug discovery (FBDD) and in silico virtual screening methods. We profiled a collection of low molecular weight compounds that were selected based on their structural diversity and ability to bind to apo‐PsaA in a virtual docking experiment. The screening resulted in two initial hits that were further optimized by structural variation to improve their potency while maintaining their ligand‐efficiency and physicochemical properties. The optimized hits were validated using cell‐based assay and molecular dynamics simulations. We found that virtual screening substantially augmented FBDD approaches, leading to the identification of novel PsaA inhibitors.
Viability of the Bilayer Bacterial Cellulose Membrane as a Biological Support for use in Tissue Engineering and Regenerative Medicine
Authors: Sartori Kamila , Lararini Silmara , Carvalho Renata , Barud Hernane , Lustri Wilton , Amaral André
Published: June 2021

Tissue engineering is an interdisciplinary scientific field that seeks to restore or improve the biological functions of damaged tissue or unable to perform its function. Some biomaterials are used as three‐dimensional structures, which act as biological media, enabling and stimulating cell proliferation and differentiation and consequent induction of tissue reconstruction. The bacterial cellulose membrane (BCm) bilayer represents a promising biomaterial for use as a biological support. However, there are no reports on their permissiveness for membership and maintenance of cellular homeostasis. This research aimed to evaluate the potential of BCm bilayer to the development and maintenance of cell viability. For this, BCm and mono bilayer were cultured for 4 days in 24‐well plates and processed cultured with osteoblast lineage (OSTEO‐I). The morphological aspects of adhesion and spreading of cells, assessed by microscopic analysis, have been established for 10 days after sowing. The results demonstrated the feasibility of BCm monolayer for membership, spreading and expanding the cell quota, causing the gradual coating the surface of the membranes. However, the BCm bilayer present in both layers, a substantially lower viability, suggesting a limited capacity for use as a biological support cellularized. Although it has promising structural features, the BCm bilayer appears to provide fewer favorable conditions for membership and maintenance of cellular homeostasis.
Effect of Acidic Tumor Microenvironment on Non‐Small‐Cell Lung Cancer Cells
Authors: Arikatla Swetha , Livesey John
Published: June 2021

In order to adapt to hypoxic stress, cancer cells switch from oxidative phosphorylation to aerobic glycolysis resulting in the metabolism of glucose to lactic acid. Increased lactic acid production and lack of removal of metabolic acids due to inefficient vasculature results in an acidic environment in the tumor parenchyma. Little is known about the effect of an acidic tumor environment on the motility of non‐ small cell lung cancer (NSCLC) cells. Our research focuses on the effect of acidic tumor microenvironment on motility and epithelial to mesenchymal transition (EMT) of NSCLC cell lines. NCI‐H1650 cells propagated in acidic pH media (pH ~ 6.5 to 6.7) showed a significant alteration in phenotype compared to the cells propagated in neutral pH media (pH ~ 7.2 to 7.4). Preliminary data on cell proliferation indicates that the acidic environment significantly decreased cell viability. No difference in motility was observed between H1650 cells propagated in acidic or neutral pH media. Expression of E‐ cadherin, an epithelial marker, was not affected by an acidic environment. Expression of vimentin, a mesenchymal marker, was observed at the mRNA level but no vimentin protein was observed, indicating altered translation, stability, or post translational modification of the protein. Examination of other EMT markers is in progress. These results indicate that unlike observations in AT‐1 prostate carcinoma cells [Pflugers Arch 466 (2014) 2127], changes in lung cancer cells by accumulation of acid in the tumor microenvironment results in modest promotion of metastatic potential.
[FeFe] Hydrogenase in Artificial Photosynthesis
Authors: Garb Jodie , Henke Noah , Wood Alex , Zibolsky Carissa , Opheim Allison , Garb Ashley , Huang Jier
Published: June 2021

The development of clean and renewable energy is critical to partially address the energy crisis and climate issues. Inspired by nature, artificial photosynthesis through water splitting by solar energy conversion is the most attractive approach for the development. The overall water splitting includes two half‐catalytic reactions, i. e. hydrogen (HER) and oxygen (OER) evolution reactions. An efficient catalyst is required to perform each of these catalytic reactions. Molecular catalysts that mimic the function of [FeFe] hydrogenase are among the most effective synthetic transition metal complexes known for HER. The Laconia SMART (Students Modeling A Research Topic) Team used 3D printing technology to model the active site of [FeFe] hydrogenase and understand its catalytic function for HER. [FeFe] hydrogenase is an enzyme that catalyzes proton reduction to bind hydrogen together. Arg265, Lys288, and Lys409 are positively charged residues that line the channel entrance. Lys 188 is at the end of the channel and may help to orient the 2Fe subcluster during hydrogen insertion. The fundamental understanding of the catalytic function of the [FeFe] hydrogenase active site in HER will provide insight into the rational design of efficient catalysts for solar fuel generation. The SMART Team program is supported by a grant from NIH‐CTSA.
Exercise Response in a New Porcine Model of Chronic Embolic Pulmonary Hypertension
Authors: Stam Kelly , de Wijs‐ Meijler Daphne , Verzijl Annemarie , van Duin Richard , Reiss Irwin , Duncker Dirk , Merkus Daphne
Published: June 2021

Chronic Thromboembolic Pulmonary hypertension (CTEPH) is a deadly disease initiated by thrombo‐embolisms persisting in the pulmonary vasculature, followed by remodelling of the pulmonary vascular bed, resulting in a sustained increase in afterload of the right ventricle (RV). Although the response of the pulmonary vasculature to the initiating thromboembolic events as well as the response of the RV to the increased afterload are known to be the most important determinants of patient outcome, the factors that underlie these processes are incompletely understood. In the present study, we developed a novel chronically instrumented swine model of CTEPH. CTEPH was induced by up to 5 weekly injections of polyethylene beads of 700 μm in combination with induction of endothelial dysfunction by eNOS‐inhibition. Swine were followed up to one month after the last embolization. Changes in mean pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) were used as indices of RV afterload and pulmonary vascular remodeling. and assessed at rest and during treadmill exercise (EX) .

Repeated embolization resulted in increases in PAP and PVR.

Control

After first embolization

Prior to sacrifice

rest
EX
rest
Ex
Rest
Ex

PAP (mmHg)
16±1
33±3*
29±3†
61±7*†
47±15†
72±16*†

PVR (mmHg.min.kg.L‐1)
86±13
98±16
160±42†
197±23†
510±237†
633±344†

* P<0.05 vs Rest,
† P< 0.05 vs Control

The increase in PAP was exaggerated during exercise, and in two animals, limited the increase in cardiac output during exercise. The increase in PVR induced by repeated embolization correlated well with RV weight (r2=0.78), indicating that swine developed RV hypertrophy. Moreover, histology revealed increased muscularization of pulmonary arterioles.

In conclusion, our new swine model of CTEPH mimics critical features of patients with CTEPH in that pulmonary vascular remodeling is present and exercise capacity is reduced.

Supported by CVON‐ PHAEDRA2012‐08
Recycling of The Kidney Anion Exchanger 1 Is Regulated by Adaptor Protein Complex 1B
Authors: Cordat Emmanuelle , Almomani Ensaf , Jung Martin , Zimmermann Richard , Alexander R.
Published: June 2021

Adaptor protein complexes 1 (AP‐1) regulate polarized trafficking of membrane proteins. Here, we investigated the role of AP‐1B in regulating the kidney anion exchanger 1 (kAE1) intracellular trafficking. kAE1 physically interacts with AP‐1B in Madin‐Darby canine kidney cells. We hypothesized that AP‐1B regulates the recycling of endocytosed kAE1 protein and that kAE1 mutants that cause distal renal tubular acidosis, fail to properly interact with AP‐1B. We studied kAE1 WT and a dRTA mutant, kAE1 R901X, which lacks the last 11 amino acids encompassing the potential canonical binding site for AP‐1B. We confirmed the physical interaction between kAE1 and heterologously expressed μ1B subunit of AP‐1B in LLC‐PK1 renal epithelial cells lacking endogenous AP‐1B. A peptide spot assay identified multiple interaction sites of AP‐1B in the kAE1 protein sequence, including residues within the second and sixth cytosolic loops, and the C‐terminus. μ1B heterologous expression decreased the amount of plasma membrane kAE1. Although kAE1 R901X mutant underwent endocytosis slightly faster than kAE1 WT, its recycling rate was decreased compared to kAE1 WT, and was independent from μ1B expression. We also observed that (i) μ1B expression disrupts the interaction of GAPDH with kAE1 WT consistent with both proteins binding to an overlapping site on kAE1, and (ii) phosphorylation of tyrosine 904 within the carboxyl‐terminal interaction motif does not affect the kAE1/AP‐1B interaction. We propose that the apically mis‐targeted kAE1 R901X mutant fails to recycle back to the basolateral membrane due to its inability to properly interact with AP‐1B.
Analysis of RVLM Gene Expression in Young, Middle‐Aged and Aged F344 Rats
Authors: Ganta Chanran , Pawar Hitesh , Garcia Anthony , Lu Nanyan , Becker Kevin , Kenney Michael
Published: June 2021

Abstract: The rostral ventral lateral medulla (RVLM) plays a critical role in regulation of basal sympathetic nerve discharge (SND). Advanced age is associated with altered regulation of SND, however, the effect of age on central medullary mechanisms regulating SND remains unknown. A series of experiments were initiated to identify cellular and molecular targets that may mediate age‐related changes in medullary regulation of SND. We tested the hypothesis that aging shifts the balance of RVLM excitatory and inhibitory influences towards excitation. Gene expression analysis was performed using Agilent Whole Rat Genome Microarray 4×44K followed by validation of microarray with TaqMan real‐time RT‐PCR and immunoblotting for specific RVLM proteins. Total RNA and protein were extracted from RVLM micropunches from young, middle‐aged and aged F344 rats. Microarray analysis revealed up‐regulation in aged compared to young and middle‐aged rats of several genes related to immune function and neurotransmitter receptors. Specifically, the chemokines CCL3, CCL4 and CCL5 were up‐regulated 3 to 4 fold in aged rats compared to young and middle‐aged rats. The results of previous studies suggest that increased chemokines in the brain can modulate NMDA receptor responses. The current preliminary results suggest that increased chemokine gene expression in the RVLM of aged rats may contribute to age‐related changes in SND regulation. Supported by NIH A6‐041948.
Increasing Spatiotemporal Control over Azidonorleucine Protein Labeling within Cells to Enable Higher Resolution Proteomics
Authors: Thomas Emily , Pandey Naresh , Silberg Jonathan
Published: June 2021

As many signaling pathways are interconnected in complex ways, it is difficult to predict the protein production changes accompanying most changes of cellular state, such as during development or disease onset. One method of resolving this issue is to use a modified methionyl tRNA synthetase (MetRS) for bioorthogonal noncanonical amino acid tagging of newly synthesized protein with azidonorleucine (Anl). Use of noncanonical amino acids with azide groups (e.g., Anl) are particularly useful because azides are uncommon within cells, thus allowing for selective “click” chemistry for imaging and sequencing. Anl labeling can be accomplished using E. coli MetRS (EcMetRS) harboring NLL (L13N/Y260L/H301L) active site mutations that direct substrate specificity from Met to Anl. Using a conditional promoter, EcMetRS activity can be spatiotemporally controlled. To expand spatiotemporal control, we built split two‐fragment EcMetRS libraries with various protein‐protein interactions and linkers with or without Tobacco Etch Virus (TEV) specific protease cleavage sites. The libraries were subject to selection and revealed diverse functional split sites across the protein sequence. Some of these split variants showed dependence on the protein‐protein interaction and on presence/absence of TEV. Overall, these findings suggest that incorporation of the NLL mutations in these new EcMetRS variants will allow for novel regulation of metabolic labeling for proteomics through a combination of transcriptional and post‐translational control. This work was funded by the Houston Area Molecular Biophysics Training Program, the Howard Hughes Medical Institute Med into Grad Program, and the Welch Foundation.
Isolation and Preliminary Characterization of Mitochondrial Spherules Involved in Flock House Virus RNA Replication
Authors: Short James , Gopal Radhika , Speir Jeff , Schneemann Anette
Published: June 2021

The replication of genomic RNA by positive‐sense RNA viruses occurs in association with host membranes. Flock House virus (FHV) is an ideal system for studying these unique interactions since much of the molecular basis for the replication of its RNA is already understood. Protein A, the viral RNA‐dependent RNA polymerase protein, is targeted to the outer mitochondrial membrane (OMM) where it is responsible for viral RNA synthesis. OMM invaginations (or spherules) containing Protein A are formed and are known to be the site of viral RNA replication. Kopek and colleagues previously generated a three‐dimensional reconstruction of these spherules using EM tomography of thin‐sectioned FHV‐infected Drosophila cells. However, these structures have not been studied in the absence of chemical fixation and staining, nor have optimal RNA replication conditions been established for the wild‐type protein in the presence of host membranes.

As part of a larger study directed at understanding the structural basis of spherule formation, we aimed to develop techniques for the robust production of replication‐induced OMM invaginations in isolated mitochondrial membranes for imaging by cryo‐electron microscopy (EM). We are now able to consistently isolate replication‐competent mitochondrial fragments and have optimized several reaction conditions enabling the imaging of spherules by cryo‐EM. Our results reveal a number of important initial insights, including the transient nature of the spherules as well as the possibility of visualizing replicated viral RNA.