bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of macromolecular crowding on the enzyme kinetics and thermodynamics of glutamate dehydrogenase with the substrate norvaline
Authors: Rundlett Emily M. , Slade Kristin M.
Published: May 2022

All living cells are packed with high concentrations of sugars, proteins, and other metabolites. These macromolecules are more likely to interact with enzymes in a crowded cellular environment, thereby influencing the kinetics and thermodynamics of catalysis. In the past, enzyme studies have been conducted under dilute conditions, which is not representative of the environment in living organisms and therefore does not account for the influence of macromolecular crowding. More recently, dextran polymers, which are as large as macromolecules have been used to mimic the crowded cellular environment. These large molecules exclude volume from other molecules thereby influencing the reaction rate in an unpredictable manner. It is believed that that the effects of crowding depend on the rate limiting step (RLS) of enzyme catalysis, since crowding slows diffusion and impedes conformation changes, but also enhances enzyme‐substrate binding and increases effective substrate concentration. To test this hypothesis, the effects of crowding on the kinetics of the enzyme glutamate dehydrogenase GDH kinetics were compared for natural substrate glutamate and an alternative substrate norvaline. The RLS of this enzyme‐catalyzed reaction with glutamate is the release of the cofactor, NADH, while the slow step with norvaline is the chemistry of catalysis. To more realistically model intracellular conditions, this GDH assay was exposed to bovine serum albumen and partially purified cell lysate, in addition to using the traditional crowding agent, dextran. The rate of the reaction was monitored by absorbance using a spectrophotometer. Repeating the assay at varying temperatures yielded an Eyring plot with thermodynamic data that allowed for the separation of the enthalpy and entropic effects of crowding. Results suggest that excluded volume has a more significant effect than chemical interaction on enzyme kinetics.
A New Family of Subnanomolar inhibitors of Soluble Epoxide Hydrolase
Authors: Escolano Carmen , Bagan Andrea , Turcu Andreea L. , Morisseau Christophe , Loza M. I. , Brea Jose , Bartra Clara , Sanfeliu Coral , Hammock Bruce D. , Vázquez Santiago
Published: May 2022

The pharmacological inhibition of soluble epoxide hydrolase (sEH) has been suggested as a potential therapy for the treatment of pain and inflammatory diseases through the stabilization of epoxyeicosatrienoic acids, endogenous chemical mediators derived from arachidonic acid that show anti‐inflammatory and analgesic effects.1

Although several potent sEH inhibitors (sEHI) have been developed, including clinical candidates AR9281, GSK2256294, and EC5026, so far no sEHI has reached the market.2

Recently, a new series of benzohomoadamantane‐based ureas endowed with potent inhibitory activity for the human and murine sEH was reported. However, their very low microsomal stability prevented further development.3

Herein, novel series of benzohomoadamantane‐based ureas were synthesized, fully characterized, and evaluated as sEHI. Most of them were endowed with subnanomolar inhibitory potencies at the human and murine enzymes. Further in vitro profiling (solubility, cytotoxicity, metabolic stability, CYP450s, hLOX‐5, hCOX‐2, hERG inhibition, permeability) allowed us to select a candidate for efficacy studies.

In summary, these novel results and the previously reported studies using other families of sEHI, strongly suggest that sEH may be a target of clinical interest for the treatment of inflammatory and pain‐related disorders.4

References:

1‐Morisseau, C.; Hammock, B. D. Annu.Rev. Pharmacol. Toxicol. 2013, 53, 37–58.

2‐Sun, C.‐P.; Zhang, X.‐Y.; Morisseau, C.; Hwang, S. H.; Zhang, Z.‐J.; Hammock, B. D.; Ma, X.‐C. J. Med. Chem. 2021, 64, 184–215.

3‐Codony, S.; Calvó‐Tusell, C.; Valverde, E.; Osuna, S.; Morisseau, C.; Loza, M. I.; Brea, J.; Pérez, C.; Rodríguez‐Franco, M. I.; Pizarro‐Delgado, J.; Corpas, R.; Griñán‐Ferré, C.; Pallàs, M.; Sanfeliu, C.; Vázquez‐Carrera, M.; Hammock, B. D.; Feixas, F.; Vázquez, S. J. Med. Chem., 2021, 64, 5429–5446.

4‐McReynolds, C.; Morisseau, C.; Wagner, K.; Hammock, B. D. Adv. Exp. Med. Biol. 2020, 1274, 71–99.
A Myosin Vb Splice Variant Regulates Coronavirus M Protein Trafficking in Polarized Epithelial Cells
Authors: Goldenring James R. , Lapierre Lynne A. , Roland Joseph T. , Manning Elizabeth , Caldwell Cathy , Glenn Honor L. , Vidalain Pierre‐Olivier , Tangy Frederic , Hogue Brenda G. , de Haan C.A.M.
Published: May 2022

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two‐hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV‐CoV) M protein with Myosin Vb (MYO5B) (Figure 1A). MHV‐CoV M interacts specifically with the alternative splice variant of cellular MYO5B including Exon D (MYO5B+D), that also mediates interaction with cellular Rab10. When co‐expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co‐localized with MHV‐CoV M protein, as well as with M proteins from porcine epidemic diarrhea Virus (PEDV‐CoV), Middle East Respiratory Syndrome (MERS‐CoV) and Severe Acute Respiratory Syndrome 2 (SARS‐CoV‐2) (Figure 2). M‐GFP chimeric proteins co‐expressed with mCherry‐MYO5B+D also co‐localized with endogenous Rab10 and Rab11a (Figure 1B,C). We identified point mutations in MHV‐CoV M that blocked the interaction with MYO5B+D in yeast 2‐hybrid assays (Figure 1B). One of these point mutations (E121K) was previously shown to block MHV‐CoV virion assembly, and it blocked interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV‐CoV, MERS‐CoV and SARS‐CoV‐2 M proteins also blocked colocalization with MYO5B+D. Knockdown of Rab10 blocked the co‐localization of the M proteins with MYO5B+D (Figure 2). Re‐expression of Cerulein‐Rab10 in Rab10 KD cells re‐established colocalization between M proteins and MYO5B+D (Figure 2). Our results suggest that the interaction of CoV M proteins with MYO5B+D may play a role in regulating their trafficking through Rab10‐containing membrane systems in epithelial cells.
Protective effects of Empagliflozin against Transforming growth factor‐beta‐induced fibrosis in renal proximal tubular cells
Authors: Bayne Sarah , Munusamy Shankar
Published: May 2022

Background

Chronic kidney disease (CKD) is the condition where the kidneys are unable to filter and clean the blood of waste properly. Hypertension, obesity, and diabetes are the leading risk factors for developing CKD. Fibrosis is regarded as a final common pathway leading to CKD. Sodium‐glucose co‐transporter 2 (SGLT‐2) inhibitors have shown to be renoprotective in type 2 diabetes patients. However, the mechanisms that underlie the renoprotective effects of SGLT‐2 inhibitors are unclear. Our study investigated whether empagliflozin (an SGLT‐2 inhibitor) treatment inhibits transforming growth factor β (TGF‐β)‐mediated fibrosis and profibrotic signaling in rat renal proximal tubular cells (NRK‐52E).

Methods

NRK‐52E cells were cultured and treated with TGF‐β with or without empagliflozin for 24 and 48 hours. The expression of fibrosis markers ‐ alpha‐smooth muscle actin (α‐SMA) and vimentin, autophagy marker – LC3II, and TGF‐b‐mediated profibrotic signaling marker ‐ SMAD 2 – were measured by western blotting.

Results

TGF‐β treatment (at 5 ng/mL and 10 ng/mL) for 24 and 48 hours induced fibrosis and activated autophagy signaling in renal proximal tubular cells. For instance, 48 h TGF‐β treatment at 10 ng/mL induced the expression of α‐SMA by 1.43‐fold and LC3II by 2.6‐fold as compared to vehicle‐treated cells. Co‐treatment with empagliflozin (100 and 500 nM) reduced the expression of the fibrotic markers (α‐SMA, vimentin), autophagy marker (LC3II) and TGF‐β‐mediated phosphorylation of SMAD‐2 in renal proximal tubular cells.

Conclusions

Our preliminary findings indicate that TGF‐β‐mediated induction of fibrosis, autophagy, and profibrotic signaling could be attenuated by empagliflozin treatment. Further studies are required to understand the molecular mechanisms that underpin the antifibrotic effects of empagliflozin against TGF‐β‐induced renal fibrosis.
WWOX regulates UV/cold shock‐mediated calcium influx and nuclear bubbling in frostbite
Authors: Chang Nan‐Shan , Tsai Cheng‐Chang
Published: May 2022

UV/cold shock‐mediated frostbite involves non‐apoptotic nuclear bubbling cell death (BCD) and participation of functional WWOX in cells (WWOXf). In contrast, cells with WWOX deficiency or dysfunction (WWOXd) undergo pop‐out explosion death (POD). Here, by time‐lapse microscopy, when WWOXf cells were exposed to UV or UV/cold shock and then incubated at room temperature, these cells rapidly and sequentially underwent: 1) loss of mitochondrial membrane potential, 2) formation of a nitric oxide (NO)‐containing nuclear bubble per cell, 3) WWOX‐dependent increase in calcium (Ca2+) influx, 4) shutdown of mRNA and protein synthesis machinery, as determined by RT/PCR and gene chip analysis, and 5) eventual cell death without caspase activation, stress fiber formation and chromosomal DNA fragmentation. In contrast, WWOXd cells exhibited a faster kinetics of stress fiber formation, explosion and death without NO production. Ectopic WWOX restored calcium influx and nuclear bubbling in WWOXd cells. In hairless mice, UV/cold shock rapidly downregulated protein expression in the skin and then liver, which may lead to organ damages. UV/cold shock induced complex formation of antiapoptotic TRAF2 and proapoptotic WWOX and their co‐translocation to the nucleus, where the complex dissociation occurred. The observations suggest that WWOX and TRAF2 dissociation is needed for nuclear bubbling and death.
Is Estrogen a Friend or Foe of the Human Microcirculation Throughout the Lifespan?
Authors: SenthilKumar Gopika , Katunaric Boran , Bordas‐Murphy Henry , Young Micaela , Buiter Nicole J. , Freed Julie K.
Published: May 2022

Cardiovascular disease (CVD) risk increases with estrogen (E2) deficiency as well as during the transition to menopause, suggesting that estrogen is protective against CVD. On the contrary, increases in plasma E2 from contraception in cis‐females or hormone therapy (HT) in trans‐females (male‐to‐female transition) is also associated with increased CVD. Microvascular endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation in response to flow (flow‐induced dilation; FID), precedes large artery disease and is predictive of future cardiac events. We hypothesize that the effect of E2 on human microvascular endothelial function is hormetic with low or elevated E2 promoting dysfunction. We further posit that the effect of estrogen on the microvascular endothelium is influenced by both age and sex. Small adipose arterioles (100‐350µm) from healthy adults (0‐1 risk factors for coronary artery disease) were prepared for videomicroscopy and constricted with endothelin‐1 prior to measuring changes in internal diameters in response to flow. Historical data was analyzed to examine changes in FID over time in healthy women, and it showed that FID is maintained over time (75.4%±4.2 of maximal dilator capacity±SEM, n=26 age<40yrs; 75.39%±3.0 age>40yrs). L‐NAME significantly inhibited FID in healthy women <40yrs of age (30.6%±9.9 n=17; p<0.0005, two‐way ANOVA*) compared to control (75.4%±4.2, n=26), however this effect was abolished when incubated with E2 (100nM, 16‐20hrs) (71.2%±10.3, n=5 vs. control 84.7%±3.8, n=5). L‐NAME also decreased FID in arterioles from women 40‐60yrs of age (46.9%±9.8, n=19) compared to control (81.3%±2.6, n=37, p<0.005), however there was a greater amount of residual dilation compared to women <40yrs. E2 also resulted in a loss of NO‐mediated FID in women >40yrs as L‐NAME had no effect (82.9%±4.3, n=4 vs. control 72.6%±8.8, n=5). In biological males, FID appears to be reduced in microvessels after E2 treatment regardless of age (52.1%±5.1 n=2, age<40yrs; ‐2.7%±9.5, n=3, age>40yrs). Together these data suggest that exposure to elevated levels of E2 may induce microvascular endothelial dysfunction in adult women regardless of age and appears to be more damaging in biological males. We conclude that elevated levels of E2 are detrimental to the microvasculature and may increase CVD risk in young women taking oral contraceptives as well as older women and trans‐females undergoing HT.
Epigallocatechin 3‐Gallate Inhibitory Effects on Inflammatory Mediators in BV‐2 Microglial Cells
Authors: Payne Ashley , Taka Equar , Adinew Getinet , Soliman Karam F. A.
Published: May 2022

Chronic neuroinflammation has been associated with many neurodegenerative diseases such as Alzheimer's and Parkinson's Disease. The chronic activation of microglia can cause neuronal damage by releasing proinflammatory cytokines and reactive oxygen mediators. Epigallocatechin‐3‐Gallate (EGCG) is a bioactive polyphenol that exhibits significant anti‐inflammatory and neuroprotective properties in green tea extract. The current study aims to examine how EGCG can prevent chronic inflammation by inhibiting proinflammatory responses and oxidative stress induction. If proven to be the case, EGCG may serve as a nutraceutical for preventing neurodegenerative disorders. In this study, we evaluated the anti‐inflammatory activity of EGCG in lipopolysaccharide (LPS) stimulated BV‐2 microglia cells. BV‐2 microglial cells were first stimulated with 1µg/mL LPS for 1hr; then incubated for 24 hrs using different concentrations of EGCG. Cell viability was assessed using resazurin (Alamar™ Blue) indicator dye. In addition, inflammatory cytokines were profiled using QIAGEN mouse inflammatory cytokines multi‐analyte ELISA array, and nitric oxide (NO) release was evaluated using the Griess Reagent Assay. Our results showed that EGCG caused a concentration‐dependent decrease in cell viability of LPS stimulated BV‐2 microglia cells for EGCG concentrations higher than 150 µM. EGCG also significantly inhibited the release of the proinflammatory cytokine IL‐6 and the inflammatory mediator NO production at 150 µM, and 175µM in LPS stimulated BV‐2 microglial cells. In conclusion, EGCG has decreased IL‐6 and NO production, but further research will be performed to elucidate the anti‐inflammatory and antioxidative attributes of EGCG on inflammation‐induced neurodegenerative diseases.
Sexually dimorphic metabolic effects of a naturally occurring flavonoid are mediated by changes in the gut microbiome
Authors: Sharma Priyanka , Silva Camila , Ye Hong , Sampath Harini
Published: May 2022

It is increasingly understood that dietary flavonoids modulate the gut microbiome which influences host metabolism. However, sex‐dependent effects of flavonoids on the gut microbiome composition and metabolic health have not been studied. In this regard, we have previously shown that an oral flavonoid 7,8‐Dihydroxyflavone (DHF), exerts sexually‐dimorphic effects on body weight and metabolic health. Specifically, oral DHF supplementation prevents HFD‐induced obesity and inflammation in female but not male mice. Interestingly, this metabolic protection was associated with early and stable changes in the composition of the female, but not male, gut microbiome. The remodeled female microbiome contributed to increased formation of the short chain fatty acid acetate, while male mice did not display a similar effect. While a causal association between the gut microbiome and metabolic protection in female mice was not previously studied, we now demonstrate using a model of antibiotic‐induced gut microbiome ablation, that an intact microbiome is required to mediate metabolic protection due to DHF in female mice. Upon ablation of the gut microbiome DHF supplementation not only did not prevent HFD‐induced obesity in female mice, it also failed to activate the TrkB receptor, whose activity has been previously ascribed to metabolic signaling by DHF. Mechanistically, we report that metabolic protection in female mice is mediated by increased thermogenic markers including UCP1, AMPK, SIRT1, and PGF‐1α in brown adipose tissue in females, but not males. This thermogenic activation also required an intact gut microbiome. These results reveal complex and novel interactions between dietary flavonoids, sex, and the gut microbiome that have important implications both to understanding the etiology of metabolic diseases and for the development of personalized nutrition strategies for their prevention.
Global Toll‐like Receptor 4 Knockout Prevents Cerebrovascular Dysfunction Associated with Chronic Stress
Authors: Burrage Emily N. , Prabhu Saina , Frazier James , Englund Phillip , Mills James , Casto Makenzie , Bryner Randy W. , Kelley Eric E. , Chantler Paul D.
Published: May 2022

Chronic stress is a risk factor for many cerebrovascular diseases including stroke and vascular dementia and our lab has previously shown that chronic stress impairs cerebrovascular function and induces a pro‐inflammatory environment. It is possible that this pro‐inflammatory environment is driven by the activation of toll‐like receptor 4 (TLR4), a potent mediator of inflammation. However, the potential role of TLR4 in chronic stress induced cerebrovascular dysfunction has yet to be examined.

Here, we utilized a global TLR4 knock out (TLR4‐/‐) combined with our unpredicted chronic mild stress paradigm (UCMS) to assess middle cerebral artery (MCA) function. At 18 weeks‐of‐age, mice underwent the UCMS paradigm (5 days/weeks for 7hrs/day) to elicit a chronic stress phenotype. On week 26, mice were euthanized and the MCAs were removed and positioned in a pressurized myobath. To test for vessel health, the MCA was exposed to increasing concentrations of acetylcholine (ACh; 10‐9M to 10‐4M), phenylephrine (PE; 10‐9M to 10‐4M), and sodium nitroprusside (SNP; 10‐9M to 10‐4M).

TLR4‐/‐ mice displayed a similar MCA response to ACh compared to controls (18.4±0.7mm to 19.4±0.5mm, respectively). Mice exposed to UCMS displayed impaired MCA response to ACh while TLR4‐/‐ mice exposed to UCMS did not display MCA impairment in response to ACh (8.1±0.4mm vs 18.1±0.4mm, respectively; p<0.05). No differences in response to SNP were noted between groups however both TLR4‐/‐and TLR4‐/‐ UCMS mice displayed an impaired response to PE compared to controls (‐16.6±2.3mm and ‐17.6±1.6mm and ‐24.2±2mm, respectively; p<0.05).

These data suggest that TLR4 may play a role in the cerebrovascular dysfunction associated with chronic stress.
Investigating Disease‐Causing Mutations in SLC4 Transporter Homologs
Authors: Hendrix Claire , Abera Tsega‐Ab , Thurtle‐Schmidt Bryan
Published: May 2022

The SLC4 family of transporters includes the archetypal transporter Band 3, or Anion Exchanger 1 (AE1), a bicarbonate and chloride ion exchanger essential for human respiration and the most abundant membrane protein in red blood cells. Mutations in SLC4 transporters such as AE1 can lead to diseases such as hereditary spherocytosis or renal tubular acidosis. The SLC4 family shares sequence and structural homology with borate transporters, which are found in plants and fungi and regulate cellular borate levels to protect against borate toxicity. Here we generated mutations in the S. cerevisiae borate transporter Bor1 homologous with known disease‐causing mutations of human AE1, and then tested for borate transport activity in a genetic complementation assay. Our results identify mutants with impaired function but wild‐type expression levels, as well as mutants with impaired function and depleted expression levels. These results are consistent with varying modes of deleterious impact of protein expression, sorting, and function that have been attributed to mutations in human AE1. These data show that mutations equivalent to disease‐causing mutations in human AE1 have major effects in ScBor1, strengthening the functional connection between SLC4 proteins and borate transporters. Additionally, we demonstrate that the ScBor1 D347 residue, which is homologous to the essential E681 of human AE1, can be complemented by the D347E but not D347N substitution, underscoring the necessity of having an acidic residue at this highly conserved functional location. Lastly, we identify several new amino acids critical for function through mutagenesis and complementation studies. These data collectively highlight similarities in the biochemical features of ScBor1 and HsAE1 and help shed light on the mechanisms of borate transporters and their SLC4 homologs.
Long‐term hypercapnia does not alter Galanin immunoreactivity in mouse brain respiratory and chemoreceptor regions
Authors: Bulbul Latifa , Power John M. , Jones Nicole M. , Kumar Natasha N.
Published: May 2022

Long‐term hypercapnia has been implicated in respiratory disorders such as sleep apnea and chronic obstructive pulmonary disease (COPD). However, the mechanisms by which long‐term hypercapnia contributes to central respiratory dysfunction in these conditions are unclear. The brainstem respiratory circuit, including the ventral respiratory column (VRC), comprises distinct neuronal populations that govern expiratory and inspiratory drive. The neuropeptide galanin is extensively distributed in brainstem chemoreceptor regions and the VRC, and microinjection of galanin into the VRC inhibits breathing and attenuates ventilatory reflex responses to acute hypercapnia. Nevertheless, the normal pattern of regional expression of galanin in the mouse brain is not known and the impact of long‐term hypercapnia on galanin immunoreactivity (Gal‐ir) in the vicinity of brainstem respiratory and chemoreceptor regions has not yet been studied. Therefore, we aimed to determine Gal‐ir across the mouse brain and investigate the impact of prolonged hypercapnia exposure on Gal‐ir in brainstem respiratory and chemoreceptor regions (pons and medulla). Adult male Phox2b‐EGFP (MGI ID: 5776545) mice (31±2 g, n=5 mice/group) were exposed to hypercapnia (8% CO2; balance room air) or room air (control) for 10 or 14 days. To determine Gal‐ir, brain slices were collected and processed for immunohistochemistry. Quantitative measurement of Gal‐ir was performed using ImageJ 1.53a (a QuPath‐integrated version of ImageJ) where a positive area fraction, reflecting the proportion of positively labeled pixels, was calculated for each region of interest [locus coeruleus, the retrotrapezoid nucleus, caudal‐nucleus of the solitary tract (cNTS), rostral‐NTS (rNTS), VRC, lateral reticular nucleus, and caudoventrolateral reticular nucleus]. Under control conditions, we found that Gal‐ir was broadly present across the medulla oblongata, and abundant in the caudal aspects when compared with the rostral levels. The cNTS had a higher average percentage of Gal‐ir area (p = 0.043, p = 0.05, 2‐way ANOVA) compared to rNTS following 10‐ and 14‐days of room air exposure, respectively. A significant difference in Gal‐ir (p = 0.011, 2‐way ANOVA) was observed between the cNTS and rNTS following 14‐days of hypercapnia exposure. Overall, there was no significant difference of Gal‐ir in all regions assessed after long‐term hypercapnia compared to controls. Collectively, we found that long‐term hypercapnia does not induce changes in galanin neuropeptide levels within respiratory and chemoreceptor regions in the mouse brain. Under control and hypercapnia conditions, cNTS contains higher Gal‐ir compared to rNTS. Further investigation is required to elucidate the involvement of NTS‐galaninergic subsets in respiratory homeostasis.
Human platelet‐mediated transfer of cardioprotection by remote ischemic conditioning is abrogated by aspirin, but not by ticagrelor
Authors: Lieder Helmut R. , Tsounami Maria , Schrör Karsten , Andreadou Ioanna , Heusch Gerd , Kleinbongard Petra
Published: May 2022

Background

Repeated ischemia/reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) protects against injury from sustained myocardial ischemia/reperfusion (I/R). Platelets are considered as mediators/targets of RIC´s protection. We have recently reported that infusion of washed platelets, taken from healthy volunteers after RIC, reduced infarct size (IS) in isolated rat hearts (FASEB Journal 2021; 35; S1; DOI: 10.1096/fasebj.2021.35.S1.03444). Anti‐platelet drugs are potential confounders of RIC.

Aim

To study the impact of anti‐platelet drugs, the cyclooxygenase inhibitor aspirin and the P2Y12 inhibitor ticagrelor, on the platelet‐mediated transfer of cardioprotection.

Methods

We included 18 healthy volunteers. As a contemporary control, volunteers were subjected to RIC through 3 x 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation. After at least 2 weeks, these volunteers were again subjected to RIC 3 hours after pretreatment with oral aspirin (500‐1000 mg) and again at least another 2 weeks later 2 hours after pretreatment with oral ticagrelor (180 mg). Venous blood samples were taken before RIC and 60 min after RIC, respectively. Washed platelets were prepared using apyrase, prostaglandin E1 and citrated Tyrode‐buffer. The platelet count was adjusted to 2.5 x 105platelets/µL. The functionality of washed platelets was tested in response to thrombin (1U/ml), aggregation measured with turbodimetric light transmission aggregometry and expressed as the increase in light transmission in percent of control. Platelet preparations with an aggregation <60% were discarded. Male Lewis rats were sacrificed, their hearts isolated and perfused at constant pressure with buffer. Washed platelets before and after RIC, respectively, were infused for 8 min (2.5 x 104 platelets/µL) into rat hearts before global 30 min/ 120 min I/R. IS was demarcated by triphenyl tetrazolium chloride staining and calculated in percent of ventricular mass.

Results

We confirm, that infusion of platelets after RIC reduces IS in isolated hearts with global I/R (Figure). This platelet‐mediated transfer of cardioprotection was abrogated by aspirin‐pretreatment of the volunteers (Figure). Platelets before RIC from ticagrelor‐pretreated volunteers reduced IS per se; nevertheless, platelets after RIC further reduced IS (Figure).

Conclusion

Human platelets serve as carriers for RIC’s cardioprotective signal through an aspirin‐sensitive and thus cyclooxygenase‐dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a cardioprotective platelet‐dependent signal, which is further enhanced by RIC. The precise signal transfer from the platelets to the myocardium and the nature of the platelet‐derived factors remain to be further identified.
Investigating the effects of the marine drug Manzamine‐A related to craniofacial development in vitro
Authors: Hardy Samantha , Hamann Mark , Cray James
Published: May 2022

Manzamine‐A is a marine‐derived alkaloid which has anti‐viral and anti‐proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV‐1) and cancers (breast, cervical, colorectal). Manzamine‐A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKTpathways. To date little work has focused on Manzamine‐A and how its use may effect craniofacial development. We hypothesize that Manzamine‐A, through SIX1, alters bone cell activity important to craniofacial skeletal development. As Manzamine‐A is a drug that has great potential in a variety of therapeutics, it is critical that we asses potential side effects that this drug could have on both patients and any developmental effects that may occur during use by expectant mothers. We aimed to assess the effects of Manzamine‐A on four cell types, pre‐osteoblasts, osteoblasts, pre‐osteoclasts, and osteoclasts. PCR, qrtPCR, MTS cell viability assays and Caspase 3/7 apoptosis assay were used to test the effects of Manzamine‐A on these cells. We also performed alkaline phosphatase (ALP) and Tartrate Resistant Acid Phosphatase (TRAP) assays to test the function of osteoblasts and osteoclasts respectively. To date we have found that Six‐1 is highly expressed in osteoblasts and their progenitors. In contrast Six‐1 has low expression levels in osteoclasts and their progenitors. Further, osteoblast progenitor cells exhibit great sensitivity to Manzamine‐A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in ALP activity. Studies are ongoing for osteoclast lineage cells. Overall our preliminary data suggests Manzamine‐A may have great effects on bone health overall and may disrupt skeletal development if exposed during pregnancy or postnatal development.
Structurally investigating a niche pathway for chemical reversal of proline hydroxylation in the pathogen Clostridioides difficile
Authors: Backman Lindsey R. F. , Huang Yolanda Y. , Andorfer Mary C. , Gold Brian , Raines Ronald T. , Balskus Emily P. , Drennan Catherine L.
Published: May 2022

The glycyl radical enzyme (GRE) family utilizes a glycyl radical cofactor, installed by AdoMet radical activating enzymes, to catalyze difficult chemical reactions in a variety of microbial metabolic pathways. Although GREs are widely encoded and expressed by bacteria found in the gut microbiome, these enzymes remain largely uncharacterized. Recently a new GRE, hydroxyproline dehydratase (HypD) was discovered to catalyze the dehydration of trans‐4‐hydroxy‐L‐proline (4‐Hyp) to 1‐pyrroline‐5‐carboxylic acid. HypD is the second most prominent GRE in the human gut microbiome and is encoded by 360 bacterial genomes, including the commonly antibiotic‐resistant human pathogen C. difficile. HypD presents a pathway for bacteria to reverse 4‐Hyp post‐translational modifications, the most common post‐translational modification in animals, which was previously thought to be irreversible. Furthermore, the bacteria that encode HypD are known to use 4‐Hyp as an electron acceptor during amino acid fermentation, their primary method of generating adenosine triphosphate (ATP). However, the enzyme responsible for assimilating 4‐Hyp into this pathway has remained unknown until now. HypD could be the missing puzzle piece to understanding how these bacteria use the abundant metabolite 4‐Hyp in energy production, while also symbiotically providing humans with a method for recycling this common amino acid. In order to elucidate the mechanism for how HypD performs the dehydration of hydroxyproline, we aimed to characterize HypD from C. difficile, in the presence of its substrate. Here, we have solved a 2.05‐Å resolution structure for HypD by molecular replacement. Subsequently, a structure for HypD with its substrate 4‐Hyp bound in the active site was solved to 2.52‐Å resolution. These structures, accompanied by site‐directed mutagenesis studies and biochemical assays with deuterated substrate, have led us to identify key catalytic residues and have provided insight into the radical mechanism for 4‐Hyp dehydration.
Deactivation of STAT3 and ERK and Activation of p38 in Ovarian Cancer Cells Treated with a Small Molecule Inhibitor of PTP4A3 Phosphatase
Authors: Lazo John S. , Isbell Kelly N. , Rastelli Ettore J. , Wipf Peter , Sharlow Elizabeth R.
Published: May 2022

The Protein Tyrosine Phosphatase of Regenerating Liver‐3, also known as PRL‐3 or PTP4A3, is among the most oncogenic known protein tyrosine phosphatases, promoting cell migration, survival, metabolism, angiogenesis, and tumor formation. These diverse cancer‐associated phenotypic actions are thought to be mediated at least in part by the unique ability of PTP4A3 to function as a positive signal transducer capable of activating STAT3 and ERK1/2 and deactivating p38 kinase. PTP4A3 is significantly upregulated in a majority of human ovarian cancers and is associated with a poor patient prognosis. Our hypothesis is that PTP4A3 phosphatase inhibitors can be effective targeted treatments for ovarian cancer. We previously described a potent, allosteric, small molecule PTP4A3 inhibitor, 7‐imino‐2‐phenylthieno[3,2‐c]pyridine‐4,6(5H,7H)‐dione, which is also known as KVX‐053 or JMS‐053. KVX‐053 shows no significant in vitro inhibition against a panel of protein kinases or protein tyrosine phosphatases other than PTP4A1‐3. KVX‐053 is cytotoxic to chemosensitive and chemoresistant human ovarian cancer cell lines in vitro and has in vivo antitumor activity. The goal of the current study was to examine the impact of a PTP4A3 phosphatase inhibitor on human ovarian cancer STAT3, ERK1/2, and p38 activation. We observed a concentration‐dependent decrease in the activated form of STAT3, namely, Y705 phospho‐STAT3, after a 4 h exposure of A2780 ovarian cancer cells to KVX‐053 in vitro. A 4 h exposure to a concentration of KVX‐053 that reduced A2780 cell viability by 25% (EC25), namely 157 nM, decreased Y705 phospho‐STAT3 levels by 46%. An EC50 (470 nM) or EC90 (4.23 μM) concentration decreased Y705 phospho‐STAT3 levels by 61% and 87%, respectively. STAT3 levels remained unchanged except for a modest decrease with 4.23 μM of KVX‐053. ERK1/2 phosphorylation was decreased 64% with a 4 h treatment of an EC90 concentration of KVX‐053 but not with the EC25 or EC50 concentrations. In contrast, KVX‐053 caused a concentration‐dependent increase in p38 kinase phosphorylation and activation. These results suggest that acute KVX‐053 exposure disrupts the PTP4A3/STAT3, PTP4A3/ERK, and PTP4A3/p38 homeostatic loops, providing additional information about the role of PTP4A3 in cancer cell signaling and survival.
Prevalence of Tuberculosis, Rifampicin Resistant Tuberculosis and Associated Risk Factors in Presumptive Tuberculosis Patients Attending Some Hospitals in Kaduna, Nigeria
Authors: Egbe Nkechi E. , Olatunji Azizat , Vantsawa Peter A.
Published: May 2022

The accurate diagnosis of tuberculosis (TB), detection of drug‐resistant TB and knowledge of the associated risk factors can reduce transmission of TB in the community. Therefore this study was carried out to determine the prevalence of Mycobaterium tuberculosis(MTB) and its resistance to Rifampicin using GeneXpert in presumptive tuberculosis patients attending some hospitals in Kaduna, Nigeria. A total of 198 sputum samples were collected and screened from participants who had been administered questionnaires using Ziehl Neelsen Acid Fast Bacilli (ZN AFB) method and GeneXpert molecular technique. Data obtained showed that using the Zn AFB staining technique, MTB was seen in 65 sputum test results but with the GeneXpert technique, MTB was further detected in 15 sputum sample, bringing the total to 80, thus confirming that GeneXpert was more sensitive in detecting TB compared to the ZN AFB method. The overall prevalence of TB and Rifampicin resistant TB (RRTB) was 40.4% and 1.25% respectively. A higher prevalence rate was reported among male subjects (47.2%), age group 31‐40 years (50.9%), HIV positive subjects (54.5%), those that smoke cigarettes (72.2%) and those that has previous contact with someone living with TB infection (82.4%). Assay for statistical significance showed that independent variables that were significantly associated with the rate of occurrence of TB (p<0.05 at 95% C.I) were Age, Gender, HIV Status, smoking behaviour and previous contact with someone having TB. The only RRTB case detected was a male who has had a previous contact with someone living with TB infection. This study indicates a low burden of rifampicin resistant tuberculosis among the participants. It is thus concluded that rapid detection of TB and Rifampicin resistant strains using GeneXpert and other molecular techniques for TB diagnosis in clinical settings is key to early access to the right therapy; it will also improve treatment outcomes and decrease transmission rates.
Inflammatory Activation and Nitric Oxide Oxidation in Exertional Dyspnea in Southwest Asia Deployed Military Veterans
Authors: Pappas Gregory , Vujic Ena , Gardner Carol , Guo Changjiang , Black Kathleen , Therkorn Jennifer , Laskin Debra , Kipen Howard , Falvo Michael , Gow Andrew
Published: May 2022

Post deployment personnel of the Southwest Asia theater of war present with significant exertional dyspnea. While deployed, these service men and women were exposed to a variety of inhaled toxicants including geological dust, burn pit smoke, and heavy metals. Upon examination, a large portion of these veterans display near‐normal pulmonary function and gas exchange in the lung, making diagnosis and treatment difficult. The aim of this study is to define the physiological and biochemical profiles of individuals with unexplained exertional dyspnea. We hypothesized that exposure to airborne toxicants has led to a chronic activated inflammatory state in the systemic vasculature, resulting in increased oxidative stress and reducing the bioavailability of nitric oxide (NO), contributing to the symptoms of dyspnea.

Eligible veterans were tested for lung function by spirometry including diffusing capacity for carbon monoxide (DLco). Resting blood samples were taken and immediately separated into plasma and cellular components. Granuloyctes were removed from whole blood by magnetic separation, and subsequent leukocytes were collected and gated on expression of the myeloid marker CD45. Within CD45+ cells, flow cytometry was used to analyze the expression of inflammatory macrophage activation markers CD87, CD11b, CD163, HLA‐DR, CD195, CD192, CD14, CD16. Circulating levels of nitrite and nitrate (oxidation products of NO) were measured in plasma via reductive chemiluminescence, and the ratio of nitrate/nitrite (NOx ratio) was used as a marker for reduced bioavailability of NO. Increased expression of inflammatory marker CD87 in CD11b+ cells was positively correlated with expression of CD163 in HLA‐DR+ and CD14 in CD16+ (rho = .54, p <.01; rho= .36, p= .047; n =30), but not CD195 in CD192+ (rho =‐.3, p =.1). Similar observations were observed in granulocytic cells regarding CD66b expression. This correlation between activation markers within the circulating myeloid‐derived cells is consistent with the presence of monocyte activation in the systemic vasculature. NOx levels were within normally observable bounds, however, the NOx ratio was negatively correlated with FEV1/FVC (a marker of lung obstruction) but not with %predicted DLco(rho =‐.56, p =.016; rho =.01, p = .96, n = 18). The reduction in FEV1/FVC with increased NOx ratio suggests potential loss of endothelial function, which may be contributing to exertional dyspnea and is currently under investigation.
Massage application to increase the spread of local anesthesia in sciatic nerve blocks: A cadaveric study
Authors: Robertson Taylor C. , Fisher Cara C. , Handler Emma C. , Nash Daniel
Published: May 2022

Introduction

Sciatic nerve blocks are essential for surgical treatment of various lower limb pathologies. Due to the complexity and variation of anatomical landmarks, ultrasound (US) guided injection of local anesthesia has become common practice. In patients with thicker thigh girth (i.e., obese patients) excess tissue may distort US penetration thereby diminishing efficacy of the nerve block and/or cause severe post‐operative pain. Dye tracing techniques have been used to test the effectiveness of nerve blocks, but there is little research on using massage to manipulate anesthetic spread. Therefore, the aim of this study is to assess the effects of massage to manipulate local anesthesia spread in sciatic nerve blocks. We hypothesize massaging after injection will increase the spread of local anesthesia compared to non‐massage post injection.

Methods

Forty un‐fixed cadaveric legs were injected with a mixture of methylene blue dye and 2% Lidocaine Hydrochloride. Specimens were divided into non‐massage (control) (n=20) and massage (n=20) groups. Sciatic nerve blocks were performed by a nurse anesthetist using US guidance at the popliteal fossa traveling proximally until the sciatic nerve was identified and the location was tagged. Immediately following, massage group specimens received five repeated proximally directed massages with the US transducer head. Specimens from both groups were then dissected to expose the sciatic nerve. Measurements of the distance traveled from marked site of injection to proximal end of dyed area were measured and compared.

Results

Spread of local anesthesia in the inferior‐superior direction was significantly higher in the massage group than the control group (p≤0.05).

Conclusions

Massaging post‐injection caused a greater spread of local anesthesia during sciatic nerve block.

Significance

Sciatic nerve block techniques often utilize nerve stimulation to identify the sciatic nerve location. This may be due to lack of US penetration through the gluteus maximus muscle. In patients with thicker thigh girth due to subcutaneous fat, imaging visibility may be more difficult as well. Our findings suggest that clinicians may block the sciatic nerve at a more distal location with US guidance and manipulate the anesthesia to the region of interest.
An E‐Box Binding Site is Sufficient for ZPA Regulatory Sequence (ZRS) Activity in the Limb
Authors: Ball Kathryn F. , Pira Charmaine U. , Oberg Kerby C.
Published: May 2022

The zone of polarizing activity (ZPA) is a cluster of mesodermal cells that secrete Sonic hedgehog (Shh) to regulate anterior‐posterior patterning during limb development. The ZPA regulatory sequence (ZRS) is the limb‐specific enhancer that controls Shh expression. ZRS microdeletion abrogates Shh expression and posterior limb development, whereas ZRS single nucleotide variants (SNVs) produce anterior ectopic Shh and preaxial polydactyly. However, the ZRS mechanism of action is not well characterized. Transcription factors (TFs) Hand2, Twist1, and Hoxd13 regulate Shh expression and ZRS contains putative binding sites for each: two Hoxd13 binding sites and three E‐boxes, sites targeted by basic helix‐loop‐helix (bHLH) TFs such as Hand2 and Twist1. Further, reports suggest these TFs may act through the ZRS: Hand2 purportedly binds the ZRS through a central E‐box; as bHLH‐TFs Hand2 and Twist1 may regulate ZRS as homo‐ or heterodimers; and both Hand2 and Hoxd13 activate the ZRS in vitro. Previously we demonstrated that disrupting all five binding sites eliminates ZRS activity. In this report, we further investigate these sites to determine the contribution of each to ZRS activity. We hypothesized that the Hand2 binding site is required for activity and that other E‐boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm.We assessed ZRS activity via an enhancer‐GFP reporter, altering all five binding sites to eliminate activity, then reinstating each site individually using site‐directed mutagenesis. Constructs were electroporated into the presumptive limbs of chicken embryos (Hamburger‐Hamilton stage 14). Enhancer activity was evaluated 48 hours post‐electroporation by fluorescence microscopy. Restoring either Hoxd13 site led to weak, focal activity. Restoring two of the E‐boxes had little to no effect, however, restoring the third, 3’ E‐box was sufficient for robust activity. This was unexpected since the central E‐box was reported as the likely Hand2 binding site. Our data suggest that the 3’ E‐box is likely the critical site used by Hand2 in ZRS‐mediated Shh regulation. Additionally, the Hoxd13 binding sites appear to impart focal activity supporting a role for localization. These findings further characterize the mechanism used to regulate focal Shh expression during limb development.
A role for BD2‐selective BET inhibitors in cocaine‐seeking behaviors
Authors: Babigian Christopher J. , Singh Mandakini , Sartor Gregory
Published: May 2022

Epigenetic mechanisms play important roles in the maladaptive transcriptional and behavioral responses to drugs of abuse. Inhibition of bromodomain and extra‐terminal domain (BET) proteins, a class of histone acetylation reader proteins, has been shown to reduce cocaine‐seeking behaviors in animal models of addiction. To date, the role of BET protein in animal models of substance use disorder (SUD) has only been evaluated using pan‐BET inhibitors, small molecules that bind to both bromodomains (BD1 and BD2) in all BET proteins. Although these initial studies are encouraging, side effects associated with pan‐BET inhibitors may limit their clinical utility as a SUD treatment. In order to evaluate safer and more selective approaches, we utilized a clinically tested BD2 selective BET inhibitor, RVX‐208. We show that RVX‐208 dose‐dependently reduced cocaine conditioned place preference in male mice, similar to the pan‐BET inhibitor JQ1. Additionally, RVX‐208 did not alter other behaviors, such as: locomotor activity, elevated zero maze, or novel object recognition memory. At the transcriptional level, RVX‐208 attenuated the expression of multiple cocaine‐induced genes in the nucleus accumbens. RVX‐208 also produced a distinct transcriptional response in stimulated primary neurons compared to JQ1 but had little effect on gene expression in non‐stimulated neurons. Together, these data indicate that targeting domain‐specific BET mechanisms may be an effective and safer strategy to reduce cocaine‐induced neurobehavioral adaptations.
Liver‐Heart Microphysiological Organoid Model for the Study and Treatment of Cardiac Amyloidosis
Authors: Williams Marcus A. C. , Jeffreys Will , Jani Vivek P. , Lin Brian , Ranek Mark
Published: May 2022

Transthyretin (TTR) amyloid cardiomyopathy (ATTR‐CM) is a form of restrictive heart disease resulting from the aggregation of amyloid fibrils, that confers substantial morbidity and mortality. Transthyretin (TTR) forms a homotetrameric protein complex generated in the liver and is involved in retinol and vitamin A transfer. However, it can dissociate into monomers, that when taken up by the heart, can lead to ATTR‐CM. A TTR valine‐122 to isoleucine (TTRV122I) mutation is the predominant hereditary form in the U.S., present in 4% of African Americans, that destabilizes the TTR homotetrameric complex. In 2019 the FDA approved tafamidis, a first in class therapy for ATTR‐CM that stabilizes the TTR homotetramer, slowing but not arresting its dissociation into cytotoxic monomers or preventing TTR monomer deposits. Treatments that achieve the latter have yet to reach clinical settings and could further improve outcomes of ATTR‐CM patients. The lack of pre‐clinical human based models has severely limited mechanistic studies of ATTR‐CM and presents a major obstacle to developing translational therapies. We have addressed this by developing a new in vitro human cell model of TTRV122I via a novel microfluidics approach to co‐culture hepatic organoids expressing TTRV122I and human iPSC derived cardiomyocyte (hiPSC‐CM) organoids. This engineered device allows for the establishment of two distinct chambers containing both hepatic or cardiac organoids that are connected via a series of microfluidic channels to simulate circulation. With this approach, we create a physiologically relevant environment where hepatocytes synthesize and secrete TTRV122I at a concentration range of 3‐7 mM, as observed in human ATTR‐CM patients, that can then be taken up by cardiomyocytes. Once taken up, the cardiomyocytes develop features of ATTR‐CM, namely oxidative stress, protein aggregation, cytotoxicity, and abormal conductance. We assess cardiomocyte function via IonOptix cytomotion. We observed diastolic impairment in cardiomyocytes incubated with TTRV122I but not in our control cells. Notably, ATTR‐CM patients exhibit marked diastolic impairment and conduction system disease. In addition, this platform allows us to pace the cardiomyocyte organoids, and using optical tracking methods, look at waveforms and calcium transience. Thus, our in vitro system accurately recaptilates many key features of human ATTR‐CM and provides a new plateform to test novel therapeutic interventions.