bluuebunny/crossref_metadata_2025 huggingface dataset:
Select Moringa oleifera Subfractions Exhibit Potent Antibacterial Properties against Escherichia coli
Authors: Orders Tanner , Nair Srijith , Smith Brandon , Slate Jamison , Moreano Noah , Oyugi Emelda , Reinker Sam , Salih Yahya , Maisura Ravi , Payne Aubryana , Emrani Jahangir , Idassi Joshua , Frank Martin , Mowa Chishimba Nathan
Published: November 2023

Bacterial infection is one of the leading causes of preterm labor and use of pharmaceuticals is not ideal during pregnancy for teratogenic reasons. In an effort to provide safer antibacterial treatments, we have previously used whole‐leaf extracts of Moringa oleifera (MO) to screen the antibacterial efficacy of whole‐leaf extracts isolated using various solvents against Escherichia coli (E. coli), a leading cause of urinary tract infections associated with preterm labor. These earlier studies found the ethanolic whole‐leaf extract to be the most potent. Here, we test the antibacterial effects of eight high performance liquid chromatography (HPLC)‐isolated ethanolic‐based leaf subfractions of M. oleifera. In the initial experiments we screened each subfraction using diffusion assays and later the more potent subfractions (numbers four, five and six) were further tested using in vitro liquid broth assays and were found to act in a dose‐dependent manner. Although all the eight subfractions exerted some antibacterial effect at a concentration of 20 μg or less, only the three subfractions cited earlier had significant activity and subfraction number five showed the most antibacterial effect at 20 μg and required the least concentration for the lethal dose fifty (LD50) and minimal bactericidal concentration. Subfraction number five was followed by four and then six in potency. We conclude that the anti‐bacterial activity of whole leaf M. oleifera is likely exerted by subfractions number five, four and six. Ongoing studies in our lab are testing the potential synergistic effects of the different subfractions.

Support or Funding Information

OSR, Appalachian State University
Oxygen glucose deprivation modulates endothelial Ca2+ signal mediated by TRPV4‐KCa channels
Authors: Lin Mike T , Choi Chung‐Sik , Zha Xiangming , Yap Fui Chi
Published: November 2023

Endothelial blood brain barrier maintenance and breakdown depend on Ca2+ magnitude and spatiotemporal dynamics. We have shown that activation of endothelial TRPV4‐KCa (transient receptor potential vanilloid 4‐calcium‐activated potassium) channels' positive feedback results in microvascular permeability increase and vasogenic edema in a Ca2+‐dependent manner. Mouse cerebral endothelial cells express two types of KCa channels—SK3 (small KCa type 3) and IK1 (intermediate KCa) channels. Both SK3 and TRPV4 channels reside at caveolae presumably via interaction with caveolar scaffolding protein, caveolin‐1. IK1 channels, however, do not reside at caveolae, suggesting differential TRPV4 Ca2+ signaling microdomains associated with SK3 and IK1 channels. In this study, we tested whether ischemia/reperfusion modulates TRPV4‐KCa microdomain Ca2+ signals. Our results showed that the Ca2+ signals mediated by TRPV4‐SK3 differed from that mediated by TRPV4‐IK1 channels' positive feedback. SK3‐associated Ca2+ events showed large amplitude with slow decay kinetic. These events were infrequent compared to that associated with IK1 channels; IK‐associated Ca2+ events had small amplitude and fast decay kinetics. In response to 60 min of oxygen glucose deprivation (OGD), Ca2+ events shifted predominately toward that resembling IK1‐associated (frequent Ca2+ events, small amplitude and fast kinetic). GSK (GSK1016790A, a selective TRPV4 channel agonist; 10 nM) increased Ca2+ event site and amplitude before OGD; however, after OGD the Ca2+ signals induced by GSK became Ca2+ wave‐like events. It is known that low [Ca2+] confined to microdomains maintains junctional protein complexes, whereas large and repetitive Ca2+ waves induced by ischemia/reperfusion enhance blood brain barrier leakage. Taken together, our results suggest that the Ca2+ dynamics underlying TRPV4‐KCa channels' positive feedback may play an important role in blood brain barrier integrity modulation.

Support or Funding Information

Supported by HL102056 (Lin) and S10 0D020149 (MS Taylor, University of South Alabama).
PGC‐1α – Dependent TFEB Expression and Activation with Exercise
Authors: Erlich Avigail Tamar , Brownlee Diane , Beyfuss Kaitlyn , Hood David
Published: November 2023

A functional mitochondrial network is required for the maintenance of healthy skeletal muscle, and this is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator PGC‐1a regulates the synthesis of new mitochondria, while the coordinated transcription of lysosomal and autophagy‐related genes is controlled by transcription factor EB (TFEB). PGC‐1a activation and expression are induced with exercise, leading to a subsequent increase in mitochondrial biogenesis. However, the effect of exercise on TEFB expression and activation has yet to be elucidated. Since biogenesis and mitophagy are closely linked, the purpose of our study was to investigate the interplay between PGC‐1a and TFEB in maintaining mitochondrial homeostasis in response to chronic contractile activity in C2C12 myotubes, and following acute treadmill exercise in WT and PGC‐1a KO mice. In C2C12 cells, TFEB nuclear localization was increased by 1.6‐fold following 2 hours of acute, electrical stimulation‐induced contractile activity. Cells transfected with a 1600 bp TFEB promoter‐reporter construct revealed that TFEB transcription was simultaneously increased by 3‐fold by contractile activity. To evaluate this in vivo, WT and KO mice were subjected to acute treadmill exercise. In WT mice, TFEB translocation to the nucleus increased 2.4‐fold. In contrast, KO mice exhibited no increases in TFEB translocation with exercise, yet showed a 4‐fold increase in mTOR activation. This suggests an increased phosphorylation and inhibition of TFEB in KO muscle following exercise. In addition, TFEB transcription, assessed through the intramuscular electroporation of the TFEB promoter‐reporter construct, was elevated by 4‐fold in response to exercise. This transcriptional response to exercise was completely abolished in the KO mice. TFEB protein levels were also reduced by 30% in the KO mice compared to their WT counterparts, however no changes were observed in most of the downstream targets of TFEB transcription. mRNA levels of the mitochondrial marker COXI were reduced by 47% in KO mice compared to WT counterparts basally, while exercise induced a 33–38% elevation in both genotypes. Furthermore, both WT and KO mice exhibited and 50% decrease in the mRNAs encoding autophagy genes. Our data suggest that acute exercise provokes TFEB expression and activation both in vitro and in vivo, in a PGC‐1a‐dependent manner. PGC‐1a controls expression of TFEB with exercise likely via transcriptional mechanisms. Furthermore, ablation of PGC‐1a seems to play a role in increased TFEB inhibition via mTOR phosphorylation, indicating a more complex role for PGC‐1a in mediating TFEB expression, as well as function.

Support or Funding Information

This work is supported by the Natural Science and Engineering Research Council (NSERC) of Canada.
Linear and Nonlinear Analysis of Heart Rate Variability in an Experimental Model of Diabetes and Obesity Submitted to Myocardial Infarction: Role of Aerobic Exercise Training
Authors: Conti Filipe F , Sartori Michelle , Santos Fernando , Dias Danielle S , Machi Jacqueline F , Souza Leandro E , Jensen Leonardo , Morris Mariana , Irigoyen Maria‐Claudia , De Angelis Kátia
Published: November 2023

This study was designed to verify the effects of aerobic exercise training (ET) on cardiac autonomic modulation in an experimental model of diabetes and obesity submitted to myocardial infarction. Ob/ob mice (10 weeks old) were randomized into (n=7): sedentary (S), myocardial infarction sedentary (MIS) and myocardial infarction + ET (MIT) groups. Myocardial infarction was induced by coronary artery occlusion. Aerobic exercise training was performed in treadmill (5x/week, 8 weeks, 40–60% maximal test). RR interval was recorded in awake animals at the end of the protocol. Cardiac autonomic modulation was evaluated by linear and nonlinear analysis of heart rate variability. At the beginning of the protocol, the groups submitted to myocardial infarction (MIS and MIT) presented lower exercise capacity in relation to the S group (S: 9.9±0.2 vs. MIS: 6.4±0.7; MIT: 4.5±0.6 min). Myocardial infarction promoted a drastic reduction of vagal modulation evidenced by linear analysis: RMSSD index (S: 5.9±0.7 vs OIS: 0.7±0.1 ms), standard deviation of pulse interval (S: 11.5±1.8 vs MIS: 1.4±0.2 ms) and high frequency band of pulse interval (S: 8.8±2.0 vs MIS: 0.1±0.05 ms2); as well as by nonlinear analysis: SD1 (S: 4.2±0.5 vs MIS: 0.5±0.1 ms). On the other hand, at the end of the protocol, the trained group (MIT) presented higher exercise capacity, while the sedentary groups (S and MIS) showed lower exercise capacity when compared to their initial values (S: 8.6±0.4; MIS: 4.3±0.5; MIT: 7.8±0.5 min). The aerobic exercise training was able to prevent these cardiac vagal dysfunctions observed in the MIS group, showing higher values for RMSSD index (MIT: 2.8±0.7 ms) and SD1 (MIT: 2.0±0.5 ms) when compared to the MIS group. Altogether, the results showed that the myocardial infarction led to a severe reduction of vagal modulation and the aerobic exercise training presented positive effects preventing this severe reduction.

Support or Funding Information

CAPES (99999.008874/2014‐00); CNPq, FAPESP and UNINOVE
Muscle Recruitment Patterns Above Critical Power
Authors: Alexander Andrew M , Hammer Shane M , Didier Kaylin D , Baumfalk Dryden R , Barstow Thomas J
Published: November 2023

Critical power (CP) represents an exercise intensity that above which exercise tolerance is limited and can be predicted. As the muscle fatigues during constant‐load exercise above CP, muscle motor unit recruitment is increased to meet the exercise demand. Our laboratory has shown that at task failure in small muscle mass handgrip exercise, muscle recruitment is not different across exercise intensities above CP.

Purpose

To test the hypotheses that 1) end‐exercise muscle recruitment is not intensity‐dependent during whole body exercises above CP, and 2) at exercise intensities above CP muscle recruitment does not reach steady state, but continuously increases until task failure occurs.

Methods

To date, four healthy, young men (23.5 ± 2.0 yrs; 92.6 ± 4.2 kg, 175 ± 4 cm) completed five exercise bouts to task failure on separate days. During the first session, subjects performed an incremental test on a cycle ergometer to determine peak power (Ppeak). The subjects then performed four constant load cycle tests on subsequent days. The power outputs during the constant load tests were selected to elicit task failure within the pre‐determined time range of 2–15 min to ensure all tests were above CP. Electromyography (EMG) was measured on the L vastus lateralis to quantify potential increases in muscle recruitment and were reported as 20 contraction averages.

Results

Average Ppeak was 300 ± 31 W. Work rate at which CP occurred was 178 ± 17W (59.4% ± 2.3 % Ppeak). There were no significant differences in end‐exercise iEMG (p=0.078) among exercise intensities above CP. Further, for each exercise intensity all subjects demonstrated a progressive increase in iEMG until task failure.

Conclusions

Confirming our hypotheses, the current data suggest that during cycling exercise above CP, muscle recruitment at task failure is similar across exercise intensities.
Whole Body Hyperthermia Enhances Angiogenesis in Tenotomized Rat Skeletal Muscle
Authors: Hirunsai Muthita , Srikuea Ratchakrit
Published: November 2023

In response to disuse, skeletal muscle is characterized by a reduction of fiber size, increase of intramuscular fibrosis, and regression of capillaries. Hyperthermia has been known to alleviate muscle atrophy and remodel extracellular matrix. Of these, the mechanisms underlying the beneficial effects of heat treatment on capillary adaptation following disuse atrophy are unknown. In the current study, we examined the effects of hyperthermia on angiogenesis and expression of the angiogenic factors in the soleus muscle following Achilles tendon ablation (tenotomy). Male Wistar rats were randomly assigned into three groups: control (CON), tenotomy for 8 day without heat treatment (TEN), and tenotomy for 8 day plus heat treatment (TEN+HEAT). Whole body hyperthermia was maintained at 40.5–41.5°C for 30 min, 24 h before and after tenotomy, and continued for five consecutive days. Following tenotomy, soleus muscle revealed a reduction in muscle fiber size and a remarkable loss of intramuscular capillary. These occur accompanied with decrease in vascular endothelial growth factor receptor‐2 (VEGFR‐2), the pro‐angiogenic factor and increase in thrombospondin‐1 (TSP‐1), the anti‐angiogenic factor, protein expressions in soleus muscle. However, tenotomy had no effect on the protein expression of vascular endothelial growth factor (VEGF). To rescue the capillary loss, whole body hyperthermia preserved the level of capillarization associated with increased VEGFR‐2 and reduced TSP‐1 protein expressions in tenotomized soleus muscle. Whereas, hyperthermia had no additional effect on VEGF protein level in soleus muscle after tenotomy. Collectively, whole body hyperthermia mitigated the effect of tenotomy‐induced capillary regression, in part, by modulating the angiogenic regulators. The results from this study suggest the potential therapeutic effect of heat treatment in relieving intramuscular capillary regression during skeletal muscle atrophy.

Support or Funding Information

The research was supported by grant from Srinakharinwirot University.
p16 and γH2AX as Markers of Cellular Senescence in Young and Old Human Skeletal Muscle
Authors: Dungan Cory , Kosmac Kate , Peck Bailey , McCarthy John , Peterson Charlotte
Published: November 2023

Cells that undergo terminal cell cycle arrest (senescence) are a hot topic in aging research due in part to their senescence associated secretory phenotype (SASP), composed of chemokines and inflammatory cytokines, which negatively affect the local microenvironment. In the mouse, muscle stem cells, satellite cells, undergo senescence with advancing age, which has been proposed to contribute to sarcopenia. Whereas loss of satellite cells in mouse muscle neither accelerates nor exacerbates sarcopenia, satellite cell SASP has been proposed to contribute to muscle wasting with age. There is currently no evidence that satellite cells in aged human muscle undergo senescence. p16 and γH2AX (markers of cellular senescence and DNA‐damage, respectively) are used to identify senescent cells in vitro and in tissues in vivo, but have not been examined in human muscle as a function of age. The purpose of our study was to examine the effectiveness of these markers in human skeletal muscle to quantify the abundance of senescent satellite cells during aging. Muscle biopsies were obtained from the vastus lateralis in young (18–25 years) and old (65–86 years) men and women, and immunohistrochemistry (IHC), combined with DAPI staining, were used to quantify p16 and/or γH2AX positive nuclei that expressed Pax7, a satellite cell marker. An antibody against dystrophin was also used to specifically localize myofiber nuclei. Surprisingly, >95% of all nuclei expressed p16 in both young and old muscle. However, labeled nuclei were preferentially localized within the muscle fiber, suggesting that in muscle, p16 is a marker of post‐mitotic, differentiated myonuclei. p16 positive satellite cell and non‐myogenic nuclei are currently being counted, as well as co‐expression with γH2AX, to attempt to identify and quantify all senescent cells in human muscle from both young and old donors. We conclude that p16 is not an ideal marker of cellular senescence in human skeletal muscle. Transient p16 expression in satellite cells may be an indicator of quiescence, while p16 expression in post‐mitotic myonuclei may be keeping them from re‐entering the cell cycle. Additional studies are needed to identify better markers of cellular senescence to determine if SASP potentially contributes to human muscle aging.
Identification of a Novel Regulator of Vasopressin Secretion
Authors: Stein Lauren Michelle , Soo Jasmine , Loewen Spencer , Hindmarch Charles , Ferguson Alastair , Yosten Gina , Samson Willis
Published: November 2023

We identified a novel hypothalamic peptide, Phoenixin, and demonstrated its hypothalamic and pituitary actions to control reproductive hormone secretion (Yosten et al. J Neuroendocrinol 25:206, 2013). We now have identified the orphan G protein‐coupled receptor, GPR173, to be the cognate receptor for Phoenixin (Stein et al. Am J Physiol 311:R489,2016). Transient compromise of either GPR173 or Phoenixin production in rat hypothalamus significantly interrupted estrous cyclicity, suggesting that this ligand‐receptor pair is essential for normal reproductive function. In situ hybridization histochemistry studies revealed that Phoenixin is expressed in neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, as is GPR173. We hypothesized that in addition to its action to control reproductive physiology, Phoenixin acts in PVN and SON to control vasopressin (AVP) or oxytocin (OT) secretion. Hypothalamo‐neurohypohysial (HNS) explants released AVP but not OT in response to Phoenixin. Intracerebroventricular administration of Phoenixin into conscious, unrestrained male rats significantly increased circulating AVP, but not OT, levels in plasma. Bath application of Phoenixin in hypothalamic slice preparations resulted in depolarization of PVN neurons indicating a direct, neural action of Phoenixin in hypothalamus. Our results suggest that the newly described, hypothalamic peptide Phoenixin, in addition to its effects on hypothalamic and pituitary mechanisms controlling reproduction, may play an important role in the regulation of fluid and electrolyte homeostasis.

Support or Funding Information

SLU President's Research Fund
Hepatic Androgen Receptor Modulates Hyperandrogenemia‐induced Glucose Metabolic Dysfunction and Hepatic Insulin Resistance
Authors: Andrisse Stanley , Wu Sheng
Published: November 2023

Hyperandrogenemia (HA) has been shown to cause impaired glucose tolerance and insulin resistance in women and in female rodent models. The mechanism of this HA‐induced metabolic dysfunction is not fully understood. We developed a mouse model that displayed pathophysiological serum androgen levels with normal body mass to ensure that the phenotypes were directly from androgens and not an indirect consequence of obesity. DHT mice demonstrated impaired glucose, insulin and pyruvate tolerance, and lowered hepatic insulin action. Our previous findings have shown that the liver is a primary target of HA‐induced metabolic dysfunction. The goal of this study was to determine the role of hepatic androgen receptor (AR) in DHT‐induced impaired glucose tolerance and hepatic insulin resistance. Molecular (Western blots, qRT‐PCR, immunoprecipitation (IP), and chromatin IP) assays were performed on the liver tissue samples obtained from the low dose DHT and control mice. Additionally, a low dose DHT cell model using H2.35 female mouse hepatocytes was developed to study androgen and AR effects on insulin signaling. Low dose DHT lowered hepatic insulin stimulated phosphoinositide‐3‐kinase (PI3K) activity, p‐AKT and p‐FOXO1 but had no effect on p‐Y‐IRS1/2 compared to control. Mechanistically, DHT increased hepatic AR binding to PI3K‐p85 resulting in dissociation of PI3K‐p85 from PI3K‐p110 leading to reduced PI3K activity and decreased p‐AKT, thus lowered insulin action. In addition, DHT increased gluconeogenesis via AR‐specific transcriptional upregulation of gluconeogenic enzymes (G6Pase and PEPCK) and co‐activators (FOXO1 and CREB). In the cell model, the DHT‐induced hepatocyte insulin resistance and increased gluconeogenesis was reversed by AR antagonist, flutamide. These findings suggest that AR in the liver is a key driver of HA‐induced whole body and hepatic metabolic dysfunction and insulin resistance. AR acts both in the nucleus and the cytosol to enact these detrimental effects on metabolism.

Support or Funding Information

NIH grant 5R00HD068130‐05 awarded to SW. JHU's Provost's Postdoctoral Diversity Fellowship awarded to SA.
Renal Olfactory Receptor 1393 May Contribute to the Progression of Diabetic Nephropathy
Authors: Shepard Blythe D , Pluznick Jennifer L
Published: November 2023

Olfactory receptors (ORs) are G protein‐coupled receptors which serve to detect odorants in the nose. It is now appreciated that ORs also have roles beyond odorant detection and serve as important chemosensors in a number of tissues including the kidney. We recently found (Shepard, et al Sci Rep 2016) that one of these renal ORs, Olfr1393, localizes to the apical membrane of the proximal tubule where it plays a role in renal glucose handling. Whole‐animal Olfr1393 knockout (KO) mice present with mild glycosuria and improved glucose tolerance despite being euglycemic with normal insulin levels. Examination of the Na+/glucose co‐transporters (Sglt1 and Sglt2) revealed a 22% decrease in the luminal localization of Sglt1, but not Sglt2, despite similar total protein expression, implicating Olfr1393 as a novel regulator of renal Sglt1. The Sglts have recently emerged as therapeutic targets for patients with diabetes and have been shown to attenuate the progression of diabetic nephropathy and diabetes‐induced hyperfiltration due to increased distal Na+ delivery. Given Olfr1393's role as a regulator of Sglt1, in this study we sought to investigate the contribution of Olfr1393 to the progression of diabetic hyperfiltration. Olfr1393 WT (n = 4) and KO mice (n = 9) were challenged with a high fat (HF) diet (60% kcal) for 16 weeks and glomerular filtration rate (GFR; transcutaneous measurement of FITC‐Sinistrin clearance) and glucose and insulin tolerance were measured. Upon the switch to a HF diet, both WT and KO mice consistently gained weight and after 16 weeks there were no differences in the kidney weight:body weight ratio, fasting blood glucose or response to glucose and insulin tolerance tests. To track diabetes‐induced hyperfiltration, GFR was measured after 0, 11.5 and 16 weeks on diet. As expected, by 16 weeks on diet WT mice presented with diabetes‐induced hyperfiltration (GFR/BW week 0 – 233ml/min; week 16 – 576ml/min, P=0.00009)). However, this hyperfiltration was blunted in the KO mice (GFR/BW week 0 – 277ml/min; week 16 – 435ml/min; P=0.005 (KO week 0 vs. KO week16), P=0.09 (Week 16 WT vs KO). Finally, in preliminary data, using Oil Red O lipid staining we detected a trend toward reduced lipid accumulation in the livers of Olfr1393 KO mice (n = 2) compared to a WT littermate (n = 1). Taken together, these data indicate that Olfr1393, through its regulation of Sglt1, contributes to the progression of diabetic nephropathy which may have extra‐renal consequences.

Support or Funding Information

NIDDK
Method for Simultaneous Measurement of Amygdala Neuronal and Sympathetic Nerve Activity in Freely Moving Rats
Authors: Shizuka Kataoka , Yoshimoto Misa , Ootsuki Fukiko , Miki Kenju
Published: November 2023

The amygdala plays a prominent role in regulating sympathetic nerve activity (SNA) in response to changes in daily behavioral states. However, to our knowledge, simultaneous measurement of amygdala neuronal and sympathetic nerve activity in freely moving animals has not been reported. In the present study, we developed a method for simultaneous and continuous measurement of amygdala neuronal activity and SNA in freely moving rats. Wistar rats were chronically instrumented with multiple electrodes (100‐μm stainless steel wire) for measurement of central amygdala neuronal activity, electrodes for measurement of renal SNA (RSNA), and electroencephalogram, electromyogram, and electrocardiogram electrodes. Changes in central amygdala neuronal activity were linearly correlated with changes in RSNA during the transition from nonrapid eye movement (NREM) sleep to quiet awake, moving, and grooming states. Thus, central amygdala neuronal activity and RSNA may be controlled by the same neural networks activated in physical activity from NREM sleep to grooming in rats. However, during REM sleep, central amygdala neuronal activity was highest, while RSNA was lowest, compared with other behavioral states. Further, there was no obvious relationship between amygdala neuronal activity and RSNA. These data suggest that the functional relationship between central amygdala neuronal activity and RSNA may be modulated by multiple control systems. JSPS Grant.
Involvement of Serotonergic Neurons in the Ventilatory Response to Hypercapnia in Birds (Chicken, Gallus gallus)
Authors: Santos Kassia M , Abreu Sheila B , Garcia Giuliana Milan Andrade , Bícego Kenia C , Gargaglioni Luciane H , da Silva Glauber S F
Published: November 2023

Respiratory system regulates acid‐base balance by eliminating carbon dioxide (CO2), with PCO2/pH variations detected mainly by central chemoreceptors. In mammals, serotonergic neurons (5‐HT) have been demonstrated to play a crucial role in the ventilatory response to CO2. It is known that birds have central CO2/pH chemoreceptors and that 5‐HT neurons are organized in the raphe. However, it is not known the neuronal phenotype involved in central chemoreception in birds. We hypothesized that 5‐HT neurons play a role in the central CO2/pH chemoreception in birds. Therefore, the present study aimed to verify the involvement of 5‐HT neurons in respiratory control in chicken (Gallus gallus) exposed to hypercapnia. Five‐day old male chicks were submitted to surgical procedures and 1μl of anti‐SERT‐SAP immunotoxin (0.1mM) was microinjected into the fourth ventricle for specific deletion of 5‐HT neurons. The control group received IgG‐SAP injections. Animals underwent to hypercapnia exposure (7% CO2) ten days after surgery. Pulmonary ventilation (VE; barometric method, whole body plethysmography) and body temperature were measured. Brainstem 5‐HT lesion was verified by immunohistochemistry. Both groups (control and lesioned) augmented VE during hypercapnia. Ventilatory response to hypercapnia of lesioned chickens was attenuated by ~30% compared to the control animals [mean values of VE during hypercapnia: 2429 and 1805 ml/kg/min, control vs lesioned group, respectively; P<0.05, (n=6)]. The attenuated response to hypercapnia was due to the lower respiratory frequency (77 and 59 breaths/min, control vs lesioned group, respectively. P<0.05). The results suggest that brainstem serotonergic neurons contribute to the respiratory response to CO2 in chicken, playing an excitatory role in CO2‐drive to breathing.

Support or Funding Information

Sao Paulo Research Foundation (FAPESP)
Insulin Resistance in Horses Varies Throughout the Year and Among Breeds
Authors: Moffatt Cheyenne N. , Paquin Zachary W. , Guild Ayla , Johnson Kathryn M.S.
Published: November 2023

In horses, insulin resistance is directly associated with laminitis, a severe lameness condition within the hoof and a leading cause of equine euthanasia. The onset of laminitis is most likely to occur in the early spring, and the risk of the condition is increased in specific horse breeds; however, it is unclear if insulin resistance is the mechanism that induces the seasonal onset of laminitis or increases occurrence in particular breeds. To determine if, 1) seasonal changes in insulin resistance occur as a possible precursor to laminitis; and 2) if insulin resistance is increased in breeds more susceptible to laminitis, horses (n=35) in southern Wisconsin and northern Illinois were classified as belonging to breeds that have an increased risk (Saddlebreds, n=7, and Welsh ponies, n=10), a decreased risk (stock horses, n=11), or an unknown risk (draft/draft crosses, n=7) of laminitis. Jugular vein blood samples were collected from all horses in September, November, January, March, and May. For all samples, plasma glucose and insulin levels were measured and insulin resistance was determined as the ratio of glucose to insulin concentrations (GIR), with lower GIR levels indicative of increased insulin resistance. There was no relationship between the time of year and glucose or insulin levels, but there was a significant seasonal effect on GIR (MANOVA, F(4,28) = 2.86, p=0.04). There was also a significant effect of breed on GIR (MANOVA, F(3,31) = 3.21, p=0.04), with the lowest GIR values attributed to breeds associated with a high risk of laminitis. Therefore, insulin resistance may be a mechanism by which breed and seasonality increase the risk of laminitis in horses. In addition, very little is known about the risks for insulin resistance and laminitis in draft and draft cross horses. Despite their greater stature and weight, often associated with insulin resistance, the draft and draft crosses had the greatest GIR, indicating the least insulin resistance. These findings suggest that despite their greater weight and likelihood of obesity, horses that belong to draft and draft cross breeds may have protective mechanism against insulin resistance, warranting further investigation.
Respiratory Control in a Rat Model of Alzheimer's Disease (TgF344‐AD)
Authors: Murphy Kevin H. , Burns David P. , Barry‐Murphy Kevin , Rae Mark G. , O'Halloran Ken D. , Lucking Eric F.
Published: November 2023

Synaptic defects seen in tauopathies such as Alzheimer's disease (AD) are known to result in the impaired function of major brain networks. It is plausible to consider that such changes can directly or indirectly affect central respiratory networks within the brainstem with consequences for respiratory homeostasis. Alzheimer's patients display indicative signs of oro‐pharyngeal complications and sleep‐disordered breathing. Upper airway dysfunction has also been shown to develop and persist in animal models of AD. The aim of this study was to characterise respiratory control at an early stage of disease progression in a novel rat model of AD.

We utilised a transgenic (Tg) rat model of AD (TgF344‐AD), which expresses the mutant human transgenes: amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9). Tg (male n=9; female n=12) and non‐transgenic wild type (non‐Tg; male n=8; female n=7) animals (34–49 weeks) were studied using whole‐body plethysmography to determine ventilation and metabolism. Cardiorespiratory reflex responses to chemostimulation were subsequently determined in a subset of animals under urethane anaesthesia (1.5g/kg). Data were statistically compared by 2‐way ANOVA (genotype × sex) with Bonferroni post hoc tests.

Transgenic mutation did not significantly alter baseline minute ventilation (VE; p>0.05) however, sex‐specific differences in breathing were evident. Exposure to 10 minutes of hypoxia (FiO2 =0.1) manifested a greater VE response in females compared with males (p<0.01); however, the ventilatory response to hypoxia was equivalent in Tg animals compared with non‐Tg animals. Similarly, baseline data recorded under anaesthesia revealed that VE was equivalent in Tg and non‐Tg animals (p>0.05) and consistent with data derived from plethysmography, sex differences in breathing were evident. No genotype or sex difference was observed in diaphragm integrated EMG activity for peak amplitude, area under the curve and maximum slope analysis (p>0.05). Baseline mean arterial pressure (MAP) was equivalent in Tg and non‐Tg animals (p>0.05), although females had a significantly increased MAP compared with males (p<0.01).

In conclusion, expression of human transgenes in this AD animal model did not significantly alter respiratory behaviour. Further analysis of respiratory stability and respiratory EMG responses to chemostimulation and airway obstruction will be carried out.

Support or Funding Information

Department of Physiology, University College Cork, Cork, Ireland
Medical School Anatomy/Pathology Workshops for High School Students Enhance Learning, Model Professionalism, and Provide Inspiration for Careers in Medicine
Authors: Zhang Guiyun , Fenderson Bruce A , Veloski J Jon , Livesey Michael , Wojdon‐Smith Tracey
Published: November 2023

“Anatomy/Pathology Workshop” is a cadaver‐based outreach program that models medical education to large groups of high school students. This study was designed to evaluate the impact of this program on students' knowledge of anatomy/pathology and interest in biomedical science. A total of 144 high school students participated in the workshop in 2015. Pre‐ and post‐ workshop assessments were administered to assess students' learning. A post‐workshop survey was conducted to solicit students' reflections and feedback. It was found that student performance on the post‐workshop examination (mean 78%) had significantly improved when compared to performance on the pre‐examination (mean 54%), indicating that this program enhances learning. Students were also inspired to consider opportunities in medicine and allied health professions: 97% indicated that they had a better understanding of medical education; 95% agreed that they had better understanding of the human body; 84% thought anatomy/pathology was interesting and exciting; and 62% of the students indicated that they looked forward to studying medicine or another health profession. Students rated the instructors highly: 95% agreed that the instructors were professional and served as role models. Medical/graduate student‐instructors were also highly regarded by the high school students: 96% thought it was valuable to have student‐instructors, and 94% thought that student‐instructors were caring and enthusiastic about teaching. In summary, this study demonstrates that outreach programs provided by medical schools help young adults during their formative years by modeling professionalism, providing role models, enhancing learning, and encouraging many to consider opportunities in the health professions.
The Klebsiella pneumoniae Gene ytfL Triggers Microtubule Disassembly in Lung Epithelial Cells through KATNAL1
Authors: Chua Michael Dominic , Siu L Kristopher , Yeh KuoMing , Guttman Julian Andrew
Published: November 2023

The Gram‐negative bacterium Klebsiella pneumoniae can cause septicemia, pneumonia, liver abscesses, and meningitis resulting in mortality rates as high as 44%. Unfortunately, the sub‐cellular interactions of these bacteria with host cells remain poorly understood. Because many bacterial pathogens target the host cell cytoskeleton, we examined the host cell cytoskeleton of K. pneumoniae‐infected lung epithelial cells. In this study, we focused on the host microtubule network and found that microtubules were severed by the microbes, which ultimately led to the disassembly of the entire microtubule network. Because severing of microtubules in lung epithelial cells are regulated by host cell mechanisms, we hypothesized that a K. pneumoniae gene product would trigger the microtubule severing events through the activation of a host microtubule severing protein. To test this hypothesis, we screened the known disease‐causing proteins of K. pneumoniae and found that expression of the capsular polysaccharide, outer membrane porins, and the type VI secretion system were not important for inducing microtubule severing. Next, we constructed and screened a library encompassing essentially all (~3000) K. pneumoniae genes to identify genes that could cause this phenotype. Using this library, we identified the gene KP ytfL that caused the microtubule severing phenotype. To further characterize this novel mechanism, we immunolocalized all known epithelial cell microtubule severing proteins in K. pneumoniae‐infected cells and found that the katanin‐like protein 1 (KATNAL1) localized precisely to the sites of microtubule severing. Taken together, our study shows that K. pneumoniae exploits a novel strategy to disassemble epithelial cell microtubules during its infections. The disassembly of microtubules is initiated by the expression of the bacterial gene KP ytfL, which ultimately induces the host protein KATNAL1 to sever microtubules and disrupt the microtubule network of host cells.

Support or Funding Information

This study was funded by NSERC, Taiwan NHRI and SFU Funds.
IP3R is localized to ER‐PM contact sites at unique endocytic structures involved in intercellular junction turnover events critical to spermatogenesis.
Authors: Lyon Kevin , Asghari Parisa , Scriven David RL , Moore Edwin DW , Vogl A Wayne
Published: November 2023

Unique endocytic structures termed tubulobulbar complexes (TBCs) are critical for mammalian spermatogenesis. They function to internalize intercellular junctions located at the blood‐testis barrier basally in the seminiferous epithelium to allow spermatocytes to move from the basal compartment into the adluminal compartment. They also function to internalize apical attachments between spermatids and Sertoli cells prior to their release into the seminiferous tubule lumen. TBCs have three distinct regions: a proximal tubule, a bulb, and a short distal tubule. The tubular regions are cuffed by dendritic actin networks and the distal tubule is capped with a clathrin‐coated pit. The bulb region lacks an actin cuff, but is closely associated with cisternae of endoplasmic reticulum (ER). The function of this ER‐plasma membrane (PM) contact is unknown but we suspect it may be involved in calcium signaling. We previously used immunofluorescence to determine whether IP3R, a calcium channel in the ER that releases calcium, is present at these ER‐PM contacts. We previously probed cryo‐sections and epithelial fragments of perfusion‐fixed rat testes with antibodies against IP3R and an appropriate fluorescent secondary. Our previous data showed that IP3R labels the distal region of TBCs which is consistent with where we would expect the bulb region to be. To gather more convincing evidence, we used thicker cryo‐sections of 10 μm compared to our standard 5 μm to ensure that entire clusters of TBCs would be present in the section. We performed 3D reconstruction on deconvolved Z‐stacks to evaluate the localization of the IP3R labeling. The labeling is seen located between the distal and proximal tubules of TBCs indicating that it is at bulb region containing the ER‐PM contacts. To determine whether or not calcium release from IP3R channels occurs at these ER contacts, Adenophostin A, an IP3R agonist, was used to induce calcium release in isolated intact spermatids with associated Sertoli cell regions containing clusters of TBCs. Calcium release was monitored using Fluo4, a fluorescent calcium indicator. Our preliminary data suggests that Adenophostin A induces calcium release from IP3R channels in regions of isolated spermatids that is consistent with the bulb regions of TBCs. The data are consistent with our hypothesis that these ER‐PM contacts are involved in calcium signaling.

Support or Funding Information

Supported by a NSERC Discovery Grant (#155397‐2013) to AWV.
Nerval danger zones for surgical procedures of the arm and the forearm
Authors: Hirtler Lena , Wlodek Vincent
Published: November 2023

Introduction

The course of cutaneous nerves at the distal forearm, the wrist joint and the hand is highly variable. This variability can cause complications in the different standardized surgical approaches for surgical interventions in these regions.

The aim of this study was therefor (1) to document the courses of the lateral, medial and posterior antebrachial cutaneous nerves, the superficial branch of the radial nerve, the dorsal branch of the ulnar nerve and the palmar branches of the median and ulnar nerve and (2) to determine their relationship to osseous and tendinous landmarks. Afterwards danger zones for surgical approaches will be defined.

Materials and methods

The cutaneous nerves were dissected on 20 formalin/phenol – embalmed forearms and hands of anatomical specimens. The position and distribution of the nerves as well as landmarks were documented and distances were measured at pre‐determined positions.

Results

Palmar danger zone in a proximal to distal course: 10 cm proximal to the wrist joint: between 0 – 20%, 40 – 66,67 %, 80 – 86,67% and 93,34 – 100% of the width of the forearm; 5 cm prox. to the wrist: 0 – 26,67 %, 53,34 – 66,67 % and 86,7 – 100 % of the width; on the line between the styloid processes: 0 – 13,33 %, 20 – 26,67 % and 93,34 – 100% of the width; on the line between the distal border of the pisiform bone and the base of the first metacarpal bone: 0 – 20 %, 46,67 – 60 % and 66,67 – 73,33% of the width. Dorsal danger zones in a proximal to distal course: 10 cm prox. to the wrist: 0 – 6,67 %, 13,34 – 33,33 %, 40 – 66,67 % and 93,33 – 100 % of the width; 5 cm prox. to the wrist: 0 – 6,67 %, 20 – 60 % and 66,67 – 73,33% of the width; on the line between the styloid processes: 0 – 20 %, 73,33 – 80 % and 93,33 – 100 % of the width; proximal metacarpus: 0 – 40 %, 73,33 – 86,67 % and 93,33 – 100 % of the width; on 50% of the metacarpal height: 0 – 6,67%, 13,33 – 33,33 %, 46,67 – 53,33 %, 66,67 – 86,67 % and 93,33 – 100 % of the width; distal metacarpus: 0 – 5 %, 25 – 35 %, 50 – 65 %, 75 – 85 % and 95 – 100 % of the width. The anatomic snuffbox and the ulnar aspect of the wrist were considered to be not safe, because of the wide distribution of nerves.

Conclusion

Aberrant and normal courses of the cutaneous nerves were documented and described in detail. Based on the results of this study, specific danger zones were defined to further help refining surgical procedures in future.

Support or Funding Information

no funding was provided.
Acetylpyrazine Thiosemicarbazone Inhibiting Topoisomerase II
Authors: Ngo Lana C , Stults Garrett
Published: November 2023

We have previously showed that Cu(II) acetylpyrazine methylthiosemicarbazone chloride ([Cu(APZ‐MTSC)Cl]) inhibits the ATP hydrolysis of human topoisomerase (Topo II) as catalytic inhibitor and share some common features as covalent poisons such as increasing TopoII‐mediate DNA cleavage which can be abolished by the presence of DTT. To further test if acteylpyrazine thiosemicarbazone metal complexes all have the similar characteristics, we synthesized a series of acetylpyrazine thiosemicarbazone metal (copper and palladium) complexes with different side chain substitutions including methyl, ethyl, turt‐butyl, benzyl, phenyl and dimethyl functional groups on the terminal nitrogen. We examined their effect on TopoII‐mediated DNA relaxation and DNA cleavage assays. Our data suggest that all metal acetylpyrazine thiosemicarbazones display similar characteristics as [Cu(APZ‐MTSC)Cl], but metal ions play a very important role in inhibiting TopoII.

Support or Funding Information

This project is funded by Faculty Development Research Grant from Tennessee Board of Regents and Faculty Research Grant from Tennessee Technological University.
Assessing DNA Cross‐Linking and Repair in Human Leukemia Cells
Authors: Le Phuong M. , Millard Julie T.
Published: November 2023

The anti‐tumor effects of bifunctional alkylating agents have been recognized for more than 50 years, notably in the frontline chemotherapeutic drug, nitrogen mustard and its derivatives. These compounds are double‐edged swords: killing both cancerous and non‐cancerous cells by forming cytotoxic DNA adducts via interstrand and intrastrand cross‐links. Intrastrand crosslinks can be readily removed by nucleotide excision repair mechanisms while interstrand crosslinks (ICLs) pose a challenging block to DNA replication, transcription, and repair and are hence a potentially lethal event. Using human leukemia (HL‐60) cells as an in vitro model, we are investigating the cytotoxicity and DNA repair of ICLs induced by nitrogen mustard (HN2), epichlorohydrin (ECH), and 1,2,3,4‐diepoxybutane (DEB)‐the active form of the anti‐ovarian cancer drug Ovastat. The median lethal dose (LD50) of each compound was determined via the AlamarBlue assay over a wide a range of drug concentrations. DNA cross‐linking activity and repair were quantified via single cell gel electrophoresis (the comet assay). Drug‐induced ICL formation retarded the mobility of hydrogen peroxide‐treated DNA in an electric field, creating a comet‐like migration pattern at the individual cell level. The shorter and brighter the comet tail was, the more crosslinks were present. Preliminary data showed a consistent cytotoxicity trend after a 12‐hour or 24‐hour drug treatment with HN2 > ECH ≥ DEB. Comet assay analysis from treating cells with equitoxic doses revealed that the percentage decrease in tail moment, a combined parameter that includes the comet tail length and fluorescence intensity, varied between different drugs, corresponding to their different cross‐linking potentials. Interestingly, cross‐linking activity was maintained in some, but not all, compounds at 24 hours compared to 12 hours, suggesting differential rates of DNA repair. With these quantitative investigations, we hope to correlate the toxicity of these bifunctional alkylating agents with their cross‐linking specificity. Our ultimate goal is to elucidate the underlying mechanism by which ICLs induced by one drug are more susceptible to removal and repair than another. These findings can improve our understanding of dose‐dependent drug cocktail administration and the long‐term effects of these chemotherapeutic drugs.

Support or Funding Information

This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.
Molecular Characterization of UHRF1 and UHRF2
Authors: Winkler Alyssa , Albaugh Brittany
Published: November 2023

UHRF1 and UHRF2 are multidomain epigenetic proteins that function to bind modified histones and subsequently regulate gene expression, particular those involved in cancer development. The Tandem‐Tudor Domain (TTD) and Plant Homeo Domain (PHD), are structural motifs that are proposed to bind trimethylated lysine‐9 and unmodified lysine 4 on histone H3, respectively. Here, we investigate the functional and structural interactions between the PHD and TTD UHRF1/2 protein domains and modified histones. Using fluorescence polarization assays, we have assessed the relative binding affinities for the linked TTD‐PHD domains of both UHRF1 and UHRF2, and found that there was no significant difference in binding affinities between the UHRF1 and UHRF2 TTD‐PHD linked domains. We continued investigating specific structural roles within the domains through mutagenesis of amino acids of interest.
Molecular Characterization of the PHD domains of UHRF1 and UHRF2 Ahmed Mohamed, Dr. Brittany Albaugh
Authors: Mohamed Ahmed , Albaugh Brittany
Published: November 2023

UHRF1 and UHRF2 are two epigenetic proteins that are involved in cancer development. These two proteins have similar amino acid sequences but play opposite roles in cancer. UHRF1 is an oncogene and UHRF2 is a tumor suppressor. Both of these proteins contained a histone reader domain called Plant Homeo Domain (PHD) which binds to Histone H3. Although previous studies determined the binding affinity of UHRF1 to H3, the binding affinity of UHRF2 to H3 was unknown. In this study, we determined the binding affinities of the PHD domains of UHRF1 and UHRF2 to both trimethylated lysine on Histone 3 (H3K9me3) and unmodified H3, by fluorescence polarization assay. We found the Kd of UHRF1‐PHD to H3K9me3 was 0.9 μM and the Kd of UHRF2‐PHD to H3K9me3 was 7.3 μM. The Kd of UHRF1‐PHD to unmodified H3 was 2.1 μM, and the Kd of UHRF2‐PHD to unmodified H3 was 22.3 μM. Theses results suggests that PHD of UHRF2 has lowered binding affinity to H3 versus UHRF1, and these proteins use a distinct mechanism of histone binding.