bluuebunny/crossref_metadata_2025 huggingface dataset:
MCT Expression in the Ventromedial Hypothalamus is Not Increased by Recurrent Hypoglycemia
Authors: Oberlin Douglas J , Fecych Mariel , Murray Coleman , Morrison Ron , Wideman Laurie , Beverly Lee
Published: November 2023

Recurrent hypoglycemia (RH) has been shown to lead to deficits in the counter‐regulatory response to subsequent hypoglycemia; however, the exact mechanisms of this effect are unknown. A proposed explanation has included increased lactate shuttling from astrocytes to neurons, which would blunt the change in glucose availability in the ventromedial hypothalamus (VMH). The objective of the current study was to measure changes in the lactate shuttling monocarboxylate transporter (MCT) proteins in the VMH after RH or control. Male Sprague‐Dawley rats (~250 g) received a subcutaneous injection of 0.9% saline or insulin (3–5 U/kg) on 3 consecutive days. Blood glucose was measured 30 minutes post‐injection via a pedal vein stick and glucometer. Animals were sacrificed 24 hr after the last episode of hypoglycemia, and the VMH was rapidly extracted, homogenized, and frozen at −80 c. MCT1, MCT2, MCT4, and GAPDH (as a loading control) were measured by western blot analysis. Quality One software was used to determine band density and the ratio of each MCT:GAPDH was calculated for each MCT in the VMH. MCT differences between groups were assessed using ANOVA (SAS 9.3).

Results

There was a significant decrease in average 30 minute post‐injection glucose in the RH group (f=402.237, p<0.001). The results showed no significant differences between the RH and control group for MCT proteins (λ=0.867, p=0.404; MCT1 F=2.87, p=0.104; MCT2 F=2.14, p=0.157; MCT4 F=0.07, p=0.796). In conclusion, these findings suggest that RH does not increase MCT expression in the VMH and that increased lactate shuttling is not likely to mediate the impaired counter‐regulatory response after recurrent hypoglycemia.

Support or Funding Information

Supported by NIH Grant DK082609
The 3D organization of the Mammalian Genome
Authors: Ren Bing
Published: November 2023

The 3D genome organization plays an essential role in orchestrating the many essential nuclear processes including gene regulation. Great strides have been made recently to characterize the 3D chromatin architecture in mammalian cells, with the development of both technologies for genome‐wide study of chromatin interactions at increasing precision and concepts such as “chromosome territories”, “topologically associating domains” and “chromatin loops”. I will discuss how these new concepts are transforming the study of gene regulatory mechanisms in mammalian cells, by giving specific examples that illustrate the role of 3D chromatin organization in developing revolutionary tools in sequencing diploid genomes, understanding molecular basis of genetic disorders caused by genome structural variations, and predicting target genes of enhancers.

Support or Funding Information

LICR, U54 HG006997, U54 DK107977, U01 CA200147
Complex Multi‐Enhancer Contacts Captured By Genome Architecture Mapping, A Novel Ligation‐Free Approach
Authors: Pombo Ana , Beagrie Robert A , Scialdone Antonio , Schueler Markus , Chotalia Mita , Xie Sheila Q , Kraemer Dorothee , de Santiago Ines , Fraser James , Dostie Josee , Game Laurence , Dillon Niall , Edwards Paul AW , Nicodemi Mario
Published: November 2023

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key elements of transcriptional control and their disruption causes disease. Technologies based on chromosome conformation capture (3C) have profoundly expanded our understanding of the role of genome architecture in gene regulation. However, 3C‐based techniques have important limitations, many of which arise from their reliance on digestion and ligation of the interacting DNA segments. We present a new genome‐wide method, Genome Architecture Mapping (GAM) for measuring three‐dimensional chromatin topology without ligation.

We use this new method to generate a genome‐wide dataset of chromatin interactions in mouse ES cells, and compare to published Hi‐C data. GAM identifies specific chromatin contacts enriched for interactions between active genes and enhancers across very large genomic distances. GAM also reveals abundant three‐way contacts genome‐wide, especially between the enhancers most highly occupied by pluripotency transcription factors and highly transcribed genomic regions. These contacts are most prominent for sequences further away from the nuclear lamina. Our results shed light on a previously inaccessible complexity in genome architecture and a major role for gene‐expression specific contacts in organizing genome architecture of mammalian nuclei.

Support or Funding Information

The work was supported by the Medical Research Council, UK (AP, RAB, MC, SQX, IdS, LG, ND), by the MRC‐Technology (AP, MC), by the Helmholtz Foundation (AP, MS, MB, DK), by Breast Cancer Campaign (PAWE) and by Cancer Research UK (PAWE). Work by JD and JF was supported by grants from the Canadian Institutes of Health Research (CIHR) [MOP‐86716, CAP‐120350].
Chronic Binge Alcohol Exacerbates Apoptotic Signaling in Simian Immunodeficiency Virus‐infected Macaques
Authors: Maxi John K. , Bagby Gregory J. , Nelson Steve , Winsauer Peter J. , Molina Patricia E.
Published: November 2023

HIV‐associated neurocognitive disorder (HAND), characterized by altered pre‐frontal cortex (PFC) mediated behaviors, affects ≈50% of persons living with HIV/AIDS. Alcohol use accelerates the development of HAND. Our studies have shown that chronic binge alcohol administration (CBA) unmasks behavioral deficits in simian immunodeficiency virus (SIV)‐infected macaques. However, the mechanisms underlying these alcohol‐induced behavioral deficits remain unknown. Alcohol administration and HIV/SIV neuronal damage result, at least in part, from excitotoxic NMDA receptor signaling stimulating mitochondrial pro‐apoptotic mechanisms. Recently, it has been recognized that loss of pro‐survival factors, specifically Brain‐Derived Neurotrophic Factor (BDNF), also contributes to alcohol and HIV/SIV‐induced neuronal damage. Thus, we hypothesized that CBA administration to SIV‐infected macaques would result in greater excitotoxic signaling and impaired BDNF‐mediated cell signaling in the PFC, suggesting a potential mechanism underlying exacerbation of HAND. Adult rhesus macaques were fitted with intragastric catheters for the infusion of alcohol (n=3, CBA/SIV, 13–14 g/kg/week) or sucrose (n=3, SUC/SIV), beginning three months prior to inoculation with SIV, and continued for the duration of the study. Animals were euthanized upon reaching end‐stage disease (13 – 23 months post SIV) and PFC tissue was isolated for mRNA and protein analysis by qPCR and Western blot, respectively. PFC tissue from non SIV‐infected macaques (SIV‐, n=3–5) was used to normalize mRNA and protein expression. PFC of CBA/SIV macaques showed increased NMDA receptor subunits NR1 and NR2B relative to dendritic marker MAP2 (1.5‐fold increase), and increased Bax:Bcl‐2 ratio (10‐fold increase), suggesting enhanced excitotoxic apoptotic signaling. Additionally, we found decreased expression of BDNF mRNA (2‐fold reduction); activated BDNF receptor (pTrkB) (15‐fold lower); and pAKT levels (3‐fold reduction) in CBA/SIV animals compared to that of SUC/SIV animals. These findings indicated that CBA exacerbates apoptotic signaling in the PFC of SIV‐infected macaques. We predict these findings implicate increased PFC neuronal apoptosis as a likely mechanism underlying accentuated neurocognitive impairment in CBA/SIV macaques.

Support or Funding Information

P60AA09803, T32AA07577, F31AA024365
Effects of water‐immersion restraint stress on some bio‐chemical parameters in Wistar rats and roles of selenium and vitamin E
Authors: Sadau Y. , Adelaiye A. B. , Magaji R. A. , Ayo J. O. , Isa A. I.
Published: November 2023

Stress is an aversive stimulus, which disturbs physiological homeostasis; it induces the strain upon both emotional and physical endurance, which has been considered the basic factor in the aetiology of a number of diseases. The aim of the present study was to determine the effects of water immersion restraint stress (WRS) on some bio‐chemical parameters and role of selenium and vitamin E in Wistar rats. (n= 35) Male Wistar rats weighing 200–220g were divided in to five groups of seven rats each: (i) passive control (non‐stress rats), (ii) active control (WRS plus distilled water), (iii) WRS plus vitamin E, (iv) WRS plus vitamin E plus selenium, and (v) WRS plus selenium. The WRS procedure lasted for 3.5 hours. Blood samples were collected through cardiac puncture for biochemical analyses. The result of the present study demonstrated that acute WRS significantly increase the activities of plasma ALT, AST, ALP and MDA concentration. WRS leads to significant decrease in the total plasma protein concentration and activities of antioxidants enzymes, SOD, GSH and Catalase. However pre‐treatment with selenium and vitamin E individually or in combination significantly lowered the activities of these liver enzymes and MDA concentration and significantly increase plasma protein concentration and the activities of the antioxidant enzymes. The effects were considerable in selenium and vitamin E co‐administration. It was thus concluded that WRS exposure causes significant alteration in on some liver function diagnostic and lipid peroxidative indices, total plasma protein and activities of antioxidant enzymes. However pre‐treatment with selenium or vitamin E ameliorate some of the recorded effects of WRS.
Acute Stress Diminishes M‐Current in Corticotrophin‐Releasing Hormone Neurons through AMPK Activation
Authors: Li De pei , Zhou Jing‐Jing , Gao Yonggang , Li De‐Pei
Published: November 2023

Acute stress activate hypothalamus‐pituitary‐adrenal gland axis and corticotrophin‐releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of hypothalamus. However, the molecular and cellular mechanisms underlying hyperactivity of PVN‐CRH neurons in acute stress are not clear. M‐channel is regulated by AMPK activity and plays important role in the regulation of neuron activity. We hypothesized that M‐channel function is impaired in PVN‐CRH neurons during acute stress. Whole‐cell patch‐clamp recording were performed on PVN‐CRH neurons identified by specific expression of eGFP driven by the rat crh premotor. Acute stress (2‐hr restraint) profoundly increased firing rate of PVN‐CRH neurons while decreased M‐current in these neurons. M‐channel blocker XE991 induced lesser increased in firing activity of PVN‐CRH neurons in acutely stressed rats compared with unstressed rats. Microinjection of XE991 into the PVN produced significantly lesser increases in plasma corticosterone concentration in acute stress rats than in unstressed rats. Furthermore, acute stress decreased KV7.3 subunit membrane‐fraction protein expression levels while increased phosphorylated AMPK level in the PVN tissues compared with unstressed rats. However, the Kv7.3 cytoplasma‐fraction protein level and Kv7.2 subunit expression level in the PVN tissue. Treatment with AMPK inhibitor compound C not only recovered the Kv7.3 membrance‐fraction expression level but also reinstalled the reduced M‐current and XE991‐induced excitatory effect on PVN‐CRH neruons in acute stressed rats. Finally, microinjection of compound C into the PVN reinstalled XE991‐induced increase in plasma corticosterone levels. Collectively, these results suggested that acute stress diminishes M‐current in PVN‐CRH neurons, which is related to AMPK activation.

Support or Funding Information

This study was supported by NIMH grants MH096086.
Angiotensin type 1A receptor expressing neurons in the NTS receive direct solitary tract excitatory neurotransmission
Authors: Carter David A , Guo Haoyao , Allen Andrew M , McDougall Stuart J
Published: November 2023

Second order neurons in the nucleus of the solitary tract (NTS) receive direct viscero‐sensory afferents from internal organs via the solitary tract (ST) to coordinate autonomic reflexes. Approximately 90% ST vagal afferents from the heart and lungs are unmyelinated c‐fibres and express transient receptor potential vallinoid type 1 (TRPV1). Activation of the angiotensin type 1A receptor (AT1AR) in the NTS modulates cardiorespiratory reflexes and is thought to potentiate ST excitatory input. Here we aimed to characterise ST input to AT1AR expressing NTS neurons, and determine what proportion of the monosynaptic afferents express TPRV1. Second, we determined the impact of angiotensin II (AngII) on ST neurotransmission to AT1AR expressing NTS neurons. We employed in vitro slice electrophysiology and whole cell recordings. Twenty‐two transgenic mice expressing green fluorescent protein in AT1AR neurons were sacrificed and 250 μm horizontal brain stem slices were prepared and superfused with artificial cerebrospinal fluid. Electrical stimulation of the ST evoked low jitter excitatory post‐synaptic currents (ST‐EPSCs) in 28 of 41 GFP positive neurons, identifying them as second order. The normalised amplitude (eEPSC/eEPSCMAX) and paired pulse ration (PPR) of ST‐EPSCs (50 Hz, 200 μs) and frequency of spontaneous (F‐sEPSCs) and asynchronous EPSCs (F‐aEPSCs) were analysed before and during the addition of 100 nM capsaicin (CAP) or 2 μM AngII to the superfusate. CAP decreased F‐aEPSCs (CSF: 13.9 ± 2.3 Hz, CAP: 0.3 ± 2.7 Hz, n = 5), however only inhibited ST‐EPSCs in 1/5 second order neurons tested. AngII had no effect on ST‐EPSC amplitude (CSF: 0.84 ± 0.03, AngII: 0.85 ± 0.05), PPR (CSF: 0.34 ± 0.05, AngII: 0.34 ± 0.04, n = 8) or F‐sEPSC. Combined, this data indicates that NTS AT1AR neurons receive direct viscerosensory signals mostly via TRPV1‐ve pathways. Further, that AT1AR is not likely to be expressed on the excitatory ST axons/terminals contacting these AT1AR expressing NTS neurons. This suggests AT1AR neurons in the NTS receive predominately Aδ‐fibre input which relay high fidelity, low threshold signals from the viscera through the central nervous system.

Support or Funding Information

This work is supported by a National heart foundation post‐graduate research scholarship.
Heparanase contributes to sepsis‐associated encephalopathy and neurovascular glycocalyx degradation
Authors: Ford Joshay , Orfila James , Perez Mario , Yang Yimu , Haeger Sarah , Herson Paco , Schmidt Eric
Published: November 2023

While there has been increasing recognition of long‐term neurocognitive dysfunction in survivors of sepsis (termed sepsis‐associated encephalopathy, SAE), little is known about its underlying pathogenesis. We developed a mouse model of septic neurocognitive dysfunction by investigating the effects of sepsis on synaptic plasticity in the hippocampus (an area important in learning and memory). As heparan sulfate (HS) is known to contribute to the regulation of hippocampal synaptic plasticity, we sought to determine the role of SAE with heparanase, a HS‐specific glucuronidase that contributes to the pathogenesis of septic lung and kidney injury. In wild‐type mice, cecal ligation and puncture (CLP) induced physical signs (piloerection, lethargy, and weight loss) of sepsis that peaked by 24–48 h; these signs abated before 7 days. Despite this apparent resolution of septicemia, CLP significantly impaired hippocampal long‐term potentiation 7 days after injury, indicative of SAE. Similar findings were noted with intraperitoneal lipopolysaccharide (LPS, 20 μg/g), a non‐infectious model of sepsis. Consistent with our previous observations in septic lung and kidney injury, CLP increased brain heparanase expression at 48 h, with induction of both proheparanase and active heparanase. Pharmacologically blocking heparanase expression attenuated the loss of hippocampal long‐term potentiation 7 days after CLP or LPS administration. Interestingly, in vivo brain microscopy revealed that endotoxemia was associated with heparanase‐mediated loss of neurovascular glycocalyx thickness, suggesting a potential vascular‐specific mechanism by which septic heparanase induction may induce SAE. In conclusion, CLP induces neurocognitive dysfunction that persists after overt sepsis resolution; this dysfunction was attenuated by heparanase inhibition. These findings identify a potential animal model of SAE as well as possible pathophysiologic mechanisms underlying its development.

Support or Funding Information

K08 HL105538
Reduction of Astrocyte and Microglial Activation in Insulin/Saline‐treated Diabetic Ketoacidosis by the KCa3.1 Blocker TRAM‐34
Authors: Glaser Nicole S , Little Christopher J , Cohen Michael D. , Tancredi Daniel J. , Wulff Heike , O'Donnell Martha E.
Published: November 2023

Previous studies have provided evidence that diabetic ketoacidosis (DKA) and/or its treatment with insulin and saline (I/S) causes brain injury in children. However, mechanisms underlying DKA‐related brain injury have been poorly understood. In recent studies using immunohistochemistry (IHC) in a streptozotocin (STZ)‐induced DKA juvenile rat model, we examined expression of glial fibrillary acidic protein (GFAP) and CD‐11b protein (OX42) in hippocampus, cortex and striatum at 4, 24 and 72 hr after I/S treatment. We found increased GFAP in hippocampus as well as increased OX42 in both hippocampus and cortex, suggesting that reactive gliosis and microglial activation occur during DKA‐related brain injury. Other studies have shown that TRAM‐34, a KCa3.1 channel blocker, reduces microglial activation in the rat middle cerebral artery occlusion model of cerebral ischemia. The present study was conducted to determine whether TRAM‐34 could also reduce DKA‐related inflammatory changes in the brain. We used IHC to investigate the effects of TRAM‐34 on GFAP and OX‐42 expression in brain sections from juvenile rats with STZ‐induced DKA before, during and after treatment with insulin and saline. As in previous studies, OX42 staining intensity was increased in the hippocampus and cortex during DKA and increased further 4 hours after I/S treatment in a manner sustained through 24 and 72 hours after recovery from DKA. GFAP staining intensity was also increased over the same time course in hippocampus but not cortex. However, in rats administered TRAM‐34 (IP, 40 mg/kg) at the start of I/S treatment, staining intensities of both OX42 and GFAP were significantly reduced at all time points after DKA. OX42 staining intensity was also decreased during DKA prior to I/S treatment in TRAM‐34 treated rats. We conclude that DKA causes inflammatory changes in the brain including reactive gliosis and activation of microglia. Treatment with TRAM‐34 not only decreases microglial activation but also reduces reactive gliosis, suggesting an overall decrease in the brain's inflammatory response to DKA.

Support or Funding Information

Supported by ADA
Inhibition of sodium bicarbonate cotransporter exaggerates ischemic penumbral cell death
Authors: Yao Hang , Azad Priti , Zhao Huiwen , Poulsen Orit , Haddad Gabriel G
Published: November 2023

Sodium bicarbonate co‐transporter (NBC) is the major bicarbonate‐dependent acid‐base transporter in mammalian astrocytes and has been implicated in ischemic brain injury. Nevertheless, the role of NBC in the ischemic penumbral cell death has not been well understood. In this work, we obtained skin biopsies from a healthy human subject and reprogrammed skin cells into induced pluripotent stem cells (iPSCs). These iPSCs were further differentiated into neuroprogenitor cells (NPCs) and then into astrocytes. These astrocytes express GFAP, S100β and Aquaporin 4 and readily propagate calcium waves. Using pH sensitive dye BCECF and PCR technique, we have confirmed that these astrocytes express functional electrogenic sodium bicarbonate cotransporter (NBCe). Using an ischemic solution (IS) that mimics the ischemic penumbral environment and a NBC blocker S0859, we have studied the involvement of NBCe1 in IS‐induced human astrocytes death. q‐PCR result shows a significant increase in NBCe1 gene expression following a 24 hour IS treatment while this does not occur in NBCn1. Cell viability was analyzed by propidium iodide stained cell counting which was normalized by the Hoechst stained cell counting and presented as the percentage of injured cell number over the entire cell counting. A 24 hr incubation of S0859 at concentration range from 0.1 to 100 μM did not induce any cell injuries in astrocytes in the normal condition. A 24 hr IS treatment resulted in a 43.6±4.5% cell death in these astrocytes (n=10). While a 24 hr IS + 30 μM S0859 elicited a 97.5±1.6% cell death (n=10), which is significantly different from that seen in IS treatment group (p<0.001). In summary, a NBC blocker exaggerates IS‐induced cell death which suggests that the activities of NBC (possibly NBCe1) are essential for astrocytes to survive the ischemic penumbral environment.

Support or Funding Information

This project was supported by NIH 5P01HL098053
Direct pathway from ventral medial prefrontal cortex to dorsomedial hypothalamus drives psychological stress‐induced hyperthermia
Authors: Kataoka Naoya , Nakamura Kazuhiro
Published: November 2023

Psychological stress‐induced hyperthermia is a basic autonomic stress response observed in many mammals including humans. We have reported that stress induces thermogenesis in brown adipose tissue (BAT), hyperthermia and tachycardia by activating a direct neural pathway from the dorsomedial hypothalamus (DMH) to the rostral medullary raphe (rMR) (Kataoka et al. Cell Metabolism, 2014). However, the mechanism by which stress signals activate the DMH neurons driving the sympathetic stress responses remains unknown. In this study, we sought for the upper neurons that provide the DMH with such psychological stress inputs. Retrograde tracing with the retrograde neural tracer, cholera toxin b‐subunit (CTb) revealed numerous projections from the medial prefrontal cortex (mPFC) to the DMH. Stimulation of mPFC neurons with a nanoinjection of bicuculline, a GABAA receptor antagonist, in anesthetized rats increased BAT sympathetic nerve activity, BAT temperature and heart rate. These bicuculline‐evoked sympathetic responses were eliminated by bilateral inhibition of DMH neurons with muscimol nanoinjections. Importantly, rats exposed to social defeat stress, a sociopsychological stress model, exhibited expression of Fos, a marker for neuronal activation, in DMH‐projecting neurons in the ventral part of the mPFC (vmPFC), and inhibition of vmPFC neurons with muscimol nanoinjections strongly reduced BAT thermogenesis and hyperthermia evoked by social defeat stress in free‐moving rats. In contrast, inhibition of neurons in the infralimbic cortex, a dorsal part of the mPFC, exhibited a weaker inhibitory effect on these stress responses. Furthermore, selective stimulation of vmPFC–DMH monosynaptic transmission using an in vivo optogenetic technique elicited BAT thermogenesis and heart rate. These results indicate that the vmPFC–DMH monosynaptic pathway provides the DMH with stress signals to activate the DMH–rMR sympathoexcitatory pathway driving BAT thermogenesis for the development of stress‐induced hyperthermia.

Support or Funding Information

NEXT Program (LS070), KAKENHI (21890114, 22689007, 24790233, 26118508, 26713009), Nakajima Foundation, Takeda Science Foundation, and Kowa Life Science Foundation
Blockade of Acid‐Sensing Ion Channels Attenuates the Skeletal Muscle Metaboreflex in Humans
Authors: Fernandes Igor Alexandre , Campos Monique Opuszcka , Mansur Daniel Elias , Mattos João Dario , Rocha Marcos Paulo , Rocha Natalia Galito , Nóbrega Antonio Claudio Lucas
Published: November 2023

Recent animal studies indicate that acid‐sensing ion channels (ASICs) play a role in evoking the metabolic component of the exercise pressor reflex. Nonetheless, it is still unclear whether these findings can be translated to humans. Our purpose was then to investigate the contribution of ASICs in mediating the metaboreflex‐induced increase in blood pressure and muscle sympathetic nerve activity in a human model. Five healthy subjects (32 ± 5 yrs) underwent two experimental conditions: (1) Control (C) and (2) Amiloride (AMD, 3 hours after oral administration of 5 mg of amiloride + 50 mg of hydrochlorothiazide), a non‐selective antagonist of ASICs. In each condition, beat‐by‐beat mean blood pressure (MBP, Finometer), muscle sympathetic nerve activity (MSNA, peroneal nerve microneurography) and femoral artery blood flow (FBF, Doppler ultrasound) were recorded during rest, static handgrip exercise (SHE, 2 min at 30% of the maximal voluntary contraction) and post‐exercise muscle ischemia (PEMI, 2 min). Blood samples were taken from the antecubital vein of the exercising arm during the last 30 s of PEMI and changes on lactate concentration were determined. MBP responses were slightly but significantly attenuated during SHE in the AMD condition (AMD +29.6 ± 7.0 vs. C +35.3 ± 8.4 mmHg, p = 0.05). A smaller increase in MSNA (N = 4) was also observed as a consequence of ASICs blockade (AMD +6.1 ± 3.5 vs. C +12.5 ± 4.2 bursts/min, p = 0.013). Similarly, AMD attenuated the PEMI‐induced increases in both MBP (AMD +18.9 ± 2.0 vs. C +31.35 ± 4.3 mmHg, p = 0.02) and MSNA (AMD +10.0 ± 4.3 vs. C +21.6 ± 1.4 bursts/min, p = 0.04). Increases in lactate concentration (AMD +1.00 ± 1.5 vs. C + 1.85 ± 0.15 mmol/l, p > 0.05) were similar between the experimental conditions. Femoral blood flow and conductance did not present substantial changes (p > 0.05). Although AMD antagonizes ASICs when administered in small doses, its influence on epithelial or voltage‐gated sodium channels and a related impairment in the impulse conduction in sensory nerves must not be discarded. To test the hypothesis that the attenuation of the skeletal muscle metaboreflex was only a consequence of non‐selective blockade of ASICs, MBP (N = 4) and MSNA (N = 2) responses were also recorded during rest and a cold pressor test (CPT, 20 min after PEMI). AMD had no impact on MBP (AMD +23.6 ± 3.7 vs. C +20.5 ± 4.2 mmHg; P > 0.05) and MSNA (AMD +22.9 ± 3.0 vs. C +23.6 ± 6.3 bursts/min, P > 0.05) responses during CPT, indicating that there was no impairment on sensory nerves ability to generate and conduct action potentials. Therefore, our findings suggest that ASICs also play a role in evoking the skeletal muscle metaboreflex in humans.

Support or Funding Information

Grant Funding Source: CNPq, FAPERJ, CAPES e FINEP
Developmental changes in breathing pattern and neuronal morphology in the respiratory centers of the neonatal rat
Authors: Williams Paul , Murray Samuel , Wilson Christopher
Published: November 2023

The autonomic brainstem generates and controls breathing rhythm. Several studies have shown that control of breathing changes during early development. However, little is known about changes in the morphology of neurons in the respiratory regions of the brainstem during early development and how these morphological changes correlate with changes in breathing pattern. We hypothesize that developmental changes in morphology of neurons in the preBötzinger Complex (pBC), the nucleus tractus solitarii (nTS), and the hypoglossal motor nucleus (XII) are correlated with changes in breathing pattern. To test this hypothesis, we used Golgi‐Cox staining, a high‐resolution bright‐field stain, to examine the somata and dendritic tree of developing neurons in pBC, nTS, and XII. Before staining, we used whole‐body plethysmography to assess the breathing pattern of animals from postnatal day 1 (P1) through postnatal day 21 (P21). We quantified changes in tidal volume, flow, and minute ventilation. We also used automated detection software to quantify breathing rate (breaths/minute), inspiratory and expiratory times, number of breathing pauses (apneas) and variability of breathing pattern (Sample and Shannon entropy). We then removed the brain from each animal in our sample cohort, sectioned the brain using a vibratome, and performed Golgi‐Cox staining to examine dendritic morphology of the neurons in the pBC, nTS, and XII. The number of apneas per minute and breathing rate increased linearly to P10. Sample and Shannon Entropy, measures of signal complexity, decreased from P1 to P10 and plateaued through to P21. The dendritic arbor of developing neurons becomes more complex from P1 through P10 with increased branching and spine numbers which correlated with the changes in breathing pattern.
Mechanisms involved in sodium depletion‐induced facilitation of respiratory responses to baroreceptor activation
Authors: de Paula Patricia Maria , Borsari Karine Cristina , Zoccal Daniel Breseghello , Menani Jose Vanderlei
Published: November 2023

Disorders in blood volume may cause behavioral, hormonal and autonomic responses in order to restore homeostasis. The model of sodium depletion elicited by furosemide promotes neuroendocrine responses, reductions in vascular reactivity and blood volume, with no changes in blood pressure and heart rate (HR) due to the activation of the renin‐angiotensin system. It was demonstrated that changes in the sodium balance modifies the cardiorespiratory responses to peripheral baroreceptor activation in anesthetized conditions. In the present study, we explored the cardiorespiratory baroreflex responses in normovolemic and 24‐h sodium depleted conscious rats and evaluated the contribution the angiotensinergic mechanisms in the reflex responses of both groups. Male Holtzman rats (290–320 g, n=19/group) that received catheter implants into the femoral artery and vein were used. Baroreceptor activation was performed by intravenous (iv) phenylephrine (phenyl) infusions (5 μg/kg/ml) and ventilation (VE) was measured by whole body plethysmograph. Subcutaneous furosemide injections (20 mg/kg) followed by 24 h of sodium‐deficient diet was used to induce sodium depletion. Sodium‐depleted animals (n=19) showed similar baseline mean arterial pressure (102±2 vs 107±2 mmHg), HR (382±9 vs 385±7 bpm) and VE (468±27 vs 447±26 ml/min/kg) compared to normovolemic control animals (n=19). Phenyl infusions in normovolemic and sodium‐depleted rats produced similar pressor (Δ = 58±2 and 59±2 mmHg) and bradicardic (Δ = −97±10 and −120±11 bpm) responses. However, a larger increase in VE during baroreflex activation was observed in sodium‐depleted animals in comparison to controls (Δ = 152±40 vs 53±26 ml/min/kg, p<0.05). This increased VE response of sodium‐depleted rats was attenuated (Δ = 77±33 ml/min/kg) after the systemic antagonism of angiotensin II type 1 receptor with losartan (10 mg/kg). The results suggest that neuroendocrine changes produced by sodium depletion, mainly the increase in angiotensin II levels, facilitate the respiratory response to baroreceptor activation in conscious sodium‐depleted rats.

Support or Funding Information

FAPESP, FAPESP/Pronex, CNPq, Prope UNESP
Repeated moderate heat exposure decreases resting sympathetic activity in humans
Authors: Cui Jian , Blaha Cheryl , Sinoway Lawrence I.
Published: November 2023

Acute whole‐body heat stress evokes sympathetic activation. However, the chronic effects of repeated moderate heat exposure (RMHE) on muscle sympathetic nerve activity (MSNA) remains unclear. We performed the RMHE with 4 weeks (5 days/week) of warm baths (39–42 °C for 30 min) in 6 elder volunteers (5 M and 1 F, 59 ± 3 yrs, 177 ± 4 cm, 88 ± 10 kg, BMI 27.6 ± 2.3). Subjects were in the recumbent position in the bathtub with water up to their mid chest. Blood pressure (BP) and heart rate (HR) were monitored with an automated sphygmomanometer throughout each bath. The internal temperature (Tcore) was measured via an ingestible pill telemetry system during the 1st, 11th (i.e. day 15th), and last bath in each subject. Hemodynamic variables and MSNA were examined prior, 1 day post 4 weeks of RMHE and 1 week after end of RMHE in a laboratory at ~23 °C. During the baths, the maximal increase in Tcore was Δ0.9 ± 0.1 °C, the maximal increase in HR was Δ12 ± 2 beats/min, and the maximal drop in mean BP was Δ15 ± 2 mmHg. Under normothermic condition, the resting MSNA significantly decreased after RMHE (prior, post, post 1‐wk: 32.6 ± 2.5, 27.4 ± 2.4, 27.6 ± 2.2 bursts/min; P<0.001; one‐way repeated measures ANOVA). The MSNA burst incidence (i.e. bursts/100 heart beats) also decreased significantly (P=0.002). The resting HR decreased in all subjects after RMHE (60.2 ± 1.6, 56.5 ± 2.1, 56.8 ± 1.8 beats/min, P<0.05), while supine BP was not significantly altered (P>0.05). These data suggest that the RMHE evoked by warm bath is a simple and safe approach to decrease baseline sympathetic activity and HR. The mechanism(s) should be examined in future studies.

Support or Funding Information

AHA 15GRNT24480051 (JC) and P01 HL096570‐05 (LIS)
Single‐cell Imaging of Wnt Palmitoylation by Porcupine
Authors: Gao Xinxin , Hannoush Rami N
Published: November 2023

Visualization of the subcellular distribution of palmitoylated protein is critical to the understanding of the molecular mechanisms regulating protein movement and function in a cell. Due to a lack of suitable techniques, the trafficking route of palmitoylated forms of the protein of interest has remained unclear. To address this unmet need, we have developed a method to image the palmitoylated form of the protein of interest with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Using this imaging method, we have shown that palmitoylated Wnt3a can be visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We demonstrate that Wnt3a is palmitoylated by fatty acids 13–16 carbons in length at Ser209 but not at Cys77. We find that porcupine (PORCN) itself is palmitoy‐lated, and this modification partially regulates Wnt palmitoylation and signaling. The data here reveal the role of O‐palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion. Our method is broadly applicable to imaging the palmitoylation of cellular proteins, such as palmitoylated Shh, tubulin or Ras, as well as other protein post‐translational modifications that are detectable by clickable chemical reporters.
Molecular insights into the regulation of expression of snake venom toxin genes in Pseudonaja textilis
Authors: Han Xia , Acerbi Enzo , Ye Quan‐zhi , Kini R. Manjunatha
Published: November 2023

Snake venom toxin genes are specifically expressed in the venom gland when it is empty (e.g. after milking). Tissue‐ and time‐specific expression of toxin genes is regulated at transcriptional level by cis‐elements and transcription factors. Recently, we have identified that multiple AG‐rich motifs in intron 1 of trocarin D, a gene encoding venom prothrombin activator in Tropidechis carinatus, silence gene expression in both mammalian and unmilked venom gland cells. Several transcription factors were identified to bound AG‐rich motifs and repress gene expression. However, the complete regulatory mechanisms of specific expression of toxin genes are still unknown. To understand this, we have sequenced the transcriptomes of both milked and unmilked venom gland tissues from a common snake P. textilis (which possesses similar AG‐rich motifs). We have compared the expression levels of different genes and performed gene ontology enrichment analysis. We also analyzed the microRNAs for their expression levels and targets of regulation in these tissues. Here I will present our findings, which will contribute to the understanding of roles of transcription factors and microRNAs in the regulation of specific expression of toxin genes.

Support or Funding Information

Source of research support: Academic Research Grants from NUS
Acrylic drawers: an alternative for teaching sectional anatomy
Authors: de Carvalho Arthur Teixeira , Borges Carlos Augusto Servato , Júnior Valdir Mazzucato , Savoldi Marcelo , Bim Waldeci Roberto , Fazan Valéria Paula Sassoli
Published: November 2023

There is no question about the importance for medical students to learn and understand sectional anatomy, since imaging methods play an important role in medical diagnosis and therapeutic planning. The interpretation of these images, particularly CT scans and MRIs, requires a comprehensive and deep knowledge of sectional anatomy. After the development of the plastination technique, plastinated sections have been widely used to teach sectional anatomy. Nevertheless, plastination is still expensive and not readily available in most of the anatomy laboratories in medical schools in Brazil. Thus, we created an alternative method to teach cross sectional anatomy in the School of Medicine of Ribeirão Preto – University of São Paulo. The method consists in building a transparent acrylic box that holds several acrylic drawers inside. The box is closed on top by a lid and filled up with the fixative solution. The acrylic drawers go inside this box. They are completely closed but perforated on the corners, so the fixative solution can get in contact with the human body cross section that will fit closely inside the drawer. When the students use the specimens, they can be washed with running water to remove the fixative from the tissue and reduce the unpleasant fixative smell. The box can be filled up with water while the specimens are being used in the laboratory, so they will not get dry. After exposing our students to the method, we understand that the acrylic drawers method is not so utterly efficient as the plastination, presenting some disadvantages. For instance, the human body cross sections used in the drawers are thicker than those used in plastination; the anatomy specimens used are wet and not as durable compared to plastination; and touching the cross sections inside the acrylic drawers is not possible. However, the acrylic drawers strategy was shown, indeed, to be an affordable and viable method which helps our future medical doctors not only to understand minute details of human anatomy, but also to scrutinize the anatomic syntopies and relative position of body structures. In spite of some specific disadvantages, the acrylic drawers method is an outstanding inexpensive solution for studying cross sectional anatomy, which is vital to the medical interpretation of imaging procedures.

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FAEPA, CNPq, FAPESP
Developing essential anatomical competencies in an osteopathic medical curriculum without cadaver dissection
Authors: Donohue Miriam L. , Rollag Mark D , Ketchum Robert J , Selinfreund Richard H , Ontiveros Steven J , Galin Claire M. , Howell Sherri J , Minugh‐Purvis Nancy
Published: November 2023

The benefits of not scheduling cadaver dissection into first year medical curricula are clear: numerous hours otherwise spent in the gross lab become available for distribution among the many other areas which curriculum designers increasingly find themselves pressured to include intoever more intense, challenging courses of study. However, the risks of eliminating cadaver dissection include depriving students of a major medium for tactile training and the development of manual skills honed by dissection. Perhaps even more significantly, eliminating cadaver dissection poses a greater challenge to students’ acquisition of a working knowledge of human gross anatomy and obtaining a competent understanding of structural organization necessary for patient examination and interpreting diagnostic imaging studies – crucial to many aspects of competent medical practice. Burrell College of Osteopathic Medicine's integrated first year curriculum, being developed for the arrival of its inaugural class in August 2016, excludes cadaver dissection by training students to examine anatomical structure first through 2‐D and 3‐D cadaver dissection image examination using virtual systems such as the Anatomedia Online and Anatomage platforms, followed closely by examination of diagnostic images, skeletal models, and model patient examination. In the Osteopathic Manipulative Medicine(OMM) lab, using imaging studies as their roadmap, students will obtain crucial tactile skills and anatomical knowledge, with the advantage of palpating tissues, anatomical features and landmarks on living tissue of model patients rather than non‐compliant cadaveric tissue. Cognitive correlations to multiple imaging modalities are incorporated into the curriculum from day one to ensure immediate student exposure to reading patient x‐ray, ultrasound, CT, MRI, and other images. The spiral design of BCOM’ sorgan system based curriculum facilitates content review while further building upon these skills throughout the first year. In the second year, anatomical imaging integrated into pathology presentations will continue to expand and refine expertise in these areas. The objective is to train a physician with a solid understanding patient anatomy for the purposes of physical exam as well as OMM, and competent in utilizing diagnostic imaging technologies dependent upon anatomical knowledge early in pre‐clinical education.
Restoring Antiendotoxin Activities of Inactive Antimicrobial Peptides
Authors: Cheng Jya‐Wei , Chu Hung‐Lun , Chih Ya‐Han , Wu Chih‐Lung , Yu Hui‐Yuan , Chou Yu‐Ting
Published: November 2023

Gram‐negative bacteria cell wall is composed of two layers: the inner phospholipid membrane and the outer membrane with a highly conserved unique lipid called lipopolysaccharide (LPS; endotoxin). LPS acts as a permeability barrier against a number of bactericidal agents. Furthermore, LPS is well known as a potent inducer of the immune system when it is released to blood and often causes septic shock syndromes in human. Some but not every antimicrobial peptide can neutralize LPS stimulated proinflammatory responses. We have developed an easy method to restore antiendotoxin activities of inactive antimicrobial peptides by linking two inactive α‐helical peptides with the AGP sequence. Antibacterial activities were determined by the standard broth micro‐dilution method of National Committee for Clinical Laboratory Standards. Toxicities of the peptides were determined from measuring cell death by the MTT assay against human fibroblast (HFW cells). Dynamic light scattering (DLS) was used to measure the size increase of LPS in the presence of the peptides. The ability of LPS neutralization in vitro was measured by Limulus Amebocyte Lysate (LAL) assay. LPS can induce nitrite oxide (NO) and TNF‐α production in macrophage cells. Experimental mouse endotoxemia model was used to test the antiendotoxin activities of the peptides. Significant antiendotoxin effects were observed in vitro and in vivo with the AGP linked variant of the short α‐helical peptides KR12 and WR6. The results will be used to help us design more potent antimicrobial peptides for clinical application in the future.

Support or Funding Information

Grant Number 103‐2113‐M‐007‐009‐MY2, Ministry of Science and Technology, Taiwan
Rapid differentiation of astrocytes from human ESCs
Authors: Han Baek‐Soo , Kim Won‐Kon , Lee Cheon Ok , Byun Jeong Su , Lee Sang Chul
Published: November 2023

Astrocytes are the most abundant cells of the human brain and have the role of biochemical support of endothelial cell, supply of nutrients to the nervous tissue and maintenance of extracellular ion balance etc. In development of nervous tissue, the differentiation of astrocytes is later than neurons. In vitro, it takes more time and more techniques to obtain mature and pure astrocytes.

In this study, we developed the protocol to gain mature and pure astrocytes from human embryonic stem cells. To keep the quality of the differentiated astrocytes and to decrease the variations of the cell properties between batch to batch, we first tried to get neural progenitor cells (NPCs) and expand the cell number. The first step of astrocyte differentiation from neural progenitor cells were performed in astrocyte induction media containing EGF and heparin. Next, the cells were attached to coated plate in astrocyte defined media containing CNTF for 23 days. And then we checked the cell property with immunocytochemistry and western blot using antibodies for astrocyte specific marker proteins. To validate the functional property of the astrocyte, IL‐6 release from astrocytes was tested. By FACS analysis, we found that the percentage of astrocytes among the cells differentiated from NPCs was over 90%. Taken together, with our protocol, we can obtain the mature and pure astrocytes within 4 weeks from NPCs and we have the plan to make micro‐environment with neurons, astrocyte and endothelial cells.

Support or Funding Information

This work was partly supported by grants from KRIBB and the Korea National Research of Foundation (No. 2012M3A9C7050101, 2011‐0030028 and 2013M3A9A7046301).
The Sterilized Fermented Feed Improves Intestinal Barrier Function and Immune Function in Chicken
Authors: Zhang Jun , Zhu Jianjin , Sun Jin , Jiang Rong , Rahman Md Ramim Tanver
Published: November 2023

The objective of this study was to evaluate the effects of dietary addition of fermentation metabolites by Sterilized fermented feed(SFF) as a replacement for fermented feed(FF) and antibiotics on expression of genes related intestinal barrier function and immune parameters in chicken. Three hundred and ninety‐six 1‐d‐old broilers with body weight of 41.27 ± 0.49 g were randomly allotted into 4 dietary treatments: 1) Positive control: PC (basal diet + bacitracinzinc80 mg/kg feed + colistin sulfate 20mg/kg feed), 2) Negative control: NC (basal diet), 3) FT (basal diet + 2.0% FF), and 4) SFT (basal diet + 2.0% SFF). After trial, 8chickens from each treatment were randomly selected to collect serum and intestinal samples. Chickens in the PC groups had a decrease (P < 0.05) in serum concentrations of lipopolysaccharide (LPS) compared with the NC, FT and SFT group, and there were no difference among group NC, FT and SFT. In the ileum, supplementation with SFF and FF down‐regulated (P<0.05) the mRNA levels of tumor necrosis factor‐α (TNF‐α) andIL‐6 compared with the NC and PC group. Besides, supplementation with SFF up‐regulated(P<0.05) the mRNA levels of Claudin‐1, Claudin‐3, Claudin‐5, zonula occludens1 (ZO‐1), zonula occludens 2 (ZO‐2), Toll‐like receptor 2 (TLR2), Toll‐like receptor 4 (TLR4), defensins AVBD‐9, mucin 2 (MUC2), secretory IgA (sIgA) and IL‐18 compared with the NC group. Furthermore, the mRNA levels of TLR2, AVBD‐9, Claudin‐3 and Claudin‐5 in group SET were lower (P<0.05) than that of group FT but higher than (P<0.05) group PC. And there were no difference on occluding among all groups. Collectively, the current results indicate that dietary addition of SFF has a beneficial influence on the health condition of chicken by improving intestinal barrier function and immune function. That indicated the probiotics is not the only ingredient that works and the fermentation metabolites should be paid more attention.

Support or Funding Information

Sanzhi Biotechnology Co. Ltd., Shanghai, China