bluuebunny/crossref_metadata_2025 huggingface dataset:
Variation in Size and Progression through Larval Stages of Drosophila melanogaster Fruit Flies in the Absence of Dietary Folic Acid
Authors: Powel Matthew B , Blatch Sydella A
Published: November 2023

Folic acid is involved in DNA synthesis and methylation, and other key reactions. Animals, including humans and fruit flies, cannot synthesize the vitamin and so require outside sources of it for proper development. Microbial symbionts alone in fruit flies can provide enough active forms of folic acid (folates) for survival on diets lacking folate. However, larvae who use folates only from microbes are less likely to survive to the pupal stage, compared to larvae fed the vitamin. In addition, the amount of folates in the tissues of these larvae are the same but vary more than larvae consuming folates. We hypothesized that this overall decrease in survival is because only some larvae are able to survive folate‐free diets well, because of individual variation in larval microbiomes that result in larvae receiving different amounts of folate. An alternative hypothesis is that most larvae survive folate‐free diets well, until the end of the larval stage when most “run out of” microbially‐derived folates. To help distinguish between these two possibilities, we aimed to see if the rate that larvae develop in media without folate varies widely or whether larvae fare similarly in this environment. Eggs were transferred to holidic media made without folic acid. Larvae were observed for 1.5 weeks and photographed under a stereomicroscope. Two‐dimensional larval size was calculated and larval instar stage was determined. The preliminary data show that both of these varied significantly in the absence of dietary folic acid. Age at which an instar stage was reached was wide‐ranging. The size of a larva for its given age and larval instar also varied. By day 10, about one‐fourth of the larvae had reached the third instar, however more than one‐third of the larvae were still first instars. The data support the primary hypothesis that some larvae cannot develop normally without dietary folic acid, while others appear to. Future studies will determine if this is due to differences in microbial flora, causing different amounts of folate to be available to the larvae. Such studies examining how individual differences in microbiomes affect the host in model organisms such as Drosophila can inform the understanding of important host‐microbiome interactions in humans and other animals.
ER Stress‐BiP Axis Regulates Endothelial Barrier Dysfunction and Inflammation in Acute Lung Injury
Authors: Leonard Antony , Rahman Arshad , Grose Valerie , Slavin Spencer , Fazal Fabeha
Published: November 2023

Recent Studies have provided evidence of associations between endoplasmic reticulum (ER) stress and human diseases. Aberrant regulation of ER stress is associated with many chronic inflammatory diseases including type 2 diabetes, inflammatory bowel disease (IBD), cancer, rheumatoid arthritis, atherosclerosis, and chronic obstructive pulmonary disease (COPD). However, the role of ER stress and ER chaperone BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. We provide novel evidence that implicates ER stress‐BiP axis as a critical determinant of EC barrier disruption and inflammation associated with acute lung injury (ALI). Our data show that stimulation of endothelial cells (EC) with thrombin caused an increase in BiP levels and pretreatment of cells with ER stress inhibitor 4‐phenylbutyric acid (4‐PBA) prevented this response. Inhibition of ER stress by this approach also decreased thrombin‐induced levels of proinflammtory mediators ICAM‐1, IL‐6, and IL‐8. Consistent with this, pretreatment of EC with AB5 cytotoxin family member subtilase (SubAB), which selectively cleaves and inactivates BiP inhibited thrombin‐induced elevation of intracellular Ca2+, a critical requirement in the mechanism of EC permeability and inflammation. Importantly, BiP inactivation also protected against thrombin‐induced VE‐cadherin disassembly and EC permeability. We determined the in vivo relevance of BiP in ALI by monitoring the effect of SubAB on lung inflammation and injury in an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI. We found a marked decrease in LPS‐induced lung PMN sequestration and microvascular leakage in mice treated with SubAB. Together these data identify ER‐BiP axis as a novel regulator of EC dysfunction in ALI.

Support or Funding Information

(Supported by: NIH (HL096907 and HL116632)
Analysis of the physical interaction between α1G T‐type calcium channel and endothelial nitric oxide synthase (NOS3) in pulmonary microvascular endothelial cells
Authors: Sellak Hassan , Chen Hairu , Alexeyev Mikhail , Wu Songwei
Published: November 2023

We have observed that, in pulmonary microvascular endothelial cells (PMVECs), activation of the site‐specific α1G T‐type Ca2+ channel (α1G) provides a cytosolic Ca2+ source necessary for endothelial nitric oxide synthase (NOS3) activation. Furthermore, the interaction between α1G and NOS3 in PMVECs has been demonstrated by in vitro Förster resonance energy transfer studies, as well as by immunoprecipitation assays. The present work was undertaken to analyze the molecular basis of the α1G‐NOS3 interaction. We constructed retroviral expression vectors harboring different deletion mutants of α1G that encompasses its cytoplasmic domains including N‐terminus (α1G‐Nter, 1–80), loop I–II (396–744), loop II–III (966–1,251), loop III–IV (1,517–1,578), and C‐terminus (α1G‐Cter, 1,823–2,254). Additionally, Rat NOS3 was cloned in retroviral expression vector and five (5) deletion mutants were generated and similarly cloned, i.e., NOS3‐F1 (1–730), NOS3‐F2 (1–450), NOS3‐F3 (400–730), NOS3‐F4 (700–1,202), and NOS3‐F5 (850–1,202). PMVECs were retrovirally transduced with full‐length α1G alone, full‐length α1G combined with full‐length NOS3, or full‐length α1G combined with different NOS3 deletion mutants. Immunoprecipitation assays using protein extracts from these transduced cells showed that fragments NOS3‐F1 (1–730) and NOS3‐F5 (850–1,202) interact with full‐length α1G. In a reverse approach, PMVECs were retrovirally transduced with full‐length NOS3 alone or in combination with each α1G cytoplasmic domains. Immunoprecipitation and immunoblot assays were performed and results revealed that α1G loop I–II, loop II–III, and α1G C‐terminus interact with NOS3. Altogether, these findings demonstrated the existence of constitutive physical interaction between α1G T‐type Ca2+ channel and NOS3, and identified the critical domains that mediate α1G‐NOS3 interaction. Thus, α1G‐NOS3 association may form a signaling complex that governs the inflammatory response of pulmonary microvascular endothelium.

Support or Funding Information

Supported by HL‐066299.
Protective role of ergothioneine from tobacco smoke‐induced oxidative stress in vitro and in vivo
Authors: Ehrhardt Carsten , Selo Mohammed Ali , Clerkin Caoimhe Geraldine , Talbot Brian Noel , Walsh John Jarlath , Nakamichi Noritaka , Kato Yukio , Lewis Joshua B , Reynolds Paul R , Nickel Sabrina
Published: November 2023

Tobacco smoke is a major contributing factor for chronic obstructive pulmonary disease (COPD) and exposure leads to oxidative stress, which contributes to alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair.

Ergothioneine (ET), an amino acid transported across cell membranes by the OCTN1 (SLC22A4) transporter, has been shown to exhibit antioxidant capacities. The objective of this study is to investigate the role of OCTN1‐transported ET on lung epithelial cells subjected to tobacco smoke‐induced oxidative stress.

For in vitro studies, human NCI‐H441 cells were cultured for at least 5 days in the presence or absence ET and then exposed to cigarette smoke extract (CSE). Reactive oxygen species were measured using H2DCFDA, enzymes involved in oxidative stress defence (e.g. thioredoxin, sulfiredoxin 1). In addition markers of xenobiotic defense (e.g. MRP1, BCRP, and PXR) were studied by qPCR and immunoblot. Moreover, octn1 knock‐out mice were exposed for 3 and 6 months to second hand smoke. The total cell numbers in broncho‐alveolar lavage fluid (BALF) were counted and smoke induced changes in lung morphology were assessed in hematoxylin‐eosin (HE) stained lung sections.

q‐PCR and immunoblot revealed elevated expression levels of sulfiredoxin 1 and thioredoxin as well as PXR after ET treatment of NCI‐H441 cells. Moreover, lower levels of oxidative stress were observed in cells, which were cultured in the presence of ET prior to CSE exposure. When exposed to room air, KO mice showed no differences in HE staining or cell numbers in BALF compared to wild type (WT) animals. However, total cell numbers in BALF as well as increased alveolar damage and immune cell infiltration were observed in KO animals compared to WT mice when exposed to second hand smoke.

These data suggest that ET can protect lung epithelial cells from oxidative damage and consequently, variants of OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ET transport.

Support or Funding Information

This work has been financially supported by a Clinical Investigator Awards from the Flight Attendant Medical Research Institute (FAMRI CIA 130016). M.A.S. is the recipient of a PhD bursary from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR) and C.G.C. is supported by a Trinity College Research Studentship Award.
Alveolar Acid Injury Causes Endothelial Mitochondrial Depolarization
Authors: Hough Rebecca F , Gusarova Galina A , Islam Mohammad N , Bhattacharya Jahar
Published: November 2023

Lung inflammation and edema, the characteristic features of Acute Lung Injury (ALI), result from lung microvascular barrier deterioration. Since ALI associates with loss of mitochondrial function, we evaluated endothelial mitochondrial responses to alveolar acid instillation. We established isolated, blood‐perfused mouse lungs at constant pulmonary artery, left atrial and alveolar pressures (n=4). To view alveoli and capillaries by confocal microscopy, we micropunctured alveoli to load the cytosolic fluorescent dyes, calcein green (CG) and calcein red (CR), respectively. We loaded capillaries with the mitochondrial potentiometric dye, trimethylrhodamine ethyl ester (TMRE). To establish acid‐induced alveolar injury, we microinjected alveoli with concentrated hydrochloric acid (HCl). HCl decreased CG by 75±4% from baseline (p<0.05, mean±SE), indicating that alveolar acid instillation injured the epithelial plasma membrane. Concomitantly, TMRE, but not CR fluorescence decreased (p<0.05), indicating that despite lack of membrane injury endothelial mitochondria were depolarized. Alveolar PBS injection was without effect. Since loss of calcein dye is an indicator of plasma membrane injury, we interpret alveolar contact with concentrated acid caused membrane injury to epithelium, but not endothelium. Nevertheless, endothelial mitochondria were depolarized and possibly dysfunctional after alveolar acid injection. We suggest, acid‐induced alveolar injury abrogates endothelial bioenergetics, leading to the microvascular deterioration underlying ALI (HL105323, HL57556).

Support or Funding Information

HL105323, HL57556
Copper manipulation of the transcriptional activity of hypoxia‐inducible factor‐1
Authors: Zhang Wenjing , Wu Zhijuan , Liu Xiaojuan , Li Pengfei , Fu Haiying , Kang Y. James
Published: November 2023

Hypoxia‐inducible factor 1 (HIF‐1) controls the expression of more than 100 genes involved in multiple cellular events, however, it is unknown how the selectivity of the expression of a certain group of genes under certain circumstances is manipulated. We previously observed that copper (Cu) is required for the expression of some HIF‐1 controlled genes (such as BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) and vascular endothelial growth factor (VEGF)), but not others (such as insulin‐like growth factor 2, IGF‐2). The present study was undertaken to understand the mechanism by which Cu manipulates the transcriptional activity of HIF‐1. Human umbilical vein endothelial cells (HUVECs) were treated with CoCl2 to induce HIF‐1α accumulation and with a Cu chelator tetraethelenepentamine (TEPA) to reduce the availability of Cu in cells. Chromatin immunoprecipitation assay (ChIP) was used to detect the binding of HIF‐1 transcription complex to hypoxia‐responsive elements (HREs) located in the promoters of Cu‐dependent genes (BNIP3 and VEGF) or Cu‐independent genes (IGF‐2) under normal or Cu‐reduced conditions. Nuclear extracts from normal and Cu‐reduced HUVECs were subjected to immunoprecipitation assay (IP) using anti‐HIF‐1α antibody and to immobilized metal affinity chromatography (IMAC) using immobilized columns loaded with Cu, followed by label free LC‐MS/MS to identify Cu binding proteins that may participate in the formation of the HIF‐1 transcription complex. Treatment with TEPA reduced Cu concentrations in the nuclei but did not change the protein level of HIF‐1α. The sequences of HRE (−550/−451) of BNIP3 and HRE (−355/−347) of IGF‐2 were defined as HIF‐1 binding sites by ChIP‐PCR assay. Cu depletion reduced HIF‐1 transcription complex binding to the HRE of BNIP3 significantly, but did not affect that of IGF‐2, suggesting that Cu manipulates the HIF‐1 transcriptional activity through affecting HIF‐1 transcription complex binding to HREs. LC‐MS/MS identified several Cu binding proteins, which will be further analyzed for their function in the regulation of HIF‐1 transcription complex binding to different HRE sequences. The results thus demonstrate that Cu may represent a means for selective manipulation of the transcriptional activity of HIF‐1.

Support or Funding Information

Supported in part by National Science Foundation of China, grant # 81230004 and 81300107
New Theory of Holistic Integrative Physiology and Medicine I: Structural Frame of Life Control
Authors: Sun Xing‐Guo
Published: November 2023

Objective

The modern systemic physiology, based on each functional system alone, has significant limitations. Human being is an organic set of all functions. We plan to discuss the life control involving all systems.

Methods

We did establish the raw functional structure of new theory to explain the control and regulation of life.

Results

After birth, independent human being's life is depended upon two core functional axis. The first one is respiration‐circulation‐metabolism, and the second one is digestion and absorption‐circulation‐metabolism. The main function of them is to maintain the homeostasis for cells’ metabolic activities in the body. The first functional axis is dominantly to keep balance between supply and demand of gases (O2, and CO2 etc.). The second is to keep balance between supply and demand of materials (glucose, fatty acid, amino acid and lactic etc.) in the body. Use circulation as a central coupling, we constructed a main control circles for life. The circulation‐metabolism is shared parts by both axis. After birth, we start our independent life. Traditionally, breathing is the only vital sign, so that we focused on the breathing control (be discussed in others abstracts) linking with all others.

The structural frame of life control

The cortex is the mind over control for almost functions. Under cortex, main breathing control circle consists of peripheral chemical/pressure receptors in arterial line (aorta arch and carotid body), afferent nerves, central integrative center (medulla and central chemical receptor), efferent nerves, N‐M connection, muscles (alternative constriction and relaxation), diaphragm and chest wall, lung and airway, pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery. The signal from receptors to muscles is transferred electronically, which dominantly neuron‐control; but signal from lung to receptors at artery is transferred by blood flow, which dominantly heart‐control. The secondary circle of breathing control is the circulation. It consists of artery, capillary (all cells metabolic activities: resting, exercise and sleeping. GI uptake, liver metabolism and kidney elimination), vein, right atrium, right ventricle, pulmonary artery, pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery. The pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery are the shared parts in both main and side circles. And the autonomic (sympathetic and parasympathetic) nerves, from central integrative center, control the heart and blood vessel for circulatory regulation.

Conclusion

We can explain that the life control through the integration of all functions of respiration, circulation, nerve, digestion, absorption and elimination, metabolism, exercise, sleeping, etc. together.

Support or Funding Information

CNSF‐Medical Science Program (81470204); NCCVD Fuwai Hospital Start Program (2012‐YJR02)
Inhibitory control of active expiration by the Kölliker‐Fuse in rats
Authors: Jenkin Sarah E. M. , Milsom William K , Zoccal Daniel B
Published: November 2023

Breathing is critically dependent on synchronized activity generated by neural respiratory centres within the pons and medulla. When respiratory drive is elevated, active expiration (AE) is observed, typically expressed by abdominal muscle activity at the end of the expiratory phase (late‐E). It is currently unknown what role the dorsolateral pons, specifically the Kolliker‐Fuse (KF), plays in generating the AE breathing pattern. Using the decerebrated in situ working heart‐brainstem preparation of juvenile male Holtzman rats (60–70 g), motor output from the vagus, phrenic and abdominal nerves were recorded during baseline conditions. The preparations were then exposed to hypercapnic conditions (8% CO2) to recruit AE. Hypercapnia resulted in a significant increase in the amplitude of abdominal late‐E activity (Δ: 118±30%, P<0.05), with little change to the other recorded parameters. During hypercapnia, the pharmacological inhibition of the KF, with bilateral microinjections of (50–75 nL) isoguvacine (10 mM), caused a significant decrease in breathing frequency (Δ: −5±2 cpm, P<0.05) due to increased inspiratory time (0.86±0.1 vs 1.37±0.1 s, P<0.05). Interestingly, while there was no change to the overall expiratory time, the duration (1.86±0.21 vs 1.47±0.13 s, P<0.05) and amplitude (3.6±0.4 vs 0.7±0.2 μV, P<0.05) of post inspiratory (post‐I) vagal activity decreased while the onset of the abdominal late‐E bursts occurred significantly earlier (1.3±0.1 vs 2.4±0.2 s, P<0.05). No changes were observed in the abdominal late‐E burst amplitude after KF inhibition. These findings suggest that the KF plays a relevant inhibitory role in coordinating the transition between post‐I activity and AE during hypercapnia.

Support or Funding Information

Financial support: NSERC, FAPESP (2013/17251‐6) and NIH/NCCIH (1R01AT008632‐01).
Acute Lung Injury Alters AMPA Receptor Subunit Density in the Dorsal Vagal Complex
Authors: Litvin David Gregory , Dick Thomas E , Jacono Frank J
Published: November 2023

Ventilatory pattern is a neurally integrated output that is altered by lung injury. Work in our lab has provided evidence that acute lung injury (ALI) promotes changes in ventilatory pattern, which persist in situ and are associated with synaptic plasticity at 2nd order nucleus tractus solitarii (nTS) neurons. Therefore, in the current study, we sought to identify evidence that brainstem autonomic circuits were undergoing changes in glutamatergic transmission following ALI. We hypothesized that ALI could be promoting changes in brainstem α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunits. To test this hypothesis, we intratrachially instilled bleomycin (Bleo) in postnatal (P) 11 Sprague Dawley rats and sacrificed them at P18 for fluorescent immunostaining of the AMPA‐R subunits GluR1, GluR2, and GluR4, followed by confocal microscopy and densitometric analysis. AMPA‐R subunit immunoreactivity was measured in the nTS, dorsal motor nucleus of the vagus (DMNX), and hypoglossal nucleus (XII). Significant increases in GluR2 immunoreactive density were observed in the nTS of Bleo (0.185±0.036AU n=5) versus PBS (0.108±0.035AU p=0.02, n=5) rats. There were no significant differences in GluR1 (p=0.31) or GluR4 (p=0.70) immunoreactive density in the nTS. In the DMNX, there were significant increases in GluR1 (Bleo: 0.071±0.017AU vs. PBS: 0.0496±0.01AU, p=0.047) and GluR2 (Bleo: 0.057±0.016AU vs. PBS: 0.028±0.0134AU, p=0.03) immunoreactivity. There was a significant decrease in GluR4 density in the DMNX (Bleo: 0.238±0.023AU vs. PBS: 0.295±0.025AU, p=0.02). Finally, there were no significant changes in GluR1 (p=0.209), GluR2 (p=0.275), and GluR4 (p=0.103) in the XII. Taken together, these results show that bleo induced lung injury promotes changes in the density of AMPA‐R subunits in the dorsal vagal complex. These findings suggest that altered glutamatergic transmission in the dorsal vagal complex may contribute to synaptic plasticity and ventilatory pattern changes in the setting of lung injury.

Support or Funding Information

VA Research Service Award Number I01BX000873 and NIH T32HL007913
The background sodium channel NALCN regulates basal excitability of chemosensitive retrotrapezoid nucleus neurons and stimulation of breathing by CO2
Authors: Shi Yingtang , Abe Chikara , Holloway Benjamin , Shu Shaofang , Kumar Natasha , Weaver Janelle , Sen Josh , Perez‐Reyes Edward , Stornetta Ruth , Guyenet Patrice , Bayliss Douglas
Published: November 2023

CO2/H+‐sensitive neurons of the retrotrapezoid nucleus (RTN) are a nexus for integration of multiple respiratory‐related inputs in service of homeostatic regulation of the rate and depth of breathing, blood gases, and arterial pH. The spike firing activity of RTN neurons controls respiratory output. Using a transgenic mouse line in which RTN chemosensory neurons are marked with GFP, we identified two populations of RTN neurons ‐‐ Type I and Type II ‐‐ with different firing activities evident over distinct pH ranges. The higher baseline firing rate in Type II cells is associated with greater inward current at −60 mV and larger NMDG‐sensitive, TTX‐resistant current. We hypothesized that NALCN, a “leak” Na+ channel might underlie this RTN current. Single cell RT‐PCR (scPCR) and in situ hybridization showed that NALCN is expressed in nearly all RTN neurons (>90%), and quantitative scPCR revealed that NALCN expression is highest in Type II cells. Using viral‐mediated shRNA knockdown of NALCN, we found that NALCN depletion favors Type I properties in vitro and blunts ventilatory responses to CO2 in vivo. Therefore, NALCN plays an important role in establishing baseline excitability of RTN neurons and contributes to control of breathing by these cells.

Support or Funding Information

NIH (HL074011, PGG; HL108609, DAB)
Characteristics of Breathing Rate Control by Neurons in the Pontine Medial Parabrachial Region (PBR)
Authors: Zuperku Edward J , Stucke Astrid G , Hopp Francis A , Stuth Eckehard A
Published: November 2023

A subregion within the medial PBR plays a key role in the control of expiratory duration (TE). Drug‐induced activation of PBR neuronal activity decreases TE, and neuronal inhibition increases TE. AMPA‐induced pontine‐mediated tachypnea is associated with increased pre‐I neuronal activity in the preBotzinger (preBC) region. This study used bipolar microstimulation to activate the PBR neurons to more precisely control the input pattern to the preBC/BC region and examine peri‐stimulus time histograms (PSTHs) for short‐time scale correlations in neuronal activity suggestive of direct synaptic connections. Multichannel recordings of preBC/BC neurons were performed in decerebrate, vagotomized dogs. The phrenic neurogram (PNG) was used to assess phase‐timing responses. PBR step frequency stimulus patterns markedly decreased TE in particular when applied during the E‐phase. The E off‐switch threshold decreased with time into the E‐phase. In preBC/BC neurons of various subtypes, PSTHs showed short duration evoked excitation, longer duration inhibition or both, suggesting fast EPSP‐ and slow IPSP‐like synaptic inputs. These results were also reflected in cycle‐triggered histograms. The data suggest that the control of TE from the PBR is mediated by inhibition of E‐neurons and excitation of pre‐I neurons.

Support or Funding Information

Merit ReviewAward # 2 I01 BX000721‐05 from the United States Dept. of Veterans Affairs, Biomedical Laboratory Research and Development Program. Edward J. Zuperku, Ph.D. (PI)
Long‐range gene regulation in the context of chromatin domains
Authors: Dekker Job
Published: November 2023

The 3D organization of the genome is critical for gene regulation, genome stability and faithful transmission of chromosomes to daughter cells. We apply Chromosome Conformation Capture‐based technologies to determine how cells fold their chromosomes and to discover the mechanisms that drive the spatial organization of genomes. I will discuss our recent proposal that in interphase nuclei mammalian chromosomes are hierarchically organized (Gibcus and Dekker, 2013). First, at the nuclear level, chromosomes are compartmentalized into large multi‐Mb domains that are either active and open or inactive and closed. These compartments themselves are composed of smaller sub‐Mb Topologically Associating domains (TADs) (Dixon et al. 2012, Nora et al. 2012). Finally, long‐range gene regulation occurs within TADs through long‐range looping interactions between genes and regulatory elements.

TAD organization, and their role in gene regulation have important implications in cancer cells. I will highlight two new studies from my laboratory that reveal different roles of TADs in chromosome‐wide gene regulation along the dosage compensated X‐chromosomes. First, in C. elegans we find that a Condensin complex mediates TAD formation and dosage compensation. Second, in mouse the dosage compensated X chromosome folds in a unique way with global loss of TADs, except around loci that escapee inactivation.
Intraneuronal Acidification with Acetic Acid, an Ethanol Metabolite, Increases Excitability of Central Nucleus of Amygdala Neurons with Axon Projecting to Rostral Ventrolateral Medulla (CeA‐RVLM)
Authors: Chapp Andrew D. , Driscoll Kyle M , Shan Zhiying , Carter Jason R. , Chen Qing‐Hui
Published: November 2023

We have reported that activation of central nucleus of amygdala (CeA) is involved in the in vivo sympathoexcitatory and pressor responses to acute local challenge of acetate, the ethanol metabolite. However, the underlying neural mechanisms have not been determined. Evidence indicates weak organic acids are capable of decreasing intracellular pH due to intracellular transport of the organic acid and alternating neuronal activity. Recently, we have found that bath application of acetate caused a dose dependent increase in the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM), which may underlie the mechanisms of increased sympathetic outflow in response to acetate challenge. We hypothesized that intraneuronal acidification due to intracellular transport of acetic acid may be a contributing factor. The role of intraneuronal acidification with the ethanol metabolite acetic acid was investigated under brain slice preparation. Specifically, we compared the action potential firing rate, depolarizing input resistance (Rinput), and voltage threshold (Vt) for firing action potentials between control intracellular recording solution (pH = 7.35) and 7.5 mM acetic acid loaded intracellular recording solution (pH = 5.20). In current‐clamp recordings, graded current injections evoked graded increases in discharge frequency among neurons from both groups. Maximum discharge evoked by +250 pA current injection in the 7.5 mM acetic acid loaded (pH = 5.20) group was 31 ± 1 Hz (n=6), which was significantly greater (P < 0.05) than that of controls group (pH = 7.35, 19 ± 1 Hz, n=7). Intraneuronal acidification with 7.5 mM acetic acid significantly (p < 0.05) increased Rinput of CeA‐RVLM neurons (731 ± 64 MΩ) compared to control (531 ± 48 MΩ). Likewise, 7.5 mM acetic acid caused a significant (p < 0.05) hyperpolarizing shift in Vt (−49.28 ± 1.37 mV) compared to control neurons (−43.83 ± 1.76 mV). In cultured neurons incubated with a pH sensitive probe, increases in bath application of acetate (7.5 mM) significantly (p < 0.05) increased the corrected fluorescence intensity of the neurons compared to control, indicative of a decrease in intraneuronal pH. This data suggests that extracellular acetate leads to intraneuronal acidification via transport of acetic acid which has profound effects on CeA‐RVLM membrane properties that enhance neuronal excitability and may contribute to the mechanism underlying alcohol associated sympathetic activation and hypertension.

Support or Funding Information

11SDG7420029 (ZYS) and R15‐HL122952 (QHC)
Changes in the Firing Frequency of Medullary Respiratory Neurons of Juvenile Female Rats Exposed to Chronic Intermittent Hypoxia
Authors: Souza George Miguel P R , Amorim Mateus R , Moraes Davi JA , Machado Benedito H
Published: November 2023

Juvenile female rats exposed to chronic intermittent hypoxia (CIH) presented a significant reduction in the time of inspiration and increase in sympathetic activity during this phase of respiratory cycle, characterizing changes in the respiratory‐sympathetic coupling. There is evidence that the firing frequency of the pre‐sympathetic neurons is modulated by the respiratory neurons activity. In this context, we hypothesized that: in CIH‐female rats, the enhanced sympathetic activity during inspiration is due to the increased inspiratory neurons activity that modulates the pre‐sympathetic neurons firing frequency. We aimed to evaluate the respiratory neurons activity in the ventral surface of the medulla in juvenile female rats exposed to CIH. For this purpose, juvenile female rats (P20–21) were exposed to CIH (FiO2=6%) for 10 days and after this protocol we performed extracellular single unit recordings of medullary respiratory neurons using the working heart‐brainstem preparation (ventral approach). The respiratory neurons were identified and classified by their activity (firing frequency) in relation to the phrenic nerve activity. All experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory/Inspiratory (Pre‐I/I) neurons of CIH‐female rats (n=12) presented a reduced firing frequency (39.3 ±5 vs 61.5 ±8 Hz, p=0.027) when compared to control (n=10). Ramp‐I, Post‐I and Aug‐E neurons presented no significant changes in their firing frequency in CIH‐female rats compared to control. We concluded that Pre‐I/I neurons presented a reduced firing frequency in CIH‐female rats, which may explain the reduced time of inspiration.

Support or Funding Information

FAPESP, CAPES and CNPq.
Decreases in Core Temperature and Sweating Onset During Whole‐body Heating in Individuals with Multiple Sclerosis Following Administration of 4‐aminopyridine
Authors: Huang Mu , Allen Dusty R , Frohman Elliot M , Davis Scott L
Published: November 2023

Multiple sclerosis (MS) is a progressive neurological disease of the central nervous system that can result in impairments in thermoregulatory function. 4‐aminopyridine (4AP), a voltage‐gated K+ channel blocker, has been postulated to improve conduction in demyelinated axons and improve MS related disabilities. The aim of this study was to test the hypothesis that administration of 4AP would improve sudomotor function in individuals with MS during passive whole‐body heating (WBH). Eleven subjects (8 females, 3 males; 39±7 years; 78.6±24.0 kg; 170±9 cm; 8±4 years since diagnosis) with relapsing‐remitting MS participated in this study. A randomized single‐blinded 2‐week crossover design was utilized with 4‐AP and placebo (PBO) including a 1‐week washout period between drugs. Subjects were outfitted in a tube‐lined water perfusion suit and exposed to a normothermic (NT) baseline (34 °C water) and WBH (48 °C water; increase in internal temperature of ~0.8 °C) condition. Core body temperature (telemetric pill) and sweat rate at the dorsal forearm (capacitance hygrometry) were continuously measured. Baseline NT core body temperature while on 4AP was lower compared to placebo (4AP: 37.3±0.2 °C vs. PBO: 37.5±0.2 °C, p=0.002). Core body temperature at the onset of sweat was also lower while on 4AP compared to placebo (4AP: 37.4±0.2 °C vs. PBO: 37.6±0.3 °C, p=0.0006). However, sweat rate (Δ from NT) on 4AP was similar compared to placebo during WBH (4AP: 0.7±0.3 mg/cm2/min vs. PBO: 0.8±0.4 mg/cm2/min, p=0.49). These initial findings suggest K+ channel blockers may have protective benefits for people living with MS by lowering resting core temperature and the sweat onset core temperature threshold. Thus, administration of 4AP may improve heat sensitivity in MS by expanding the range for core temperatures increases prior to symptom worsening.

Support or Funding Information

Project funded by NIH grant (R15 HL117224) and National Multiple Sclerosis Society grants (RG4043A, RG4696A, PP1485)
A monofunctional nonribosomal peptide synthase might be involved in the biosynthesis of marine toxins of the palytoxin family
Authors: Yamazaki Tomoko
Published: November 2023

The aim of this study was to identify in the transcriptome of the dinoflagellate Ostreopsis ovata genes, particularly such encoding nonribosomal peptide synthases (NRPSs), that are possibly involved in the biosynthesis of ovatoxins. Polyketide synthases (PKSs) and NRPSs are multienzyme complexes of mainly bacterial and fungal secondary metabolism. They comprise various enzymatic activities and are often organised in so‐called modules, each of which inserts one building block. The minimal NRPS module consists of an adenylation domain (A domain), which selects and activates the substrate, a condensation domain (C domain), which catalyses the formation of the new peptide bond, and a thiolation domain for binding intermediates. In the last decade, PKS and NRPS genes have also been identified in various protists, including some dinoflagellate species. A strain of the dinoflagellate O. ovata that was sampled in Okinawa, Japan, produces several ovatoxin variants. Ovatoxins belong to the same structural family as the extremely poisonous compound palytoxin. The biosynthetic scheme of palytoxin and its congeners is completely unknown. Sequencing of the O. ovata transcriptome by means of RNA‐Seq revealed a wide range of genes encoding PKSs and NRPSs. Most interestingly, O. ovata possesses a new type of NRPS, which is characterised by an unusual monofunctional architecture. This monofunctional architecture resembles a special type of monofunctional PKSs that have been found exclusively in dinoflagellates. The same type of monofunctional NRPSs found in O. ovata was also identified by screening the published draft genome of the dinoflagellate Symbiodinium minutum. These enzymes form a distinct dinoflagellate‐specific clade in comprehensive phylogenetic reconstructions including NRPSs from bacteria, fungi and other eukaryotes. Expression of those genes on the RNA level was confirmed by RT‐PCR, and the sequences of the transcripts demonstrated that the transcriptome‐based prediction of monofunctional NRPSs was not an artefact caused by assembly issues. Therefore, this study has experimentally demonstrated for the first time the existence of a new type of NRPSs, which at present seems to be limited to dinoflagellates. Structural modelling revealed differences between the active centre of A domains of dinoflagellate origin and those from bacteria and fungi. This new type of NRPS found in O. ovata might be involved in the biosynthesis of ovatoxin and related compounds, as the ovatoxin structure hints to incorporation of an amino acid as part of the biosynthesis pathway. The finding presented here will make it possible to initiate the elucidation of the biosynthesis scheme of palytoxin and related compounds.

Support or Funding Information

This study was supported by internal funds of the Okinawa Institute of Science and Technology Graduate University (OIST).
COnsortium for METabolomics Studies (COMETS): Leveraging Resources to Accelerate Scientific Discovery
Authors: Albanes Demetrius , Moore Steven , Ulrich Cornelia , Stolzenberg‐Solomon Rachael , Poole Elizabeth , Temprosa Marinella , Verma Mukesh , Boerwinkle Eric , Casas Juan , Clish Clary , Gerszten Robert , Gunter Marc , Harris Tamara , Herrington David , Langenberg Claudia , Le Marchand Loic , Lotta Luca , Matthews Charles , Menni Cristina , O'Donnell Christopher , Pereira Alexandre , Rexrode Kathryn , Shah Svati , Shu Xiao Ou , Stevens Victoria , Yu Bing , Zanetti Krista
Published: November 2023

Metabolomics is a systems approachto the biology of health and disease and an ‘‐omics’ discipline that measuressmall metabolites representing end products of a variety of metabolic andcellular processes as reflected in available biological specimens (e.g., blood,urine, saliva, feces and tissue). As an emerging approach used forepidemiologic and clinical studies, metabolomics has the potential to improvedisease risk assessment, screening, diagnosis, prognosis and predictiveresponse to therapy, as well as provide disease mechanistic insight and help toestablish criteria for causation. The number of publications of epidemiologicalstudies that use metabolomics technology has rapidly increased over the past 10years, including application to a wide variety of exposures and disease states.

It is timely to establish mechanisms for leveraging existing resources and data for novel biomarker discovery using metabolomics approaches. To this end, the National Institutes of Health COnsortium of METabolomics Studies (COMETS) was established in 2014 (http://epi.grants.cancer.gov/comets/),and currently includes 25 prospective cohorts and 3 established consortia from the United States, Europe, South America, and Asia. The COMETS mission is topromote collaborations among prospective cohort studies that follow participants for a range of outcomes and perform metabolomic profiling of individuals. COMETS aims to facilitate an open exchange of ideas, knowledge, and results to accelerate a shared goal of identifying metabolomic profiles associated with chronic disease phenotypes (e.g., heart disease, diabetes, cancer). Here we describe the COMETS structure and activities which aim to advance the use and impact of metabolite profiling in population‐based research.

Support or Funding Information

Consortium support: Intramural and Extramural Research Programs of the National Institutes of Health along with other individual cohort funding.
DNA Aptamers Blocking Activity of Anthrax Lethal Toxin
Authors: Ryabko Alena K , Kozyr Arina V , Kolesnikov Alexander V , Marin Maxim A , Zeninskaya Natalia A , Lisitskaya Lidia A , Shemyakin Igor G
Published: November 2023

Background

Anthrax has been recently pinpointed as „deliberately emerging disease”. Apart from natural outbreaks occurring constantly around the world, lethality of anthrax and high resistance of B. anthracis spores to envioronment and disinfectants made it the most feared bioterrorist threat.

Anthrax three‐component binary toxin consists of receptor subunit, known as protective antigen (PA), and two effectors, edema factor (EF), and lethal factor (LF). The PA‐LF complex known as lethal toxin (LeTx), causes macrophage death and, ultimately, fatal organ dysfunction. Until recently, no effective remedy existed to protect organism from LeTx action.

Therapeutic antibodies (mAbs) neutralizing LeTx have been developed, however, all clinically approved mAbs are specific to PA with activity limited to the extracellular milieu, whereas LeTx already entered the cell is impune to the antibody‐mediated neutralization.

Diagnostic and therapeutic potential of DNA aptamers, a new prospective class of affinity molecular tools, is actively explored in different fields, including pathogen detection and neutralization.

Objective

The purpose of the present study was to develop LeTx‐specific DNA aptamers capable to neutralize anthrax lethal toxin inside the mammalian cell.

Results

We identified DNA aptamers binding LF by employing new technique tailored to avoid carryover of target‐specific DNA molecules with aptamers binding to the solid phase. For this purpose recombinant LF was produced as fusion protein with His6 affinity tag and cleavable SUMO peptide.

Random oligonucleotide library (1014) was subjected to 6 rounds of panning and proteolytic separation of LF with bound aptamers from His6‐ SUMO peptide attached to IMAC magnetic beads. Selected aptamers were analyzed for capability to block proteolysis of internally quenched fluorescent LF substrate. Positively acting aptamers were tested for blocking LeTx intoxication in mice. Co‐administration of some selected aptamer species and LeTx to mice substantially increased their survival compared to the control groups injected with LeTx alone.

Conclusion

The data obtained outlines DNA aptamers as prospective tools for the development of the new generation of therapeutic agents, combatting anthrax.

Support or Funding Information

The work was supported by Russian Science Foundation research grant No 14‐15‐00630.
Rational design of potent histidine kinase inhibitors with antimicrobial activities
Authors: Zschiedrich Christopher , Boibessot Thibaut , Lebeau Alexandre , Bénimélis David , Benfodda Zohra , Meffre Patrick , Szurmant Hendrik
Published: November 2023

The excess use of antibiotics has resulted in the appearance of multidrug resistant pathogens, described as one of the most eminent threats to human health in the 21st century. To address this problem many unique fundamental processes for bacteria are being probed for the development of new antimicrobial drugs. In bacteria signaling via the two‐component system is a widespread mechanism, with bacteria typically featuring in excess of 10 such systems per genome. The two‐component system consists of a sensor histidine kinase and a response regulator. A conserved two‐component system (YycFG aka WalRK) essential for viability has been discovered in most gram‐positive pathogens suggesting that inhibition of these systems would lead to cell death. However, targeting a single system can be expected to quickly result in drug resistances. It would thus seem desirable to discover compounds with the ability to inhibit multiple two‐component systems simultaneously. Synthetic compounds have been described as potent competitive inhibitors of DNA gyrase and one such compound has entered phase 1 clinical trials. Gyrases share a common catalytic ATP‐binding domain with histidine kinases; redesign of these compounds to bind the catalytic domain of the latter thus seemed possible. We utilized available high‐resolution structures of the inhibitor/gyrase complex and of a histidine kinase catalytic domain to rationally design novel compounds and probe histidine kinase binding in silico. The most promising in silico hits were synthesized and tested for their ability to inhibit several histidine kinases of Bacillus subtilis. A series compounds were found to be potent inhibitors of all tested histidine kinases but did not inhibit the structuraly distinct ser/thr kinases in vitro. Several of these compounds showed antimicrobial activities against methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant Enterococcus faecalis (VRE) and other Gram‐positive pathogens. Hemolytic assays on sheep erythrocytes ruled out that these compounds act by simply lysing cells, a problem encountered with many previously discovered histidine kinase inhibitors. In summary, the discovered compounds are promising leads for the development of new antibacterial drugs.

Support or Funding Information

This work was supported by grant GM106085 from the US National Institute of General Medical Sciences, National Institutes of Health
RNA‐Seq Analysis of the Salt Tolerance Response in Prairie Cordgrass
Authors: Robben Michael , Gonzalez Jose
Published: November 2023

The food vs fuel debate has created a demand for non‐cereal bioenergy plants that can be grown on lands not suitable for food crops. We examined the potential bioenergy crop Spartina pectinata, Prairie Cordgrass (PCG), for its salt tolerance by performing RNA‐seq on plants treated with 400 mM NaCL against those treated with water to find upregulated genes and genetic markers for salt tolerance. As a result we show a genetic component for salt tolerance through heritability studies and link it to the overexpression of genes in PCG when exposed to high salt concentrations. Future genetic studies could reveal the mechanism by which PCG survives poor soil conditions.
Immune modulator effects of hydroalcoholic extract of Matricaria chamomilla in mouse macrophages
Authors: Torres‐Tirado David , Leon‐Buitimea Angel , Perez‐Flores Gabriela
Published: November 2023

In México is common the use of medicinal plants to treat different illness and symptoms. One of the most commonly used plant used is Matricaria chamomilla or chamomille which is a traditional plant with digestive activity and others effects test partially as antinflammatory and anesthetic that need to continue being studied. The presence of polyphenols in the hydroalcoholic extract suggests the ability to block signal transduction pathways like Mitogen Activated Protein Kinases (MAPK), this could have an important implication in the activation of the immune cells such as macrophages also strengthen the ability to react to external agents to organism. In this work we tested the effects of exposure to one hour of the chamomille hidroalcoholic extract in the J774 cell line and its effects in the viability, bacterial phagocytosis and the pathway activation of MAPK, ERK 1/2‐P. Preliminary results show that the viability of macrophages did not affected in one hour but decrease at the 6 hours of the expose, the phagocytosis of bacteria increase slightly to 5 % and ERK 1/2 expression has not change expression but phosphorylation of ERK 1/2 decrease importantly. Our preliminary conclusions are that the chamomile extract induce sensibility in phagocytosis to bacteria without affect its the viability but decreasing the MAPK pathway activation.

Support or Funding Information

PROMEP UASLP‐DSA/103.5/14/11016 and FAI‐UASLP
Identification and testing of inhibitors of kinases secreted from malaria parasites
Authors: Lin Benjamin , Xu Lingyin , Winston Dennis , Brandt Gabriel
Published: November 2023

Small molecule inhibitors of a family of secreted kinases from the malaria parasite Plasmodium falciparum were identified and shown to arrest parasite growth in blood culture. P. falciparum is primarily an intracellular parasite in its human host. During the blood stage of a malaria infection, the parasite dramatically alters the surface of its host red blood cell, by exporting proteins (accounting for roughly 10% of its genome) into the erythrocyte. Among these proteins is a family of 18 secreted Ser/Thr kinases. Recombinant expression of a number of these kinases and their Plasmodium vivax ortholog has permitted the screening of a library of potential kinase inhibitors. Several of these compounds were found to attenuate kinase activity in vitro. In order to ascertain whether inhibiting kinase activity affects the ability of parasites to remodel the red blood cell surface, the five best inhibitors were incubated with parasites in human blood culture. Scanning electron microscopy was used to assess the extent of red blood cell surface remodeling. Rather than an overt effect on remodeling, initial results showed an overall growth arrest of treated parasites, with no progression to trophozoites in the treated cultures.

Support or Funding Information

American Philosophical Society Franklin Research Grant