bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific oral amino acids induce a protective response
Authors: Jordi Josua , Camargo Simone , Boyle-Neuner Christina , Lutz Thomas , Verrey François
Published: June 2021

Food intake is controlled by the central nervous system that uses multiple signals to drive food selection and meal termination. To test the hypothesis that specific amino acids (AAs) may act as satiation signals, all 20 proteogenic AAs were administered separately at isomolar doses by gavage. Only Arg, Lys and Glu reduced 1h food intake in rats by 50%, 47% and 35%, respectively. Arg and Lys evoked neuronal activity visualized by cFOS staining in the area postrema and the nucleus of the solitary tract. In a two‐bottle conditioned taste aversion test Arg and Lys provoked saccharine rejection, suggesting that their administration triggers a protective response. At the level of the intestine, these AAs induced a strong secretion leading to changes in plasma Cl− (−8% for Arg) and albumin (+9% for Arg, +12% for Lys) concentrations. Furthermore, delayed gastric phenol red release (25% Lys) and accelerated transit into the caecum (428% with Arg, 322% with Lys) indicated changes in intestinal motility. Taken together, these results suggest that oral Lys and Arg administration trigger a protective mechanism. By their dilution and retention in the stomach and accelerated transit through the small intestine their efficient acute absorption might be prevented and thereby the milieu interieur protected from an acute overload. This project is supported by Zurich Center for Integrative Human Physiology.
Insulin, C‐peptide, Glucose, and Heart Rate Responses to Acute Intermittent Hypoxia in the Neonatal Rat: Body Temperature and Chemical Sympathectomy
Authors: Guenther Mitchell Andrew , Bruder Eric David , Chintamaneni Kathan , Raff Hershel
Published: June 2021

Apnea due to immature respiratory control is a common cause of neonatal intermittent hypoxia (IH). We hypothesized that IH disrupts glucose homeostasis and is a metabolic stressor. Rats at postnatal days (PD) 2–3, 7–8, or 11–12 were exposed to 6 @ 30‐sec cycles of IH (3% O2) over 1 hr. An additional group of PD7‐8 pups underwent IH for 3 cycles over 0.5 hr, but were pretreated (on PD3, 5, and 6) with guanethidine (chemical sympathectomy). In PD2‐3 rats, plasma glucose, insulin, and C‐peptide were increased at the 6th cycle of IH. In PD7‐8 rats, plasma glucose, insulin, and C‐peptide increased by the 3rd cycle, but by the 6th cycle, glucose had returned to baseline. PD11‐12 pups experienced a similar increase in plasma glucose, but a much larger increase in insulin and C‐peptide compared to PD7‐8. IH caused bradycardia and a decrease in body temperature. Pretreatment with guanethidine in PD7‐8 rats augmented the increase in insulin and C‐peptide; heart rate at baseline, and heart rate and body temperature at their nadirs during IH were lower after guanethidine pretreatment. We conclude that IH alters glucose homeostasis and induces bradycardia in neonates. The sympathetic nervous system restrains the insulin response and maintains heart rate during IH in the neonate.
Combined acute intermittent hypoxaemia and a high glucose/lipid load augments oxidative stress and exaggerates postprandial hyperglycaemia
Authors: Griffin Harry Sebastian , Pugh Keith , Wadley Alex J , Turner James E , Aldred Sarah , Taheri Shahrad , Kumar Prem , Balanos George M
Published: June 2021

In obstructive sleep apnea (OSA), intermittent hypoxemia (IH) induces oxidative stress (OxS), mediating an increased risk of cardiovascular disease. Hyperglycemia increases OxS during ischemic reperfusion injury, which shares a similar pattern of hypoxia and re‐oxygenation with IH. Postprandial hyperlipidemia alone, is also associated with increased OxS. Therefore, we hypothesized that OxS during IH would be increased by postprandial hyperglycemia and hyperlipidemia. 9 males undertook 3 trials. Trial 1: High glucose/lipid meal + 6h IH. Trial 2: High glucose/lipid meal + 6h room air. Trial 3: Low glucose/lipid meal + 6h IH. Blood samples were assessed for biomarkers of OxS, and glucose/lipid levels. Simultaneous, but not individual exposures to IH and high glucose/lipid ingestion increased plasma lipid peroxides (baseline 6.68 ± 0.66 vs. 6h 8.34 ± 0.58 nM/ml) accompanied by increased antioxidant capacity (baseline 417 ± 23.9 vs. 6h 477 ± 23.2 μM/ml). Despite consumption of identical meals, blood glucose levels at 1 and 1.5h were significantly higher during IH vs. room air (7.58 ± 0.59 vs. 6.52 ± 0.45 and 9.04 ± 0.57 vs. 7.25 ± 0.68 mM/L). Hyperglycemia and/or hyperlipidemia increase OxS during IH and it is associated with a compensatory increase in antioxidant capacity. IH also augmented postprandial hyperglycemia. These findings are relevant to all OSA patients and in particular those with coexisting diabetes.
Optogenetic stimulation of C1 neurons activates breathing in mice
Authors: Guyenet Patrice G , Depuy Seth D , Stornetta Ruth L
Published: June 2021

The C1 neurons regulate sympathetic tone and blood pressure, contribute to glucose homeostasis and regulate the CRF/ACTH/glucocorticoid cascade. Here, we tested whether the C1 cells also regulate respiration. We used a mouse line in which the Cre recombinase is expressed by dopamine‐beta hydroxylase synthesizing neurons. A Cre‐dependent AAV2 that expresses ChR2‐mCherry under promoter ef1‐alpha was injected unilaterally into the C1 region. The transgene was exclusively expressed by C1 neurons (>98% co‐localization with TH‐ir somata). We found that mouse C1 neurons express VGLUT2. Their projections were indistinguishable from those of the rat and included several regions involved in breathing regulation (pre‐Botzinger complex, retrotrapezoid nucleus, dorsal parabrachial and locus coeruleus). Photostimulation of the ChR2‐expressing C1 neurons with 473nm laser light (30 s; 5–20Hz; 1–10 ms pulses) increased diaphragm EMG frequency and/or amplitude in anesthetized mice (N=7) and increased breathing frequency in unanesthetized mice (N=7; plethysmography). These observations indicate that the C1 neurons have reciprocal connections with the respiratory network. They imply that breathing stimulation elicited by hypotension (barorespiratory reflex), carotid body stimulation, central hypoxia and nociception could also be partially relayed via C1 neurons. Support: HL 028785.
Expression of deiodinases in the hippocampus of epileptic rats
Authors: Nassif Renata Jorge , Cysneiros Roberta Monterazzo , Scorza Fulvio Alexandre , Ribeiro Miriam Oliveira
Published: June 2021

Recent studies show that thyroid disorders compromise the integrity of brain tissue, such as hippocampus and parietal cortex, influencing behavior and process of learning and memory. The deiodinase type III (D3), prevents activation of T4, converting it into reverse T3 which are considered inactive forms of the hormone. D3 is increased in critically ill patients, primarily in tissues with hypoxic‐ischemic injury. Sudden death has high incidence in individuals with chronic epilepsy. Respiratory abnormalities such as apnea and hypoxia could be the major cause of death in these individuals. Following this reasoning we evaluate the effects of epilepsy on the local reduction of T3 in brain tissue. For that we studied rats with epilepsy induced by pilocarpine and quantified the activity of deiodinase type II (D2) and D3 in the hippocampal regions, using the method of Immunohistochemistry and Western Blot. D3 expression was dramatically increased in the CA3, Dentate Gyrus and Hilo when compared to control. D2 activity increased in the same region but less than D3 and in regions CA1 and CA2. Our data shows that epilepsy reduces local T3 by increasing the expression of D3 and could be a neuroprotective effect.
Evaluating a possible role for persistent inward currents in firing rate saturation
Authors: Revill Ann Louise , Fuglevand Andrew J
Published: June 2021

During increasing isometric force contractions, motor unit firing rates (FRs) tend to increase rapidly and then saturate (that is, reach a plateau), despite continued augmentation in force and, presumably, synaptic drive. The mechanism underlying this behavior is presently unknown, although persistent inward current (PIC) activation may provide one possible explanation. PICs contribute a non‐synaptic excitation source that activates around recruitment threshold and boosts synaptic input. Furthermore, PICs appear to be inactivated by inhibitory input. Therefore, we reasoned that if we artificially delivered synaptic inhibition to motor neurons before a ramp contraction, then this might prevent full expression of PICs and prevent the initial rapid increase in FR. To test this hypothesis, human subjects performed isometric triangular ramp contractions using tibialis anterior in the absence and presence of additional inhibition provided by surface sural nerve stimulation. We compared single motor unit FR profiles, expressed as a function of force, by fitting the data with rising exponential and linear functions. During control trials, ascending FR profiles were best fit by a rising exponential function but became more linearly related to force with sural nerve stimulation. Overall, our results are consistent with the hypothesis that PIC activation contributes to FR saturation. Funding from NIH NS070897
Hibernation‐dependent Alterations in Hypothalamic Synaptic Markers
Authors: Oeltgen Peter R. , Sparks Larry D. , Brown Stephen A.
Published: June 2021

To examine the possible involvement of the pineal‐hypothalamic neuroaxis during hibernation, we determined serotonergic, dopaminergic and cholinergic synaptic markers in the hypothalamus of hibernating 13‐lined ground squirrels compared to summer‐active animals. We also determined the effect of the pineal gland products, melatonin and N‐acetyl‐serotonin, on serotonergic binding in the hypothalamus of four groups of 13‐lined ground squirrels: Summer‐active, winter‐hibernating, recently aroused, and summer‐induced hibernating animals. In winter‐hibernating animals, hypothalamic content of dopamine, 5‐ hydroxyindolacetic acid (5‐HIAA), and the turn over of serotonin were decreased. The serotonin content and the activities of cholinergic enzymes were increased during hibernation. Moreover, there was no difference in homovanillic acid content or in the total serotonergic type‐2 binding. The modest decrease in serotonin type‐1 binding in hibernating animals was not statistically significant. Melatonin had no displacement effect on serotonin type‐1 binding and little effect on serotonin type‐2 binding in any animal. Although serotonin type‐2 binding was unaffected by N‐acetyl‐serotonin, the effect on serotonin type‐1 binding was state dependent. In summer‐active 13‐lined ground squirrels, N‐acetyl‐serotonin significantly inhibited serotonin type‐1 binding, while the inhibition was more pronounced in hypothalami from hibernating animals. The data suggest that the pineal gland may modulate hypothalamic function via serotonergic receptors.
Potentiation of gastric carcinoma cell invasion by TNF‐α and MMP secretion from peritoneal mesothelial cells
Authors: Tsuji Tsutomu , Shimada Kentaro , Kenmotsu Hiroki , Sekine Wakana , Saito Yuta , Oku Teruaki , Tsuiji Makoto
Published: June 2021

Adhesion and invasion of gastric carcinoma cells to the peritoneum are crucial processes of metastatic dissemination. These processes involve a variety of factors including cell adhesion molecules and cytokines, and are promoted by inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α). In this study, we utilized reconstituted peritoneal membranes consisting of Matrigel and peritoneal mesothelial cells in the Boyden chamber system to examine effects of TNF‐α on carcinoma cell migration. The transmigration of MKN1 human gastric carcinoma cells through reconstituted peritoneal membranes was potentiated in the presence of TNF‐α. TNF‐α also promoted integrin‐meditated adhesion of MKN1 cells to monolayers of mesothelial cells. We then analyzed matrix metalloproteinase (MMP) production by gelatin zymography, and found that both MKN1 cells and mesothelial cells secreted higher levels of MMP‐2 and MMP‐9 upon the treatment of these cells with TNF‐α. These results suggest that the secretion of MMPs stimulated by TNF‐α plays an important role in the peritoneal invasion of gastric cancer.
Decreased expression of Wnt9b and Ube2ql1 in the face of CL/Fr mouse embryos at E11.5 based on microarray analysis
Authors: Takagi Brennan Nicholas , Hynd Thomas , Somponpun S. Jack , Nonaka Kazuaki , Lozanoff Scott
Published: June 2021

The CL/Fr mouse demonstrates heritable bilateral and unilateral cleft lip and palate (CLP) at a rate of approximately 35%, generally above the background “A” strain mouse. Using classical mouse breeding strategies, it has been suggested that at least two disease loci, clf1 and clf2, are involved in the defect and candidate genes have been identified. Additionally, gene‐targeting analyses strongly suggest that Wnt9b contributes to CLP in the “A” strain mice. The aim of this study was to test the expression of clf1 and clf2 candidate genes in the facial prominences of CL/Fr embryos, utilizing microarray analysis. Medial nasal, lateral nasal, and maxillary prominences of phenotypically normal as well as cleft E11.5 CL/Fr mice were dissected and RNA was extracted using standard techniques for Agilent‐microarray protocol. Results indicate that expression of the clf1 candidate gene, Wnt9b, and the clf2 candidate gene, Ube2ql1, are significantly reduced (−3.11 and −1.83 fold, respectively) in the CL/Fr cleft tissues, suggesting these genes may be involved in the CLP mutation in CL/Fr mice. Future gene expression studies through quantitative RT‐PCR and regional expression analyses through immunohistochemistry and whole mount in situ hybridization will be performed to further test the expression of these clf1 and clf2 candidate genes.

Grant Funding Source: NIH/NCRR 5P20RR024206 (S.J.S) and R01‐DK‐064752 (SL)
Effect of Thyroid Hormone Exposure on Murine Calvarial Derived Pre‐Osteoblasts
Authors: Cray James John , Weinberg Seth M , El-Salanty Mohammed
Published: June 2021

Craniosynostosis (CS) is the premature fusion of one or more of the fibrous joints of the skull, which normally allow for the growth of the neurocranium. The incidence of CS is one in every 2000–3000 births. CS involves the overgrowth of bone at the osteogenic fronts of the developing cranial bones. The etiology of CS, whether genetic or environmental, is unknown for the majority of cases. Little is known about the role of environmental agents or gene‐environment interactions in modulating CS phenotypes. The clinical and epidemiological literature suggests that maternal hyperthyroidism is a risk factor associated with CS in offspring, however the mechanism of action is unclear. Here we provide a preliminary report of the effects of exogenous thyroid hormone exposure on cells derived from murine calvaria. Murine derived calvarial cells were assayed for proliferation and osteogenic differentiation after 24,72, 168 hours in culture with exogenous thryoxine at doses ranging from 10^−4 and 10^−16 mol. Exogenous exposure to thyroid hormone appears to stimulate cells to express higher levels of alkaline phosphatase (ALP) in a dose dependent manner. These differences reach 5 times the amount of ALP activity after exposure to the highest doses. There does not appear to be a pattern of difference for proliferation. Thus, it appears thyroid hormone may exert its effects on sutures by increasing the activity of osteoblasts.

Grant Funding Source : None
Amelioration Of Congenital Toxoplasmosis Effects By Maternal Immune Stimulation
Authors: Hrubec Terry C , Goodwin David G , Klein Bradley G , Lindsay David S
Published: June 2021

In mice, peri‐conceptual maternal immune stimulation can reduce the incidence and severity of structural birth defects caused by a wide variety of physical and chemical agents. Maternal immune mediated protection against malformations caused by an infectious agent has not previously been examined. We investigated the ability of interferon gamma (IFN) to protect against birth defects caused by the parasite Toxoplasma gondii. We assessed structural and behavioral deficits in offspring of control, Toxo only, IFN only, and IFN+Toxo treated mice. Offspring were assessed structurally on prenatal day 17 and functionally at 4 and 8 weeks of age using the Barnes maze, motor activity and function observational battery assessments. Infection with T. gondii reduced fetal viability to 23%; however, viability was increased to 58% in dams treated with IFN prior to infection. Congenital toxoplasmosis decreased memory and learning at 4 weeks of age. Treatment of dams with IFN altered spatial memory, coordination and processing of stimuli. By 8 weeks of age, all the deficits observed at 4 weeks had disappeared for both sexes, but male T. gondii infected mice acquired deficits involving spatial memory and rate of learning. IFN treatment prior to infection eliminated negative effects of congenital toxoplasmosis in male offspring. Supported by the Harvey Peters Foundation.
Presence of Carbamazepine in Drinking Water Does Not Appear to Cause Neural Tube Defects in Mice
Authors: Melin Vanessa , Hrubec Terry , Magnin-Bissel Geraldine , Blodgett Dennis , Etzkorn Felicia
Published: June 2021

In May of 2006, our lab suddenly began to observe neural tube defects (NTDs) in embryos of untreated control mice. Exposure to a teratogenic agent in tap water was identified as the cause. Pharmaceuticals have emerged as ubiquitous contaminants of drinking water. One such NTD inducing drug, carbamazepine (CBZ), was analyzed. CBZ was detected in several ground, surface, and tap waters, ranging in concentrations from 0.28 ng/L to 9.5 ng/L. To test whether CBZ was responsible for NTDs, we exposed mice to CBZ in 2 water types. In well water, CBZ concentrations were at 15 and 1500 ng/L. In double distilled water, CBZ concentrations were at 6 and 1500 ng/L. Both studies found no significant differences in NTD rates among the dose groups. As no dose effect was observed, we concluded that CBZ was not directly responsible for the malformations; however it is possible a mixture of contaminants, including CBZ, may be involved.

Grant Funding Source : NIH‐NIHSR21E5016886‐01
Analytic view of simultaneous occurrence of sacralisation and congenital anomalies
Authors: Singh Rajani
Published: June 2021

Lumbar and coccygeal sacralisation may be defined as fusion of fifth lumbar vertebra with first sacral vertebra at the cranial end and apex of sacrum with first coccygeal vertebra at the caudal end respectively. The modulated mutation of HOX11and PAX1/9 families caused defective sacralisation with simultaneous occurrence of anomalies in organs like hindlimb, forelimb, thymus, parathyroid etc in mice. Therefore an attempt has been made to extend the study of sacralisation and associated anomalies from mice to men by conceiving a Hypothesis to be established by future experimentation.

66 dry human sacra were studied in the Osteology laboratory of CSM Medical University Lucknow, UP, India. The defective sacra were genetically analysed and corroborated with experimental results from mice. These sacra were correlated with the mutation of the HOX11and PAX1/9 genes expression and complemented with the associated anomalies as in mice.

The study may revolutionize the clinical practice and diagnosis.

No financial support could be obtained from any source by author.

Grant Funding Source: N/A
Negative Pressure Accelerates Wound Healing by Activating Cdc42‐regulated Proteins
Authors: Yu Shin-Ying , Hsu Chih-Chin
Published: June 2021

Negative‐pressure wound therapy (NPWT) is a treatment to keep wound healing at certain subatmospheric pressure. Biological effects of NPWT on cell migration in wound‐healing process at a molecular level have not been explored. The study attempted to compare cell behaviors and Cdc42‐regulated protein expressions of injured human keratinocytes between ambient pressure (AP) and negative pressures of 75 mmHg (NP75), 125 mmHg (NP125), and 175 mmHg (NP175). An electric cell‐substrate impedance sensing technique was used to record wound‐healing courses in a negative‐pressure incubator. The Cdc42‐regulated protein expressions on cell membranes at different pressures for 6 and 12 hours were measured. Wounded cells at NP125 had decreased complete wound closure time, an enlarged diameter with the mean strain of 11.2%, and obvious cell podia. Cdc42‐dependent proteins in cells at NP125 for 12 hours increased non‐significantly than those in cells at NP125 for 6 hours. Although significant (p< 0.05) protein expression was only observed in N‐WASp at NP125 for 12 hours, similar trends were detected in other related proteins at NP125 for 12 hours. Therefore, the integrated system is helpful to analyze negative pressure effects on biological reaction in wound‐healing process. NP125 can facilitate actin polymerization by stimulating the Cdc42‐dependent pathway on the cell membrane.
Primary osteoblasts colonize and mineralize 3D nanofibrous gelatin scaffolds in 14 days
Authors: Sachar Ashneet , Strom Amanda , Serrano Maria , Miguel Symone San , Liu Xiaohua , Svoboda Kathy K.H.
Published: June 2021

To enhance the outcome of bone tissue engineering, it is important to understand the interactions between osteoblasts and scaffolds. In this study, we analyzed and quantified cell‐matrix interactions and tissue formation by primary cultured osteoblasts on 3D nanofibrous gelatin scaffolds (3D‐NF‐GS) using confocal microscopy. 3D‐NF‐GS were cultured with osteoblasts for 5 or 14 days and immunolabeled with specific antibodies: Integrin b1, vinculin, tyrosine phosphorylated paxillin and collagen type I. A Leica Confocal SP5 microscope was used to analyze the protein distribution. Alizarin red staining was used for mineral detection. Osteoblasts on 3D‐NF‐GS exhibited uniform migration by 5 days and the cells proliferated to an increased confluence by the 14th day. They adhered to 3D‐NF‐GS via focal adhesions by 5 days demonstrated by integrin b1, vinculin and phosphorylated paxillin. Integrin b1 and phosphorylated paxillin adhesions increased in number by 14 days and vinculin adhesions turned more fibrillar. The osteoblasts started arranging into nodular aggregations by 14 days and abundant mineralization of 3D‐NF‐GS was documented. In conclusion, osteoblasts’ adhesion, migration and proliferation on 3D‐NF‐GS lead to secretion of the primary osteoid protein, type I collagen and matrix mineralization. This demonstrates favorable osteoblast behavior on this 3D matrix material.

Grant Funding Source: NIH/NIDCR P30 DE020742
Hyperactivity and Altered Social Attention Following Concussion in Triple Transgenic Mice Predisposed to Alzheimer‐like Neuropathology
Authors: Clough Rich W , Viscomi Natalie S , Thompson Kirk M , Rose Gregory M
Published: June 2021

Modeling Concussive Brain Injury (CBI) in laboratory mice affords opportunities to study mechanisms of neural compromise, genetic influences on behavioral recovery, and interventional therapeutics. Closed skull CBI was induced using a 55 g piston, a 55 cm guide tube with the end positioned on the skull surface and an impact velocity of 3.28 m/sec. Comparisons between groups on latency to righting reflex and open‐field ambulation immediately following CBI indicated an acute concussion in CBI mice. Subsequent periodic behavioral testing through 3‐months post CBI revealed: 1) hyperactivity, manifest as significantly increased overall locomotor activity in all behavioral tests conducted, and, 2) a social attention alteration, manifest as significantly increased incursion/excursion interactions in a social interaction test. No apparent changes were observed in 6 other behavioral measures. Psychological stability is usually seen after mild concussion in humans and C57B6 (normal) mice subjected to single CBI (EB, 2010). In contrast, a single CBI in transgenic mice produced subtle but long‐term adverse effects that persisted through 3 months duration. This work suggests a subtle but increased susceptibility to behavioral change after CBI in mice genetically predisposed to Alzheimer's pathology. Support: SIU CIRCNS and SOM Center for Alzheimer Disease and Related Disorders.
Chromatin Dynamics During Activation and Pseudo‐differentiation of an Innate Immunity Gene
Authors: Adamik Juraj , Su Anjey , Auron Philip E.
Published: June 2021

IL1B is an immediate‐early gene expressed in monocytes upon induction by microbiological molecules, such as lipopolysaccharide (LPS). Chromatin immunoprecipitation studies reveal distinct spatial and temporal epigenetic patterns of this gene during poising, induction and termination. Prior to LPS stimulation, ILB is transcriptionally inactive, but poised for rapid induction. In resting monocytes the promoter region of IL1B is moderately marked by H3K9Ac and H3K4me3 near the transcription start along with elevated levels of H3K9me1 and H3K27me3, denoting a repressed euchromatic state. Upon stimulation, inhibitory methylation is replaced by acetylation and a release of RNA polymerase II (Pol II) from a paused state to one of elongation. This is kinetically followed by an increase in H3K36me3, primarily at the end of the gene, further supporting the argument for elongation. Analysis of several non‐hematopoietic cell lines (HEK293, MG63 and ST2T) that do not express IL1B, reveal differences in chromatin architecture for these non‐competent cells. One such cell line, HEK293, exhibits heterochromatization of an upstream distal enhancer and is rendered partially competent for IL1B transcription by ectopic expression of a transcription factor (Spi‐1) previously reported to be essential for monocyte differentiation. Examination of chromatin at the IL1B locus in the presence and absence of Spi‐1 reveals minimal inhibitory chromatin changes. However, Spi‐1‐ induces de‐novo pausing of Pol II, that is accompanied by transcription in the presence of an inducer. This argues for additional regulatory mechanisms beyond the simple release of inhibitory histone modifications.
Characterization of the cell morphology differences between pre‐diapause and diapause embryos of the killifish Austrofundulus limnaeus
Authors: Toni Lee , Padilla Pamela
Published: June 2021

Austrofundulus limnaeus is a freshwater annual killifish from Venezuela that produces embryos capable of entering three distinct stages of diapause. Diapause II is a state of developmental arrest that can last over 100 days and confers extreme anoxia tolerance. Diapause I and III are inducible while II is obligate. Diapause II occurs in ideal conditions (25oC) whereas incubation at 30oC induces a bypass of diapause II. Diapause II provides an opportunity to study an arrest of development in a vertebrate animal without exposing the organism to compounding stresses. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state. We hypothesize that down regulation of gene expression is central to inducing diapause II. Our aim is to use assays that are principally based upon immunohistochemistry to characterize at the cellular level differences in cell morphology between pre‐DII and DII embryos. We are looking at changes in chromatin modifications, which are known to be involved in the repression or activation of developmental genes, as well as changes in mitotic cell division. Preliminary results show a change in the signal of H3K27 chromatin modifiers as the animal transitions between stages of pre‐DII and DII. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.
Development of vitamin D‐resistance in breast cancer cells through SLUG‐mediated coordinate repression of CYP2R1, CYP27B1 and VDR gene promoters
Authors: Mittal Mukul Kumar , Misra Smita , Chaudhuri Gautam
Published: June 2021

Metastatic breast and other cancers are often found to be refractory to vitamin D therapy. We report here that the metastasis modulator protein SLUG, which is often over‐expressed in metastatic breast cancer cells, coordinately represses the levels of CYP2R1, CYP27B1 and VDR proteins in the breast cancer cells to induce vitamin D‐resistance in these cells. SLUG inhibits CYP2R1, CYP27B1 and VDR gene promoter activities in these cells. ChIP assays revealed that SLUG is recruited at the CYP2R1, CYP27B1 and VDR gene promoters along with the co‐repressor CtBP1 and the effector HDAC1. Inhibition of HDAC1 activity by Trichostatin A increased acetyl histones H3 and H4 at the CYP2R1, CYP27B1 and VDR gene promoters and abolished the repressor activities of SLUG. Knockdown of SLUG in highly invasive MDA‐MB‐231 and BT549 cells increased their CYP2R1, CYP27B1 and VDR gene expression and decreased their resistance to VD3 in vitro. Our data establish that SLUG regulates vitamin D metabolism and contribute to the induction of VD3‐resistance in human breast cancer cells through the inhibition of CYP2R1, CYP27B1 and VDR gene promoters epigenetically through chromatin remodeling. Supported in part by the NIH grant 1U54RR026140‐01 to SM and DOD‐CDMRP IDEA Grant# BC050641 to GC.
Discovery and characterization of an L‐kynurenine responsive transcriptional regulator of the oxidative tryptophan degradation pathway in Burkholderia xenovorans
Authors: Hall Richard S , Unkefer Clifford J , Unkefer Pat J
Published: June 2021

To uncover the transcriptional regulation of oxidative tryptophan degradation, comparative genomics were used which focused on the operon containing the genes kynA, kynU and kynB. In β and γ‐proteobacteria, including Burkholderia, Ralstonia, Collimonas, Cupriavidus and Pseudomonas species, there is a conserved asnC/lrp family transcriptional regulator (TR) gene located upstream of the operon on the opposite strand. In Burkholderia xenovorans the TR is Bxe_A0736 and we designate it as kynR. Using DNAse footprinting with fluorescence capillary electrophoresis, we determined that the KynR protein binds a set of four degenerate, palindromic sequences within the intergenic region from 35 bp upstream of kynB to over the start site of kynR. The optimal consensus sequence is ATATTCCGAATAT. KynR binds L‐kynurenine with greater affinity than any other amino acid or tryptophan degradation product and does so with a dissociation constant of 67 ± 14 μM, as determined by frontal affinity chromatography with ESI‐MS detection (FAC‐MS). Using fluorescence anisotropy, we determined that L‐kynurenine increases the affinity of KynR for a 39 bp section of the regulatory site from 145 +/− 2 nM to 97 +/− 1 nM. We propose that KynR promotes the transcription of kynA, kynU and kynB in the presence of L‐kynurenine while inhibiting the transcription of its own gene, kynR. This research was funded by the DOE/LANL LDRD program.
Long non‐coding RNA NEAT 1 & 2 regulates phosphorylation of SR proteins and PKCβII splicing during 3T3 L1 adipogenesis
Authors: Cooper Denise R , Carter Gay , Li Pengfei , Watson James , Patel Niketa A
Published: June 2021

The alternative splicing (AS) of PKCβII on day (D)6 of adipogenesis allows for 3T3‐L1 cells to develop full insulin responsive glucose transport and accumulate lipid droplets. PKCβII activates Akt via mTORC2 and increases glucose transport via membrane fusion of GLUT4 vesicles (BBRC 388 (2009) 554). The regulation of AS is poorly understood during this process. Long non‐coding (lnc) RNAs are key regulatory molecules in differentiating cells. They can organize splicing proteins in the nucleus for phosphorylation. Here we demonstrated the highly regulated expression of NEAT1 & 2 during adipogenesis by RT‐PCR. On D6, NEAT1 & 2 were elevated 2‐ fold vs D0 in union with increased Clk1 kinase and PKCβII expression. Cells were treated with siRNA for NEAT1. On D0, phosphorylation of SR proteins, SRp40 and 55, by Clk1 kinase decreased. Remarkably, PKCβII AS was dysregulated (elevated) by D2. This suggests that lncRNAs may organize serine/arginine rich “SR” splicing proteins via their RRM domains for Clk phosphorylation in AS during differentiation. Regulation of lncRNA levels during adipogenesis may further determine the level of AS activity in combination with Clk1 expression.
Suppression of MazF toxicity by fusing a C‐terminal segment of MazE to MazF, and its activation by sequence specific HIV‐1 and HCV proteases
Authors: Park Jung-Ho , Yamaguchi Yoshihiro , Inouye Masayori
Published: June 2021

The genomes of human immunodeficiency virus and hepatitis C virus consist of a single‐stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus‐encoded specific proteases. Thus, these proteases are essential for viral infection and have been used as targets for drug development. Here, instead of inhibiting these proteases, we took an opposite approach by using the protease activity to prevent viral infection. We engineered the MazE‐MazF antitoxin‐toxin system of Escherichia coli to fuse a C‐terminal 41‐residue fragment of antitoxin MazE to the N‐terminal end of toxin MazF with a linker having a specific viral protease cleavage site for HIV PR (HIV‐1 protease) and NS3 protease (HCV protease) as well as the cleavage site for factor Xa. These fusion proteins formed a stable dimer (instead of the MazF2‐MazE2‐MazF2 heterohexamer in nature) to inactivate MazF mRNA interferase (endoribonuclease) activity. When the fusion protein was incubated with the corresponding proteases, the MazE fragment was cleaved off to release MazF. MazF thus released functioned as an ACA‐specific mRNA interferase to cleave single‐stranded MS2 phage RNA. The activation of MazF toxicity by proteases of single‐stranded RNA viruses such as HIV‐1 and HCV may provide a novel approach for preventive and therapeutic treatments of infection by HIV‐1, HCV and other RNA viruses.
The pro‐inflammatory but not protective effects of protease‐activated‐ receptor‐2 in the airway require β‐arrestin‐2
Authors: Nichols Heddie , DeFea Kathryn
Published: June 2021

As asthma prevalence, health care use, and mortality increase therapeutics with a distinct target are needed. One attractive target is a G Protein Coupled Receptor (GPCR) known to play both protective and pro‐inflammatory roles in the airways. Namely, Proteinase Activated Receptor‐2 (PAR2) is activated by serine‐like proteases such as trypsin, mast cell tryptase, and those found in cockroach frass and Alternaria alternata. Given the dual and apparently opposing actions, both inhibitors and activators of PAR2 have already been proposed as therapeutics for asthma. PAR2 can signal through two independent pathways: a G‐protein‐dependent and a β‐arrestin‐dependent/G‐protein‐independent one. The β‐arrestin‐dependent pathway promotes leukocyte migration, while bronchiolar smooth muscle relaxation requires G‐protein signaling intermediates. We use a mouse ovalbumin model for PAR2‐modulated airway inflammation to address the hypothesis that inflammatory responses to PAR2 activation are β‐arrestin dependent while ‘protective’ anti‐constrictor effect of bronchial epithelial PAR2 are β‐arrestin‐independent. We show that PAR2‐ induced lung inflammation, mucus production, Th2 cytokines, airway responsiveness and cellular inflammation were abolished in β‐arrestin‐2−/− mice while tracheal smooth muscle relaxation and PGE2 production were equivalent in wild type and β‐arrestin‐2−/− mice.
High‐pressure Adaptation in Piezotolerant Enzymes Studied with Cytochromes P450 from Deep‐Sea Bacteria
Authors: Davydov Dmitri R , Sineva Elena V
Published: June 2021

We have explored the adaptation of the enzymes of deep‐living organisms (piezozymes) to high hydrostatic pressures. Due to the role that water plays in enzymatic catalysis, this adaptation must prevent uncontrolled hydration during the catalytic cycle. Strict conservation of the protein fold and functional importance of protein‐bound water make cytochromes P450 a unique subject for these studies. We cloned, expressed and purified fatty‐acid binding cytochromes P450 from the deep‐sea bacteria Photobacterium profundum SS9 (CYP261C1) and Moritella sp. PE36 (CYP261D1), as well as from the shallow‐water P. profundum 3TCK (CYP261C2). Analysis of pressure‐induced transitions revealed a striking peculiarity of the piezozymes. Both CYP261C1 and CYP261D1 exhibit an apparent conformational toggling that takes place at the physiological pressure of habitation of the host bacteria. This conformational transition, which results in decreased water accessibility of the active site, is not observed in the non‐piezophilic CYP261C2. According to our concept, the changes in the energy landscape evolved in piezozymes must stabilize less‐hydrated, closed conformers, which may only be transient in the catalytic mechanisms of non‐piezophilic enzymes. The studies of piezozymes should help unravel the mechanisms that control water access in the P450 catalytic cycle. (Supported by NIH grant GM054995).
Content in the Educational Era of Process
Authors: Voet Judith G. , Voet Donald
Published: June 2021

We have spent our academic careers focusing on biochemistry content delivery, both as lecturers and as textbook authors. Our philosophy has been that if the professor provides an enthusiastic and interesting presentation of the material that s/he believes is most important for the student to master, the student will be motivated to master that material. We have depended on prior education to provide the student with the appropriate study skills. As editors of BAMBED we have noticed changes in the educational focus from content delivery to the process by which students learn. In addition, both the amount and the specific nature of the content that students are expected to learn are changing. As editors, we have encouraged those who maintain a concern for content to develop ways to provide this content through innovative processes, both in the classroom and in assignments. We will talk about how we see both the content and process of biochemistry and molecular biology evolving.
High‐throughput screening for Plasmodium falciparum glucose‐6‐phosphate dehydrogenase‐6‐phosphogluconolactonase inhibitors
Authors: Preuss Janina , Hedrick Michael , Sergienko Eduard , Pinkerton Anthony , Jortzik Esther , Rahlfs Stefan , Becker Katja , Bode Lars
Published: June 2021

During malaria infections, there is a significant increase in the pentose phosphate pathway activity of Plasmodium falciparum (1). In addition, its key enzyme, glucose‐6‐phosphate dehydrogenase‐6‐phosphogluconolactonase (PfGluPho), shows structural differences to the human enzyme, thus making PfGluPho an ideal target for antimalarial drug design (2). Recent cloning and overexpression (2) of recombinant PfGluPho allowed investigations in a high throughput format. We were able to develop a high‐throughput screening assay to identify inhibitors for PfGluPho. Three small molecule compound libraries were screened, namely LOPAC™ (Sigma‐Aldrich, 1,280 compounds), Spectrum™ (Microsource discovery system, ~2,000 compounds), and DIVERSet™ (ChemBridge, ~50,000 compounds), which resulted in ~900 compounds that inhibited PfGluPho activity for at least 50%. Selected compounds were subjected to follow‐up studies, including IC50 determination and mechanistic characterization. The availability of recombinant PfGluPho, its major differences to human G6PD, and the identified inhibitors facilitate further studies on PfGluPho as excellent drug target candidates for the search of new antimalarial drugs.

Supported by NIH 1R21AI082434‐01
Biochemical characterization of PqsD activity in alkylquinolone biosynthesis in Pseudomonas aeruginosa
Authors: Zhang Yong-Mei , Yang Mo Wei , Zhou Alvin , Szulc Zdzislaw , Davies Christopher
Published: June 2021

Pseudomonas aeruginosa (Pa) is a versatile opportunistic pathogen. Quorum sensing (QS) is essential for Pa pathogenicity by activating virulence factor production. Among the three known QS systems in Pa, Pseudomonas Quinolone Signal (PQS) is an alkyquinolone, one of >50 quinolone compounds produced that differ in alkyl chain length and desaturation. The condensing enzyme PqsD catalyzes the branching point of quinolone synthesis, utilizing either malonyl‐CoA or 3‐oxo‐fatty acids of various chain lengths. We showed that PqsD exhibited higher activity using fatty acid substrates with 10, 12 or 14 carbons than those with 6 or 8 carbons, suggesting the fatty acid chain length is critical in positioning the carboxylic group for efficient catalysis. In addition, PqsD showed preference for malonyl‐CoA over 3‐oxo‐decanoic acid in vitro. Alanine mutants of the active site triad (C112, H257, and N287) were assayed for PqsD activity. All 3 residues were essential for condensation, whereas N287 was not required in the formation of anthraniloyl‐PqsD intermediate. Inhibitors of PqsD were identified by virtual screen and the IC50s of the top‐scoring hit compounds were determined. Our results suggest that regulation of the substrate supply in vivo may be important in determining the composition of quinolone compounds, which in turn affect the virulence factor production in Pa. (Supported by NIH P20 RR017677 and UL1 RR029882)
Yeast Aging Proteome Unveiled a Novel Aging Regulation Pathway Mediated by the Chromatin Remodeling Complex ISW2
Authors: Dang Weiwei , Jansen Pascal , Dorsey Jean , Cao Kajia , Perry Rocco , Kaeberlein Matt , Kennedy Brian K , Vermeulen Michiel , Berger Shelley
Published: June 2021

Calorie restriction is known as the most robust intervention to extend lifespan for eukaryotes, from yeast to primates. Although numerous conserved aging regulators and pathways involved in calorie restriction have been identified over the years, the overall proteomic changes during aging or under calorie restriction conditions remain largely unexplored. We compared proteomes between young and replicatively aged yeast cells under normal and calorie restricted conditions using the quantitative proteomics method, SILAC. Analysis of proteomic changes under these conditions unveiled distinct signatures in the aging proteome and the similarity between the aging and calorie restriction proteomes. These discoveries provided clues to a novel aging regulation pathway mediated by the chromatin remodeling complex ISW2. Disruption of ISW2 mimics the calorie restriction conditions by elevating cellular response to stress and extends replicative lifespan. This study presents the first evidence that chromatin remodeling plays an important role during aging and that yeast cells exploit the benefits of calorie restriction in the later stages of their lives.
Hyperactivation of Mitochondrial Metabolism in Cancer Cells In Situ
Authors: Lisanti Michael , Sotgia Federica
Published: June 2021

We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. Cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, via the secretion of hydrogen peroxide. Oxidative stress in the tumor microenvironment then leads to autophagy‐driven catabolism, mitochondrial dys‐function, and aerobic glycolysis. This, in turn, produces high‐energy nutrients drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new “parasitic” cancer model is that epithelial cancer cells should increase their oxidative mitochondrial capacity. Here, we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over‐abundance of COX activity, while adjacent stromal cells remain essentially negative. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells.
Strong antioxidant activity of the novel selenium‐containing imidazole compound ″selenoneine″
Authors: Yamashita Michiaki , Imamura Shintaro , Hossain Md. Anwar , Touhata Ken , Yabu Takeshi , Yamashita Yumiko
Published: June 2021

The novel selenium (Se)‐containing strong antioxidant selenoneine, 2‐selenyl‐Nα, Nα, Nα‐trimethyl‐L‐histidine, has recently been discovered to be the predominant form of organic Se in tuna blood (Yamashita & Yamashita, JBC, 285, 18134–18138, 2010). This compound is thought to play a key role in the Se redox antioxidant mechanism in animal cells. Here, we report evidence that the uptake of selenoneine was mediated by organic cations/carnitine transporter‐1 (OCTN1). Although OCTN1 has been known to transport some kinds of organic cations, such as ergothioneine, triethylamine, and carnitine, our data indicate that selenoneine in culture medium is incorporated into human embryonic kidney HEK293 and zebrafish embryo cells by OCTN1. The Km value of selenoneine uptake was determined to be 13.0 μM in HEK293 cells and 9.5 μM in zebrafish erythrocytes, respectively, indicating that selenoneine is the most specific substrate for the OCTN1 in the human and zebrafish cells. When we added selenoneine into culture media of HUVEC and zebrafish embryonic ZE cultured cells, cell growth was promoted and GPx1 gene expression was induced, and cellular ROS level was reduced. Therefore, selenium redox status, mediated by incorporation of selenoenine via OCTN1, might be important in inflammatory and chronic diseases, and methylmercury detoxification caused by Se deficiency and/or oxidative damages.
Using Drosophila as a model system to study cold tolerance
Authors: Luor David , Parikh Kosha , Shain Daniel , Yakoby Nir
Published: June 2021

The simple genetics yet high homology of Drosophila genes to their orthologous counterparts in higher organisms and the availability of genetic perturbation toolkits make Drosophila species a powerful system for cold tolerance tests. Particular enzymes including adenosine monophosphate phosphatase have previously been shown to underlie cold tolerance in ice worms by regulating the body energy levels. The mechanism is assumed to be linked to rates of mitochondrial oxidative metabolism; this, in turn, causes the shutdown of molecular thermostats and allows for cold tolerance. To evaluate for cold tolerance in Drosophila, we developed a mobility test of third‐instar larvae under different temperatures. Our results indicate that significant differences in mobility across species are present between certain specified temperature intervals. Remarkably, adult flies survived more than three times longer in lower temperature than that in room temperature. Our findings of tolerance in lower temperatures provide tools to continue studying the effects of lower temperatures on the wild type and genetically perturbed flies, contributing ultimately to the research on extending the tight 24‐hour period shelf life of donated human organs. This research is supported by Dean's Undergraduate Research Fund and Travel Fund and by NIH‐NIGMS R15 GM093685‐01 awarded to DS and NY.