bluuebunny/crossref_metadata_2025 huggingface dataset:
Jadomycin B Acts Synergistically with NSAIDs to Produce Cytotoxic Effects in Human Breast Cancer Cells in vitro
Authors: McKeown Brendan T. , Goralski Kerry B.
Published: May 2022

Background

Breast cancer is the most prevalent cancer in North American women. Jadomycin B is a novel compound that is effective in killing triple negative human breast cancer cells that have become resistant to anthracycline and taxane chemotherapies. The mechanism through which jadomycin B exerts its cytotoxic effect remains to be fully characterized. A quantitative PCR (qPCR) array of cancer drug targets identified cyclooxygenase‐2 (COX‐2) as a gene of interest in jadomycin B resistant breast cancer cells.

Objective

To explore the effects of COX‐2 signalling on jadomycin B cytotoxicity in MDA‐MB‐231 cells as a model of invasive triple‐negative, human breast cancer requiring more effective treatments.

Methods and Results

Control MDA‐MB‐231 (231‐CON) cells exposed to increasing concentrations of jadomycin B (0‐2.2 µM) over 7 months developed a 3‐fold resistance to jadomycin B as determined using methyl‐tetrazolium (MTT) cell viability assays. By qPCR, there was a significant increase in COX‐2 (40 fold, p < 0.05) but not cyclooxygenase‐1 (COX‐1) (0.17 fold, p < 0.05), mRNA expression in jadomycin resistant (231‐JB) versus 231‐CON cells. A similar increase in COX‐2 was not observed in mitoxantrone resistant MDA‐MB‐231 cells (231‐MITX). We therefore hypothesized that inhibition of COX‐2 signalling would increase the susceptibility of breast cancer cells to jadomycin B. The 231‐CON and 231‐JB cells were treated with jadomycin B with or without the COX‐2 selective inhibitor celecoxib (CXB), or COX‐1/2 inhibitors ibuprofen (IBU) or Naproxen (NAP). The SynergyFinder 2.0 web application was used to analyze the cytotoxicity (MTT assays) of these combinations and calculate synergy scores using the Bliss model. IBU and NAP were found to act synergistically with jadomycin B in both control and jadomycin resistant cells, while CXB acted additively (Table 1).

Conclusions

Jadomycin B acts additively or synergistically with inhibitors of COX‐2 in vitro, resulting in increased cytotoxic effect. These results justify further investigation into the COX‐2 signaling pathway as a means to elucidate the mechanism of action of jadomycin B. Additionally, our research has the potential to contribute to an improved understanding of breast cancer relevant COX‐2 signalling, facilitating the use of COX‐2 inhibitors as chemosensitizers.
Effect of increased ventilation during feeding on broiler breeder biochemical, enzymatical, and electrolytes parameters
Authors: Olanrewaju Hammed A. , Collier Stephanie , McCafferty Klint , Magee Christopher , Fussell Leonard , Purswell Joseph
Published: May 2022

Breeders exhibit increased heat stress during and after meal feeding, thus increasing ventilation during and immediately post‐feeding may result in improved productivity and health as well as minimizing physiological stress. This study investigated the effect of increased ventilation during feeding on broiler breeder biochemical, enzymatical, and electrolytes parameters. A total of 288 breeder pullets were obtained from a commercial farm at 21 weeks of age (woa) and assigned to 12 pens (24 birds/pen). Each pen was equipped with two tube feeders, nipple drinkers, fresh pine shaving, and LED lighting. Temperature and lighting were adjusted according to primary breeder recommendations. Feed was provided by a commercial feed mill and meal fed once daily, while water was available ad libitum. Feed was presented at 0730 each day concurrently with the beginning of the photoperiod. Treatments included still air (S, negative control), conventional ventilation with no cool‐down (C, positive control), and variable air speed to mimic a cool‐down ventilation program during and immediately post‐feeding (V). Air speed in the V treatment wind tunnel was increased for three hours to offset the increase in metabolic heat production due to feeding and nesting activity, before returning to the baseline ventilation rate. Three sentinel birds in each pen were identified for physiological monitoring at the beginning of the study. On d 21 (start of treatments), 63 (midpoint), and 105 (end of the treatment period), blood samples (3 mL) were collected between 0800 and 0900 h from the brachial wing vein from each of the three sentinel birds into heparinized syringes within 45 s after birds were caught. The collected blood samples were centrifuged to separate the plasma and used to determine the electrolytes levels, concentration of biochemical (ALB, TBILI, BUN, CREAT, TP, UA, CHOL, LDL‐C, HDL, TRIG, GLUC) parameters and enzyme (ALT, ALP, AMYL, AST, CK, GGT, LDH, LIP) activities using an ACE‐AXCEL automatic analyzer. Results show that ALT, BUN, GLUC, and LDL‐C were affected by treatments. However, woa had significant effects on most examined variables along with few interactions. Plasma CORT was not affected by treatments and time, indicating absence of physiological stress. The results are in broad agreement with those reported in the literature and contribute to our knowledge of increasing ventilation during and immediately post‐feeding on blood metabolites variation in broiler breeders.
Laminar Shear Stress Enhances Cytosolic PINK1 Expression: Increased Mitophagic Sensitivity Toward Mitochondrial Dysfunction
Authors: Hong Soon‐Gook , Shin Junchul , Saredy Jason , Choi Soo Young , Wang Hong , Park Joon‐Young
Published: May 2022

PURPOSE

Phosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise‐mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.

METHODS & RESULTS

First, we measured full‐length PINK1 (FL‐PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL‐PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL‐PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL‐PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC‐1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL‐PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS‐preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS‐enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise‐preconditioned mice were more protective to angiotensin II‐induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1‐dependent manner.

CONCLUSION

LSS increases a cytosolic pool of FL‐PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1‐dependent cell protection mechanisms.
Acute Respiratory Muscle Fatigue in Patients with Obesity Following Peak Aerobic Exercise
Authors: Severin Richard S. , Alshammari Saleh , Sherman Sara R. , Phillips Shane A.
Published: May 2022

Introduction

Respiratory muscle (RM) performance has been theorized to contribute to exercise‐induced dyspnea, exercise intolerance, and low exercise capacity in patients with obesity. However, the impact of acute impairments in RM performance on exercise intolerance and exercise capacity in patients with obesity remains uncertain. We hypothesized, that following peak aerobic exercise, impairments in RM performance would be observed in patients with obesity.

Methods

We prospectively recruited 7 participants (recruitment is ongoing; 1 male, 6 female, Age: 42 ± 9 yrs; BMI: 47.7 ± 5.9 kg/m2; Waist Circumference: 126 ± 12.04 cm; Body Fat [%]: 50.91 ± 3.6). Diaphragm Thickness at expiration and Thickening Ratio (thickness at end‐inspiration – thickness at end‐expiration)/thickness at end‐expiration) were assessed via B‐mode ultrasound at the right 8th intercostal space while patients were in a semi‐recumbent position elevated 30 degrees from supine. Participants performed a cardiopulmonary exercise test (CPET) using a ramp treadmill protocol to peak exercise capacity. Measurements of RM performance were measured at baseline and repeated at 0‐minutes and 5‐minutes following CPET; which included Maximal Inspiratory Pressure (MIP), Sustained Maximal Inspiratory Pressure (SMIP), Inspiratory Duration (ID), Slope of the SMIP Plot (SMIP Slope), and Maximal Expiratory Pressure (MEP). Two‐Sided Paired T‐Tests were used to compare RM performance data before and after testing; statistical significance was set at p<0.05.

Results

Mean CPET Results: VO2peak: 19.37 ± 2.4 mL/kg/min; RER: 1.01 ± 0.11; Age Predicted Heart Rate Max [%] 87.57 ± 0.14; Ventilatory Reserve [%] 25.25 ± 18.32; VE/VCO2 Slope: 28.84 ± 4.50. Diaphragm Thickness: 2.8 ± 0.24 mm and Thickening Ratio: [%]: 75.47 ± 25.97. When comparing Baseline to 0‐minues following CPET significant reductions in MIP, SMIP, and MEP were observed: MIP (94.57 ± 25.01 cmH2O vs. 72.29 ± 22.09 cmH2O p = 0.02); SMIP (524.14 ± 198.85 PTU vs. 418.43 PTU p = 0.04); MEP (73.71 ± 17.10 cmH2O vs. 62.29 ± 12.83 cmH2O p= 0.02). A reduction in ID (10.6 ± 2.30 vs 8.99 sec p= 0.06) and an increase in SMIP Slope (9.2 ± 3.39 vs. 9.74 ± 3.30 p= 0.74) were observed at 0‐minutes following CPET compared to baseline but were not significant. At 5‐minutes following CPET, significant reductions in MIP (67.3 cmH2O ± 12.6 p= 0.01) and MEP (62.57 ± 14.47 cm H2O p= 0.01) were observed but not for other measures of RM performance.

Conclusion

Our results suggest that patients with obesity demonstrate significant reductions in RM performance following peak aerobic exercise which may persist even after recovery. Interestingly, at baseline, the patients with obesity in this study demonstrated relatively normal RM performance, diaphragm thickness and thickening ratio. Acute fatigue of the respiratory muscles may contribute to the exercise induced dyspnea, exercise intolerance and low exercise capacity observed in patients with obesity.
Correlation Between Liver Weight and Dimensions to Patient Demographics
Authors: Alimohammadi Majid
Published: May 2022

Background

Treatment of hepatocellular carcinoma (HCC) often involves the use of targeted radiation therapy, chemotherapeutic agents, and percutaneous ethanol injections, but issues of underdosing and overdosing still prevail and dosimetric calculations are often suboptimal.

Methods

33 cadavers donated to the University of British Columbia (UBC) Body Donation Program were examined, and data was collected on liver weight and dimensions. The individual’s demographics (height, weight) were obtained from the department’s database, and the liver demographics were correlated with the individuals’ demographics.

Results

Data was analyzed for correlations between body weight and liver weight, person’s height and the height of the liver along the right surface, body weight and the liver’s length along the superior surface, and body weight and the distance the liver extended left of the midline. A strong positive correlation was identified between body weight and liver weight, with r=0.7475 and p < 0.00001. All other results either concluded weak correlations, or were insignificant.

Discussion

The strong correlation identified between body weight and liver weight can be used as a potential additional tool during initial clinical examinations to aid dosimetric calculations of radiation and chemotherapy treatments.
Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics
Authors: Ferrero Kimberly M. , Chuprun Kurt , Pfleger Jessica , Tilley Douglas , Gao Erhe , Koch Walter J.
Published: May 2022

G protein‐coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non‐GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post‐injury phenotype. Stress‐induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography‐mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post‐injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.
N‐Glycosylation Inhibitor Interferes with WNT Signaling and Inhibits Angiogenesis
Authors: Banerjee Dipak K. , Lebron Arelis S. , Collazo Irizarry Diego K. , Baksi Krishna
Published: May 2022

Neovascularization, a normal physiological process orchestrates through vasculogenesis from mesoderm cell precursors. In healthy adults, the vasculature is quiescent but pathological angiogenesis is evident during wound healing and in tumor growth. Thus, angiogenesis becomes a fundamental step for tumors transitioning from a benign state to malignancy where endothelial cells proliferate 45 times faster. Our laboratory has recently shown that asparagine‐linked (N‐linked) protein glycosylation is essential for enhanced capillary endothelial cell proliferation in situ. Inhibiting N‐glycosylation with Tunicamycin causes G1 arrest and induces cell death by apoptosis due to “ER stress” and is not reversible by VEGF. Tunicamycin also inhibits angiogenesis in vivo in Matrigel™ implants in nude mice as well as in a pre‐clinical mouse model of double negative (ER‐/PR‐/HER2+) breast tumor.

Wnt pathway is involved in cellular differentiation to organogenesis, and supports angiogenesis. In “canonical” Wnt/β‐catenin signaling pathway β‐catenin translocates to nucleus and activates Wnt/β‐catenin target genes, including vascular endothelial growth factor A (VEGF‐A). Irrespective of the Wnt ligand and Frizzled being glycoproteins, the cross‐talk between protein N‐glycosylation and the Wntpathway has not been addressed. This led us to hypothesize that decreased expression of Wntprotein(s) in Tunicamycin treated endothelial cell would decrease cellular proliferation. We have used capillary endothelial cells as a model and observed down regulated expression of Wnt proteins in Tunicamycin treated cells. Our results support Tunicamycin‐induced “ER stress” interferes with Wnt signaling and inhibits angiogenesis. We, therefore, conclude molecular understanding of the process would provide a novel target to inhibit angiogenesis and breast tumor progression.
Generation of isofemale lines from hypoxia tolerant Drosophila melanogaster population
Authors: Zhou Dan , Xue Jin , Stobdan Tsering , Haddad Gabriel G.
Published: May 2022

Hypoxia is a common pathological element in many human diseases. Understanding the mechanisms that underlie hypoxia tolerance or susceptibility is essential for developing novel strategies for treatment or prevention. Through experimental evolution, we obtained a Drosophila melanogaster population that can live perpetually in severe, normally lethal, hypoxic environments. We generated a Panel of Low Oxygen Tolerant (PLOT) isofemale lines (total 79 lines) from this hypoxia tolerant population to study or identify the specific genetic mechanisms that are responsible for the hypoxia tolerant trait. These isofemale lines showed significantly higher hypoxia tolerance as compared to their wildtype parental isofemale lines and the wildtype DGRP (Drosophila melanogaster Genetic Reference Panel) isofemale lines. For example, 95% of the PLOT lines showed hypoxia tolerance with eclosion rate >50% at 5% O2 level. In contrast, none of the parental lines and only 15% of the DGRP lines had similar eclosion rates under the same hypoxic condition. Furthermore, under the lethal condition with 4% O2, our PLOT lines exhibited a wide range of eclosion rate from 1% to 80% with a clear pattern of Gaussian‐Distribution, demonstrating that these PLOT flies may provide us a unique opportunity to study the molecular basis of stress‐directed evolution, such as the role of genetic variations in the protein coding regions and functional DNA elements in hypoxia adaptation. These PLOT lines can also be used to study phenotypic plasticity as well as genetic‐epigenetic interactions in hypoxic, or other stressful, environments.
Identifying Novel Molecular Genetic Markers in Attention Deficit Hyperactivity Disorder and Glioblastoma Multiforme
Authors: Kim David T.
Published: May 2022

Attention deficit hyperactivity disorder (ADHD) and glioblastoma multiforme (GBM) are two neurological disorders with high rates of incidence and mortality respectively, both capable of inflicting long‐term physiological damage. Although prior research has indicated that brain tumor survivors exhibit increases in susceptibility to ADHD, the underlying genetic components still remain relatively unknown. The primary purpose of this investigation was to identify genetic associations between ADHD and GBM by single‐nucleotide polymorphisms (SNPs) of target genes. NCBI’s Gene Expression Omnibus was accessed, and genes of interest were selected by differential gene expression analysis in GBM patients. Sequence data of ADHD and GBM patients were obtained from the Sequence Read Archive (SRA), followed by the construction of analysis pipelines to map the sequences against human chromosome 12, followed by indexing and variant calling. This study highlights 18 variants (including 7 reported SNPs) that contribute to the genetic overlap of ADHD and GBM and may provide a useful tool for precision medicine.
PCID2 Facilitates the Nuclear Export, but not the Centrosomal Localization, of BRCA2 in Hs578T Breast Cancer Cells
Authors: Pollio Noah C. , Resendes Karen K.
Published: May 2022

PCI‐domain containing protein 2 (PCID2) assists in the export of proteins from the nucleus to the cytoplasm. PCID2 also localizes to the centrosome, where it is present more frequently in cells exhibiting an abnormally high number of centrosomes, an aberration associated with the development of cancer. Several proteins that negatively regulate the centrosome cycle are typically found in the nucleus, and we have previously discovered that PCID2 is involved in the nuclear export and centrosomal localization of one key centrosome cycle regulator, BRCA1. Therefore, PCID2 may play a role in the regulation of the centrosome cycle by delivering key regulators of duplication. BRCA2 is a nuclear tumor suppressor that travels to the centrosome, where it helps to arrest the centrosome cycle and prevent overduplication. BRCA2 mutations are associated with the development of breast and ovarian cancers, both of which are also associated with centrosomal overduplication. We proposed that PCID2 aids in the transport of BRCA2 from the nucleus to the centrosome. Using siRNA knockdown of PCID2 and subsequent immunofluorescence studies of BRCA2 and γ‐Tubulin, we found that the loss of PCID2 significantly decreased the amount of BRCA2 able to be exported from the nucleus to the cytoplasm, with Hs578T breast cancer cells depleted of PCID2 demonstrating a 37% increase in nuclear BRCA2 retention. However, the loss of PCID2 demonstrated no significant impact on the ability of BRCA2 to localize to centrosomes. Our results indicate that while PCID2 affects the cellular localization of BRCA2 by assisting its nuclear transport, it likely does not directly influence the recruitment of cytoplasmic BRCA2 to the centrosome. Ongoing studies are exploring the relationship of PCID2 to BRCA2 export and centrosomal localization in ovarian cancer cells and are attempting to confirm the presence of a PCID2‐BRCA2 nuclear export complexes in breast and ovarian cancer cells. These findings will shed light on the process by which PCID2 role in nuclear transport and the centrosome cycle, leading to a better understanding of centrosomal amplification and tumorigenesis in a variety of cancer types.
Soft silicone‐based neural interface to modulate bladder function: chronic studies in awake behaving cats
Authors: Kumar Ritesh , Gopinath Chaitanya , Simpson Tyler W. , Weir David M. , Thiessen Alexander , McDonnall Danny , Gaunt Robert A.
Published: May 2022

Direct bladder wall stimulation has been attempted for decades to restore bladder function in people with spinal cord injury and other voiding dysfunctions. However, these efforts were limited by co‐activation of the urethra, legs and other pelvic organs at stimulus intensities that evoked bladder contractions. Neural interfaces for the detrusor muscle itself face several challenges due to its structure and the volume changes it undergoes in normal function. We designed a stretchable silicone net that can be placed around the bladder body to anchor a soft electrode array that interfaces directly with the base of bladder to generate bladder contractions.

We created implantable versions of the electrode nets and tested them in chronic experiments. We implanted 5 healthy cats (4 females, 1 male) and tested them with and without anesthesia for 2‐3 months. A pressure catheter was implanted into the bladder to control volume and measure pressure.

Bladder wall stimulation through various electrode configurations (monopolar, bipolar), temporal patterns (single electrode, sequential stimulation of multiple electrodes) and stimulus intensities were able to generate complete bladder emptying. In behaving cats, bladder wall stimulation at many different stimulus intensities elicited efficient voiding at physiological bladder pressures (med: 41, IQR: 27‐60 cmH2O). However, there was little relationship (r<0.28) between stimulation amplitude and bladder pressure itself. Rather, stimulation amplitude had an effect on the time between stimulation onset and bladder contraction onset. At low stimulation amplitudes, there was a longer time between stimulation onset and voiding (med: 35s, IQR: 13.5‐68.2s) than occurred at higher amplitudes, which evoked more rapid voiding (med: 23s, IQR: 10‐45s). This contrasts with anesthetized tests, where stimulation at 3 ± 2 mA evoked increases in bladder pressure up to 35 cmH2O. These results suggest that stimulation of the bladder wall recruits sensory pathways leading to an urge to void, but that can be suppressed by the animals. For most of the duration of the implants, stimulation evoked functional voiding with consistent electrical stimulation thresholds. However, after several months there was a failure of an implanted bond that limited the overall duration of the implants.

Chronic experiments demonstrate that these electrode nets can be used as a neural interface to generate or initiate comfortable, complete bladder emptying in awake behaving cats. Future efforts include understanding the effects of evoked sensations on voiding and improving the integrity of the bonding between the flexible conductors and the lead wire.
Central nervous system 5‐HT plays a role in ventilatory and arousal responses to hypoxia and hypercapnia in spinal cord injured mice
Authors: Qiu Qingchao , Kissane Dylan , Kuhn Donald , Mateika Jason
Published: May 2022

Purpose

The present investigation was designed to explore the impact of serotonin (5HT) on the arousal and ventilatory response to hypercapnia and hypoxia following spinal cord injury (SCI) in mice.

Methods

Telemetry transmitters were surgically implanted in wild type mice (Tph2+/+) (n =7 for SCI, n=7 for Sham) and tryptophan hydroxylase 2 knockout mice (Tph2‐/‐) (n=6 for SCI, n=6 for Sham) to measure electroencephalography, electromyography (EMG) of the genioglossus (GG) muscle, core body temperature and gross motor activity. Following recovery, the Tph2+/+ and Tph2‐/‐ mice were placed in whole body plethysmographs and measures of the ventilatory and arousal response to hypoxia and hypercapnia during non‐rapid eye movement sleep were obtained. Following these measures, a C2 hemi‐section of the spinal cord was completed and the physiological measurements were repeated 4 and 21 days following SCI. The ventilatory data is presented as a fraction of baseline.

Results

The breathing frequency, tidal volume, and minute ventilation response to hypercapnia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Breathing frequency: 1.37 ± 0.12 vs. 1.02 ± 0.10, p = 0.036, Tidal volume: 1.70 ± 0.21 vs. 0.91 ± 0.09, p = 0.002, Minute Ventilation: 2.87 ± 0.48 vs. 0.93 ± 0.12, p = 0.001). In addition, the tidal volume and minute ventilation response to hypoxia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Tidal Volume: 1.27 ± 0.09 vs. 0.94 ± 0.08, p = 0.009, Minute Ventilation: 1.45 ± 0.14 vs. 0.98 ± 0.09, p = 0.012). The arousal latency to hypercapnia was reduced in Tph2+/+ compared to Tph2‐/‐ mice at 4 days post SCI (16.22 ± 4.15 vs. 73.83 ± 15.78 s, p = 0.013). The number of arousals during exposure to hypercapnia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (5.31 ± 0.50 vs. 3.40 ± 0.51, p = 0.021). The magnitude of the GG response to hypercapnia and hypoxia was greater in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (Hypercapnia: 47.42 ± 10.17 vs. 0.73 ± 0.18, p = 0.012, Hypoxia: 43.82 ± 11.25 vs. 3.30 ± 1.65, p = 0.019). The fraction of episodes in which there was no EMG response to hypercapnia and hypoxia was lower in Tph2+/+ compared to Tph2‐/‐mice at 4 days post SCI (Hypercapnia: 0.43 ± 0.10 vs. 0.92 ± 0.08, p = 0.001 Hypoxia: 0.19 ± 0.12 vs. 0.85 ± 0.15, p = 0.008). The fraction of episodes in which there was no EMG response to hypercapnia was lower in Tph2+/+ compared to Tph2‐/‐ mice at 21 days post SCI (0.19 ± 0.06 vs. 0.79 ± 0.10, p = 0.001).

Conclusion

The ventilatory, arousal and GG response to hypercapnia and hypoxia is greater in Tph2+/+ compared to Tph2‐/‐ mice following SCI. Our results suggest that deficiencies in serotonin have a role in regulating respiratory and arousal responses to hypercapnia and hypoxia following SCI.
The Role of Glutamatergic Signaling in Central Noradrenergic Neurons in Respiratory Control
Authors: Chang Yuan , Ray Russell
Published: May 2022

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS) and Rett Syndrome, in which abnormal chemosensory function is thought to play a role. However, which NA populations modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Histological evidence suggests that glutamate is co‐transmitted in subsets of brainstem NA neurons, some of which may contribute to regulating chemoreflexes. However, the extent of glutamate co‐transmission in the central NA system remains uncharacterized. Further, it remains unclear what role NA‐based glutamatergic signaling plays in various aspects of respiratory homeostasis.

To anatomically map potential glutamate‐expressing NA neurons, we characterized the expression profile of all three known of Vesicular Glutamate Transporters (Vglut1, Vglut2 and Vglut3) in NA neurons by intersectional genetic fate mapping. For each Vglut1/2/3 transporter, acorresponding Cre knock‐in recombinase line was combined with DBH‐p2a_Flpomice. These Vglut1/2/3_Cre; DBH‐p2a_Flpocompound lines were crossed with the RC::F_TdTomato_P_GFPintersectional reporter mice. In each of the three intersectional reporter crosses, NA neurons co‐expressing either Vglut1, Vglut2, or Vglut3 are labeled by green fluorescent protein (GFP) while NA neurons without any Vglut1/2/3 expression are labelled by red fluorescent protein (tdTomato). Our preliminary fate maps, which reflect the cumulative expression profiles of glutamate markers in NA neurons throughout the development and lifetime of the mouse, found that about 80% NA neurons express Vglut2 at some point during their development and posterior medullary NA neurons express Vglut3 as well.

Prior studies suggest that Vglut2‐based glutamatergic signaling may play a role in respiratory control. To determine what physiological role, if any, Vglut2‐based glutamatergic signaling may play in adult respiratory homeostasis, we conditionally ablated Vglut2 in central NA neurons by crossing DBH‐Cre/+with Vglut2flox/flox mice. Mutants and sibling controls (6‐8 weeks) were assessed for respiratory function by whole‐body barometric plethysmography. Ventilation and VO2 were assessed under room air (21%O2,79%N2), hypercapnia (21%O2, 74/72/69%N2, 5/7/10%CO2) and hypoxia (10% O2, 90% N2). Our preliminary data indicates that loss of Vglut2 in NA neurons does not significantly impact breathing under room air, hypercapnia, or hypoxia. Thus, our data suggests that Vglut2‐based glutamatergic signaling within the central NA system is dispensable for normal baseline breathing and hypercapnic, and hypoxic chemosensory reflexes.
Conservation of the trblGene Across Drosophila Species: How Does Structure Support Function?
Authors: Raab Sarah , Hark Amy T.
Published: May 2022

In collaboration with the Genomics Education Partnership (thegep.org), we seek to understand how genes functioning in the insulin signaling pathway evolve. In Drosophila melanogaster, the gene tribbles(trbl) codes for a protein that binds to Akt (also known as Protein Kinase B) and blocks Akt‐mediated insulin signaling. Trbl misexpression also affects the Akt target, Foxo (Forkhead box transcription factor). In humans, expression of trblorthologs is associated with degradation of substrates through E3 ligase‐dependent ubiquitination. Misexpression of TRIB2, one of the three Tribbles pseudokinases in humans, has been found to be associated with certain cancers. Our initial research consisted of annotating orthologs of trbland more recently foxoacross the genus Drosophilato look at conservation within the fly species. In order to identify orthologs, we considered synteny of the genomic neighborhood and determined conservation with the D. melanogasterprotein using BLAST. We then determined the location of the coding exons using data available in the UCSC Genome Browser, creating a gene model to evaluate how well our proposed gene matches the D. melanogaster ortholog. In addition to this process, we also used sequence alignments as well as structural databases to gather information about conservation of the Trbl orthologs among seven species. Trbl orthologs across Drosophilashow 60‐70% amino acid similarity. The highest degree of conservation is within the pseudokinase domain, which takes up the majority of the structure of the gene, for both Drosophilaand humans. Multiple sequence alignments reveal that the N‐ and C‐termini show less conservation and are shorter in human orthologs as compared to Drosophila proteins. This is significant as those regions in human TRIBs have documented functions in protein degradation. Continuing this work, we hope to gain a better understanding of how the structure of Trbl/TRIB proteins supports their function, including with other players in the insulin signaling pathway.
Morphometric Analysis of the Foramen Magnum in a Brazilian Population
Authors: Rocha Paulo V. P. , Pedroza Rafaella H. S. , de Oliveira Vanessa F. , Ribeiro Rodrigo M. , Carioca Levi C. , e Silva Renata S. , Cerqueira Gilberto S. , de Miranda João A. L.
Published: May 2022

The foramen magnum is an opening located at the base of the skull, more specifically in the occipital bone, and it has a remarkable anatomical significance, as many important structures, such as nerves, arteries and the medulla oblongata, pass through it. A small, large or asymmetric foramen magnum can end up causing clinical complications, such as compression, or increasing the risk of herniation. By becoming aware of the most frequent sizes and shapes in the population, it also becomes easier to detect diseases that change its morphology, such as achondroplasia, which makes it smaller, and Chiari I and II malformations, which makes it larger. In this study, we aim to observe gender‐related anatomical differences of the foramen magnum and report the frequency of the most commonly observed shapes. To achieve this, we studied the morphometry of the foramen magnum in 40 skulls. Anteroposterior and transverse length measurements were manually obtained with the aid of a digital caliper. Digital images were used to analyze the shapes of each available foramen magnum, which were then subdivided according to the following categories: oval, diamond, hexagon, egg‐shaped, pentagon and round. The differentiation between male and female was based on sexual dimorphism, using anatomical landmarks as a reference. The results obtained showed that among the 40 skulls observed, 32 were male and 8 were female. Our study revealed a statistically significant (p=0.0462) sex‐related difference between the anteroposterior diameters of the foramina, which is often larger in men and smaller in women. In contrast, the transverse diameter was not statistically significant. As for the shapes, 39 were analyzed: 12 (30.76%) of them in oval shape, 10 (25.64%) in diamond shape, 8 (20.51%) in hexagon shape, 4 (10.25%) in egg shape, 3 (7.69%) in pentagon shape and 2 (5.12%) in round shape. Therefore, we conclude that the anteroposterior length of the foramen magnum can be used as a tool to help differentiate between skulls of different sexes for academic and forensic purposes. Knowledge of the most common possible shapes for the foramen magnum can also be an important predictor, useful for clinical and anatomical knowledge. The data obtained has potential use in forensic techniques, in addition to providing information on variations in the Brazilian population.
Prospects of transient elastography’ using in the veterinary hepatology
Authors: Ponamarev Vladimir , Andreeva Nadezhda , Popova Olga
Published: May 2022

Elastography is the qualitative and quantitative analysis of tissue’ elastic characteristics. The diagnostic of hepatopathies is based on the puncture biopsy of the liver. At the same point, the low biochemical activity of liver diseases is not always associated with the analogic histologic pattern. There were not excluded the advance of hepatic pathology. The primary hypothesis of this research was the prospects of transient elastography used in the diagnosis of hepatic disorder in animals. The methodology is about the formation of rotational waves in the determinate organ and measuring their velocity. The rotational wave has generated by a special detector which has the source of mechanical vibrations. The detector is located on the patient’s body surface in the zona of the organ’s projection. The studies were carried out in the laboratory animals, which were rabbits (n=20). The tool was used iLIvTouch. Standard methods also performed the liver biopsy and histological examination. Thus, we have detected the correlation between parameters of liver stiffness and the biopsy’ results performing transient elastography. The histology was pointing to the specific diagnosis: the fatty hepatosis with liver stiffness in 3.8‐4.2 kPa (n=10, the normal range was 5.8 kPa). Liver fibrosis was also diagnosed with liver stiffness in 6.2‐6.8 kPa (n=5) and toxic hepatitis with liver stiffness in 3.2‐3.6 kPa (n=5). The results of our research on laboratory animals after hepatopathies’ induction with different pathogenesis showed that the parameters of liver stiffness using transient elastography correlated with the results of the morphological evaluation. So, we can assume prospects of transient elastography using equally with standard methods.
The role of CGPR ‐ receptor antagonist in the plastic process on the hepatopathies with toxical genesis
Authors: Ponomarev Vladimir , Lunegov Alexander , Andreeva Nadezhda , Kovalev Sergey
Published: May 2022

Many research results showed that the CGPR concentration in the blood plasma has significantly increased by hepatopathies with different genesis. The hyperkinetic portal blood flow has caused this. This research's primary hypothesis was the potentiation of CGPR ‐ receptor antagonist to the liver's plastic process by toxical hepatopathies provocation. The study aimed to evaluate the influence of combination the hepatoprotective agent “Hepaton‐vet” (dose ‐0.5 mg/kg per rat during 7th days) and monoclonal antibodies G2 (IgG2, dose ‐0.75 ml/kg per rat one‐time before experimental start) on the hepatopathy's regenerative process which was 1,2‐dichloroethane induced during 7th days with following orthothanasia (the method by Malcom A., Steele M.D., 1991). White outbred rats were used (n=20). The body mass of rats was ranged from 180 to 220g. The liver histological examinations were performed by a visual method using Image J Software. Thus, the histologic pattern of rats' liver was included in the connective tissue membrane, which was obduced the liver externally. The hepatic lobules of rats have had shapes of hexagon prism without clean‐cut peripheries. The interlobular tissue in the lobule's feather included the interlobular vena, the interlobular artery, and the bile duct lined with cuboidal epithelium. The lobular base was sited central vein with hepatic plates, which were formed hepatocytes. The rats' hepatocytes had a wrong multiangular shape were was 60 % of all in the liver cells pattern was typical for liver with high regenerative level without signs of vassal‐ mesenchymal reactions, which was correlated with CGPR ‐ receptor antagonist. So, we can assume that the CGPR ‐ receptor antagonist drugs have a great variety in their administration, including the potentiation of other drugs used in the pharmacorrection of different pathologies. The balanced influence on the CGPR – receptors significantly influence the homeostasis maintenance and the nociception of the hepatobiliary system.
Evaluation of Small Molecule Inhibitors of Pseudomonas Virulence factor LasB as Non‐Traditional Immunotherapeutics
Authors: Chou Sophia R. , LaRock Doris L. , Sun Joshua , Nizet Victor , Bohannon Julia K. , LaRock Christopher N.
Published: May 2022

Pseudomonas aeruginosa can cause severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis. Due to its multidrug resistance, it is a priority pathogen for therapeutic development. Inflammation during such infections, which causes destruction of lung architecture and function, is activated in part through the maturation of IL‐1β by the virulence factor LasB. Previous results in mouse models suggest that matrix metalloprotease inhibitor drugs also inhibit LasB, preventing cell death and inflammation caused by caused by P. aeruginosa. We hypothesized that other inhibitors of matrix metalloproteases, including tetracycline‐family antibiotics, could be used to target LasB. We evaluated small molecule inhibitors against LasB activation during infections of macrophages, neutrophils, and monocytes and the synergy of these inhibitors with conventional antibiotics and anti‐inflammatories. We first optimized the drug concentration by testing the ability of the various drugs to inhibit LasB at various dilutions. Using an effective concentration, we then infected the various cell types with P. aeruginosa and analyzed the impact of the inhibitors on cell death and maturation of IL‐1β in infected cells. The results indicate that several tetracycline‐family compounds inhibit LasB during P. aeruginosa infections and decrease cell death. This was true for compounds that had no antimicrobial activity yet still inhibited LasB. In addition, the results suggest that the drugs target different cell types. Together, we find that tetracyclines may have therapeutic benefits, even against resistant bacteria, due to their ability to decrease inflammation. This may serve as an effective adjunctive therapy during infection to limit tissue damage and give the immune system and antibiotics more time to respond.
The Yeast Glycogen Synthase Kinase Homolog Rim11 Phosphorylates the Phosphatidic Acid Phosphatase Pah1 to Inhibit its Catalytic Activity
Authors: Khondker Shoily P. , Han Gil‐Soo , Carman George M.
Published: May 2022

The yeast phosphatidic acid (PA) phosphatase, Pah1, regulates the synthesis of triacylglycerol (TAG) and phospholipids by controlling the relative amounts of its substrate PA and product diacylglycerol. Pah1 phosphorylation, which is mediated by multiple protein kinases, has been shown to protect the protein against proteasomal degradation and regulates its localization and catalytic activity. Phosphorylated Pah1 is cytoplasmic, but it translocates to the nuclear/endoplasmic reticulum membrane for catalytic function through its dephosphorylation. In this study, we show that Rim11, a yeast homolog of glycogen synthase kinase phosphorylates Pah1. The rim11Δ mutant shows alterations in lipid composition with elevated TAG levels during exponential growth, the phase in which Rim11 is the most abundant. Rim11 purified from yeast phosphorylated Escherichia coli‐expressed Pah1, which lacks endogenous phosphorylation, in a manner that is dependent on reaction time and the amounts of Rim11, Pah1, and ATP. Phosphoamino acid analysis and phosphopeptide mapping showed that Pah1 phosphorylation by Rim11 occurs at multiple serine and threonine residues, which was supported by mass spectrometry analysis which indicated that Rim11 has a major phosphorylation site that is shared with multiple kinases (Ser‐602) and two unique minor phosphorylation sites (Thr‐163 and Thr‐164). Furthermore, in vitro PA phosphatase assays show that Pah1 catalytic activity is inhibited following phosphorylation by Rim11.
Virus‐Like Vesicles (VLVs) as Therapeutic Vaccine Vectors for Hepatocellular Carcinoma
Authors: Yang Lei , Pham Kien , Yarovinsky Timur O. , Defina Samuel M. , Rose John K. , Liu Chen
Published: May 2022

Hepatocellular Carcinoma (HCC) is the most common form of liver cancer and is the leading cause of cancer‐related mortality globally. Despite the substantial progress that has been made in current treatments, HCC remains malignancy with a high recurrence rate due to a poor prognosis. Therefore, developing novel immunotherapy approaches to treat HCC is crucial to solving this global public health problem. Anti‐tumor vaccines are promising strategies to elicit immune responses for anti‐tumor immunity. In this study, we designed and cloned an alphavirus‐vesiculovirus hybrid vaccine vector VLV‐GPC3 that can generate virus‐like vesicles (VLVs) as self‐amplifying RNA replicons to additionally express GPC3, a cell‐surface heparan sulfate proteoglycan is highly expressed in HCC. The potency of the VLV‐GPC3 as tumor vaccine was assessed in vivo by immunization of C57BL/6 mice with 108 PFU of VLV‐GPC3, followed by subcutaneous implantation of Hepa1‐6 HCC cell line cells into C57BL/6 mice. The results showed that VLV‐GPC3 immunization could elicit robust GPC3‐specific antibody responses and effective protection by increasing CD8+ T cells and minimizing the tumor size. Further, the immunized mouse splenocytes co‐cultured with GPC3 could significantly promote IFN‐r production and CD4+ T cell proliferation. Our data indicate that VLV‐GPC3 could be developed as a novel vaccine candidate and offer a new treatment strategy against HCC.
Effect of Uracil DNA Glycosylase Activity on the Efficacy of Thymidylate Synthase Inhibitor/HDAC Inhibitor Combination Therapies in Colon Cancer
Authors: Kulkarni Rashmi S. , Weiser Brian P.
Published: May 2022

Human uracil DNA glycosylase (UNG2) is responsible for removal of uracil bases from DNA and initiates base excision repair pathways. Accumulation of uracil or its fluorinated analogs in DNA is one of the killing mechanisms of thymidylate synthase (TS) inhibitors in cancer cells, and depletion of UNG2 often enhances the toxicity of these anticancer drugs. We used CRISPR to knockout UNG2 from HT29 colon cancer cells and confirmed the absence of protein by western blot and uracil excision assays. We tested the effect of UNG2 KO on the efficacy of multiple TS inhibitors (5‐fluorouracil, fluorodeoxyuridine, pemetrexed, and raltitrexed), and we determined that only fluorodeoxyuridine and raltitrexed were significantly more potent in UNG2 KO cells compared to wild‐type HT29 cells (fluorodeoxyuridine IC50: 2 mM (wt) vs. 3 nM (KO); raltitrexed IC50: 14 nM (wt) vs. 2 nM (KO)). Interestingly, UNG2 protein levels can also be depleted by the HDAC inhibitors SAHA and MS275, providing a pharmacologic strategy to reduce UNG2 activity in cells. Unexpectedly, the HDAC inhibitors synergized with 5‐fluorouracil, but not fluorodeoxyuridine, in both wild‐type and UNG2‐knockout cells. This suggested that HDAC inhibitors sensitized cells to 5‐fluorouracil through an UNG2‐independent mechanism. Moreover, cell death pathways activated by fluorodeoxyuridine and regulated by UNG2 activity are not sensitized by HDAC inhibitors. Our combined genetic and pharmacologic strategies targeting UNG2 activity in cells are defining cell death mechanisms for combination therapies of TS inhibitors and HDAC inhibitors. Future work will examine these drug combinations in additional cell lines to understand optimal therapeutic combinations and to further refine mechanisms of cell death.