bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Chlamydomonas reinhardtii’s Cia7 gene in E. coli Utilizing pMAL‐c5x and pCold I Vectors
Authors: Gonzalez David I. , Ynalvez Ruby A.
Published: April 2020

With the increase in industrialization, heavy metal pollution has become prevalent in the environment posing a danger to public health. Sustained exposure to heavy metals can induce mutagenic effects in genetic expression and in biochemical processes in the body. With an attempt to survive in heavy‐metal pollution, some organisms have developed detoxifying mechanisms that utilize metal‐binding proteins. Chlamydomonas reinhardtii is a green haploid microalga that is used as a model organism in the study of metal homeostasis and tolerance. C. reinhardtii’s Cia7 gene encodes for the protein, CIA7. The CIA7 protein is composed of 104 amino acids consisting of several cysteine residues. Cysteine residues are found to have a high affinity towards metal ions. Thus, we hypothesized that CIA7 might play a role in heavy‐metal homeostasis. The objective of this research is to (1) isolate Cia7 from C. reinhardtii, (2) compare the over‐expression of the CIA7 protein in Escherichia coli using the pMALTM‐c5x vector and the pColdTM I vector, and (3) establish the potential role of the Cia7 gene. The synthesized cDNA library was used to amplify and isolate the target gene, Cia7. The amplified product was purified and ligated using the maltose‐binding vector, pMALTM‐c5x, and the cold‐shock expression vector, pColdTM I. Recombinant DNA were transformed into E. coli and thirty clones per vector were isolated. Presence of the insert was verified by restriction enzyme digests and by overexpression of the CIA7 protein. This study will report the comparison of protein over‐expressions in two overexpression vectors, namely pMALTM‐c5x and pColdTM I. By overexpressing the CIA7 protein from C. reinhardttii we can further characterize the gene and establish its involvement in metal tolerance.
Disruption of Metal Homeostasis and its Effect on Bacterial Growth and Antibiotic Resistance
Authors: Padyab Nika , Gallas Genna , Lozoya Maria , Veltri Charles , Hernandez Jose
Published: April 2020

As effective as antibiotics have been since they were first discovered, antibiotic resistance proves to be a worldwide threat, endangering their efficacy. Over the years, there has been a significant decline in development of novel class antibiotics. These two factors have made once easily treated infections difficult to manage. Carbapenem‐resistant Enterobacteriaceae (CRE) are an emerging threat due to their high levels of antibiotic resistance. CRE have become resistant to almost all antibiotics, and cause 23,000 infections per year in the United States. Metals are a vital nutrient in all living organisms including bacteria and, have thus been targeted by antimicrobial agents. Bacteria, like all living organisms, must regulate their metal homeostasis in order to survive and replicate. Therefore, mechanisms able to diminish the availability of the metals to bacterial pathogens can be used to develop new therapeutic drugs.

An example of a mechanism of bacterial resistance to antibiotics is the production of inactivating enzymes, such as carbapenemases, an example of which is seen in Klebsiella pneumoniae. Carbapenemases play an important role against b‐lactam antibiotics such as imipenem in Enterobacteriaceae. Carbapenemases are categorized based on their functional and molecular properties. Class B carbapenemases are metallo‐b‐lactamases (MBL) which require zinc in their active site, and are therefore considered metalloenzymes. MBL can hydrolyze all b‐lactams and are not inactivated by b‐lactamase inhibitors.

In this study we analyzed antimicrobial susceptibility of K. pneumoniae isolates, KPC 2146, KPC 1705 and KPC 1706 which contain class B, class A and no carbapenemase, respectively. Metal homeostasis of isolates was disrupted by inducing zinc deficiency using TPEN, a metal chelator with high affinity for zinc, and preparing a zinc deficient minimal media. Carbapenemase activity was assessed using CARBA‐NP test and LC‐MS. Bacterial susceptibility of zinc‐deficient isolates to carbapenems was analyzed using spot plating, growth curve, minimum inhibitory concentration and ring of inhibition.

Our findings demonstrate zinc dependency of class B carbapenemase in the KPC 2146 isolate, while class A carbapenemase in the KPC 1705 isolate was not affected. Spot plating and growth curve analyses demonstrated that zinc deficiency lowers bacterial growth in KPC 2146 in the presence of imipenem. Zinc deficiency via TPEN addition lowered imipenem, doripenem, ertapenem and meropenem MIC in KPC 2146. Ring of inhibition of KPC 2146 with imipenem was greatly affected by zinc chelation. In conclusion, we demonstrate that disrupting zinc homeostasis affects class B carbapenemase activity, which in turn increases bacterial susceptibility to carbapenems.

Support or Funding Information

Nika Padyab was supported by funds from the Biomedical Sciences Program at MWU. Drs. Veltri and Hernandez were supported by MWU intramural funds.
Rectovaginal septum and influencing factors in its morphology
Authors: Salinas-Alvarez Yolanda , Rodriguez-Abarca Maria Alejandra , Quiroga-Garza Alejandro , Guzman-Lopez Santos , Elizondo-Omaña Rodrigo Enrique , Jacobo-Baca Guillermo , de la Fuente-Villarreal David , de la Garza-Castro Oscar
Published: April 2020

Introduction

The rectovaginal septum (RVS) is a layer of dense fibro‐connective tissue, smooth muscle, and elastic and collagen fibers containing a neurovascular bundle of the inferior hypogastric plexus, providing support, innervation, and irrigation. It allows for mobility between the rectum and vagina and helps in limiting the spread of infections or tumors, as well as a guide for several types of surgeries. Any damage may predispose to rectocele, enterocele, fistula, constipation, incontinence, or sexual dysfunction.

Currently, there is no consensus regarding the morphology (thickness or length). The objective of this study was to determine morphometric characteristics and variations in correlation to the number of pregnancies, method of delivery, parity and estrogenic exposure.

Materials and methods

An observational, cross‐sectional, retrospective, descriptive and comparative study was performed. Pelvic RMIs of Hispanic women from the northeast of México were obtained, with an age of 15 years and older. Those with gynecological pathology were excluded. Age, obstetrics and gynecology history was registered and the population was categorized by the number of pregnancies (nulligravida, 1–3 pregnancies, or ≥ 4 pregnancies), type of delivery (vaginal, cesarean section, or mixed), parity (primiparous or multiparous) and estrogen exposure (premenopause and postmenopause).

Results

A total of 102 RMIs were included, the mean age was 41.03 ± 15.23 years (range 15 to 77). A quarter (24.5%) of the patients were nulligravida, the rest (75.5%) being primi‐ or multigravida. Of those with previous pregnancies, vaginal delivery was the most common type (49.35%); 16.88% had a cesarean section, and 31.17% had a history of both types of delivery. Within the women who had a vaginal delivery, 19.6% were primipara and 41.2% were multipara; only 25.5% were postmenopause.

The mean RVS length was 73.16 mm, with a thickness of 2.76, 2.24, and 2.53 mm for the upper, middle and lower thirds. In women with vaginal delivery, multiparous had a statistically significant longer RVS than primiparous (p 0.011), as well as a tendency of higher thickness, although the latter without statistical significance. Pre‐menopause women had statistically significant longer RVS (p 0.031) with a tendency to lose thickness towards post‐menopause.

Conclusion

The morphology of RVS can be modified by different factors such as age, number of pregnancies, number of births and estrogenic exposure.
Factors That Limit Hispanic Representation in Medical Education: A Review of Hispanic Underrepresentation in Medical Education
Authors: Alaina Omar I. , Ynalvez Ruby A. , Ynalvez Marcus A.
Published: April 2020

The underrepresentation of Hispanics in medical education poses a looming problem for the United States. The inability to produce a physician population that mirrors their patients leads to inadequate healthcare for individuals of all ethno‐racial backgrounds. By 2045, Hispanics are projected to make up the majority of the population. The U.S. is 30% Hispanic but only 4% of all current medical students are Hispanic. The objectives of this research were to: [1] analyze key academic, socioeconomic, and psychosocial factors that hinder Hispanics in entering medical schools, and [2] highlight the benefits of increasing Hispanic representation within medical education. A library research using electronic databases such as JSTOR, Proquest, and Medline was conducted. Fifty peer‐reviewed articles were selected, analyzed, and evaluated for their relevance. Relevancy was judged based on how well the articles addressed our objectives. The identification of key factors was done by surveying how often frequently these occurred in the literature.

Our review identified multiple key factors. In terms of academics, Hispanics were limited by low GPA, MCAT scores, and gateway courses. In terms of socioeconomic factors, Hispanics were limited by household income and the high cost of obtaining a medical education. Lastly, key psychosocial‐related factors identified were a lack of Hispanic mentors, negative stereotype, stigmatization, micro aggression, and limited exposure to real life role models and to health careers. Our review shows that at any point in time, one or more of these factors play a limiting role in Hispanic students gaining admission into a medical school. To counter these hindrances, the following recommendations are in order: [1] create mentoring programs supported by medical schools for students in primary, secondary, and undergraduate levels; [2] devote resources to socially and economically disadvantaged areas by running summer internship programs, [3] create pipeline programs that are focused on URMs, and [4] encourage institutional change within medical education itself by seamlessly integrating cultural competency aspects into programs, curricula, courses, student orientation programs, and faculty training.

Support or Funding Information

This research was funded by the Texas A&M International University College of Arts and Sciences and ACT On IDEAS.
Early Development of the Heart and Great Vessels
Authors: Weinhaus Anthony J.
Published: April 2020

The cardiovascular system is one of the first organ systems to develop in the embryo. By day 21, three germ layers: endoderm, mesoderm, and ectoderm are formed by the gastrulation process. Cells in the mesoderm coalesce through a process called vasculogenesis to form two parallel endocardial tubes. Folding of the gut tube brings these two parallel endocardial tubes towards the midline enabling them to fuse into a single linear‐shaped heart tube. By day 18, the lateral plate mesoderm splits to form two layers – splanchnic (visceral) and somatic (parietal). The cells of the splanchnic mesoderm will develop into the myocardial cells of a Primary (PHF) and Secondary Heart field (SHF). By day 22, the heart begins to beat and lengthen by the addition of cells from the SHF to the PHF. This develops into the outflow tract ‐ aortic and pulmonary vessels, and the right ventricle. By day 28, the linear‐shaped heart tube contains regions called the sinus venosus, primitive atrium, primitive ventricle, and bulbus cordis (outflow). The proximal and distal ends become “fixed” by the pericardium, the bulbus cordis and primitive ventricle grow faster than the other regions of the heart tube, and the heart bends upon itself to form a u‐shape. The primitive atrium and sinus venosus are moved dorsally, and the heart begins septation into 4 chambers. As early as day 14, two actively growing ridges called Endocardial Cushion (of the PHF) nearly divide the single lumen into two separate cavities connected by a singular (atrioventricular) canal. Subsequently, a septum grows from the cushions toward the roof of the common atrium, and from the cushions toward the apex of the primitive ventricle. Continued addition of cushion cells to the atrioventricular canal divides a single canal into a left and right canal, as well as valves attached to the ventricular wall by muscular cords. Abnormalities of Endocardial cushion can affect structures derived from the PHF. For example, Tricuspid Atresia involves the malformation of the right Atrioventricular Canal and is associated with: (i) Atrial, and (ii) Ventricular Septal Defect; (iii) hypotrophy of the right ventricle; and (iv) hypertrophy of the left ventricle. Cells of the SHF are involved in the formation and lengthening of the outflow tract. Interestingly, this growth is regulated by neural crest cells. Insults to neural crest cell development or migration can affect structures derived from the PHF. For example, Tetralogy of Fallot involves an unequal division and laterality of the bulbus cordis associated with: (1) narrow right ventricular outflow; (2) interventricular septal defect; (3) over‐riding aorta directly above the septal defect; (4) hypertrophy of the right ventricular wall because of high pressure in the right side of the heart. Neural crest cells also contribute to craniofacial development leading to facial and cardiac abnormalities in the same individual.
G protein‐coupled receptor 37L1 participates in epigenetic gene regulation in human renal proximal tubule cells
Authors: Konkalmatt Prasad R. , Fan Caini , Cuevas Santiago , Armando Ines , Jose Pedro A.
Published: April 2020

G protein‐coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormality in the renal luminal sodium transport plays an important role in the pathogenesis of essential hypertension. There are a growing number of GPCRs and their interacting protein components that are localized on the nuclear membrane or within the nucleus, where they retain their signaling functions. In most cases, nuclear GPCRs are constitutively expressed on the nuclear membrane. The functions of these nuclear GPCRs are not well understood. Recently we showed that G protein‐coupled receptor 37L1 (GPR37L1), expressed on the apical membrane of renal proximal tubules (RPT), regulates luminal sodium transport and blood pressure via modulating NHE3 function. However, the GPR37L1 signaling pathway in the RPT has not been studied. Co‐immunoprecipitation (Co‐IP), combined with mass spectrometry analyses, revealed the association of GPR37L1 with nuclear import proteins: IPO7, IPO8, KPNA2, KPNB1; nuclear export proteins: XPO1 and XPO7; nuclear pore complex proteins: NUP210; kinases involved in signaling: mTOR, PRKDC, IRS4; and a DNA (Cytosine‐5‐)‐Methyltransferase 1 (DNMT1) in the GPR37L1 protein complex. Immunoblot analyses of nuclear preparations from the human RPT cells (hRPTCs) confirmed the presence of GPR37L1 in the nucleus. Confocal fluorescence imaging of hRPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1‐GFP) showed distinct nuclear membrane expression of GPR37L1. Association of GPR37L1 with mTOR, PRKDC, and DNMT1 proteins was confirmed by Co‐IP followed by immunoblot analyses. Overexpression of GPR37L1 in hRPTCs increased the phosphorylation of PI3, and AKT and decreased the expression of DNMT1, and NHE3 promoter methylation. Our data show that GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNMT1. Decreased DNMT1 function reduces CpG methylation on the NHE3 promoter that in turn increases NHE3 transcription. This is the first report showing the direct role of a nuclear GPCR in the regulation of renal sodium transport through epigenetic gene regulation.

Support or Funding Information

R56DK116828: Prasad Konkalmatt
Selective Peripheral Chemoreceptor Hyperreflexia Evoked after Sympathetic Stimulation
Authors: Felippe Igor Simoes Assuncao , Paton Julian F. R.
Published: April 2020

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P<0.05; differences were tested using paired student‐t test.

The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.

Support or Funding Information

Health Research Council of New Zealand
Abnormal Nasal Passage Morphology in an Adult Gharial (Gavialis gangeticus) Implications for Airflow and Nasal Septum Development in Longirostrine Crocodylians
Authors: Fontenot Nicole , Bourke Jason M.
Published: April 2020

Gharials are unique among extant crocodylians for their extremely longirostrine skull morphology. The extensively elongate rostrum in this group is comprised of contributions from the premaxillae, maxillae, palatines, vomers and pterygoids. Unlike some brevirostrine crocodylian taxa, the vomers do not support the nasal septum throughout its course, but terminate approximately 1 cm distal to the prefrontal‐maxillary suture. Nasal septum integrity is maintained via a septal sulcus produced by the sharp ventral dip along the midline of the two maxillae as they join together to form the floor of the nasal cavity. In other longirostrine crocodylians such as Alligator and Crocodylus, this septal sulcus is pronounced and holds the septum in place for most of the nasal passage. However, in Gavialis the septal sulcus is weakly developed and the septum largely rests on the suture‐line of the maxillae instead. Here we describe a unique case of a large adult female gharial with an extensively deviated septum. Deviation of the nasal septum produced a strong sigmoidal shape to the nasal passage rostral to the orbit. Segmentations and 3D renderings made in Avizo 7.1 (Thermo Fisher Scientific) and Computational Fluid Dynamic analysis using ANSYS Fluent (ANSYS Inc.) were run on this deviated airway to better determine the effects this wavy septum had on airflow. Results revealed a slightly elevated resistance in nasal airflow with the production of a few, small vortices within the bends of the nasal passage. These secondary flow patterns were not enough to induce turbulent flow and the majority of the nasal passage maintained laminar flow pattern similar to other crocodylians. Our results suggest that in spite of the heavily deviated septum, this animal was able to breathe with little extra effort. We compared this specimen to two other Gavialis specimens and discovered similar, albeit weaker developed, deviations of the nasal septum in these specimens as well. We suspect that weak development of the septal sulcus in Gavialis leaves this species susceptible to septal deviations and this type of developmental abnormality may be fairly common for this species and potentially other species of crocodylians with extensively elongated rostra.
Understanding PM2.5‐Induced Oxidative Stress In Alveolar Macrophages
Authors: Kaur Mandeep , Raval Kosha , Flores David , Olivas Micah , Waterston Anthony , Hasson Alam , Dejean Laurent
Published: April 2020

In situations of oxidative stress, the antioxidant processes of any eukaryotic organism can eventually become overwhelmed. Exposure to ambient particulate matter 2.5 (PM2.5), composed of transition metals and organic compounds 2.5μm or less in diameter, has been shown to cause oxidative stress in alveolar macrophages (AMs). This can lead to various adverse health effects, including cardiovascular and pulmonary diseases. In this study, we examine the capacity of ambient PM2.5 samples collected during periods of peak air pollution in Fresno and Claremont, CA to trigger ROS production in AM cells. These PM2.5 samples were characterized for their chemical content using chromatography and mass spectrometry. A DCF‐fluorescence based microplate assay was used to estimate the potential of the PM2.5 samples to trigger ROS production in NR8383, rat AM cells. Two types of quinones (1,2‐Naphtaquinone and 1,4‐Naphtaquinone) and two transition metals (Cu2+ and Fe2+) were associated with samples that had a high capacity to induce ROS generation in NR8383. While establishing a cytotoxic link between PM2.5 constituents and ROS, we also seek to understand the underlying biological mechanisms through which PM2.5 interacts with AM cells to produce excessive ROS. Previous observations suggest that the upregulation of NADPH Oxidase (NOX1), an enzyme that enhances ROS production, may contribute to cellular oxidative stress in macrophages. In this study, NOX1’s potential role in a PM2.5‐ induced oxidative stress pathway is investigated in NR8383 cells incubated with concentrations of iron and copper relevant with those found in the collected PM2.5 samples. The change in NOX1 expression was quantified using fluorescence‐based western blotting, with zymosan, a well‐known inducer of oxidative stress in macrophages, used as positive control. Our preliminary results indicate that 4‐hour treatments with increasing relevant iron concentrations triggered both an increase of ROS and NOX1 protein levels in NR8383. Our current experiments consist of testing the potential of relevant concentrations of other chemical constituents of PM2.5 suspected of triggering increased ROS production and NOX1 expression (these include naphtaquinones, phenanthraquinones, and copper). Understanding which chemical components of PM2.5 are able to induce significant amounts of ROS will allow us to understand how pollution is pernicious and advocate for the global reduction of PM2.5 emission.

Support or Funding Information

This work is supported by Fresno State ASI, the Division of Research and Graduate Studies, and Fresno State’s CSU‐LSAMP program funded by NSF under grant #HRD‐1826490.
Serum Extracellular Vesicles Derived From Obese Mice Control Energy Expenditure and Promote Liver Steatosis in Lean Mice
Authors: Cunha e Rocha Karina , de Mendonça Mariana , Rodrigues Alice Cristina
Published: April 2020

Cells can communicate with neighboring or distant cells trough extracellular vesicles (EVs). EVs have been shown to participate in many diseases, however, little is known about their role in metabolic diseases. Obesity is a major risk factor for noncommunicable diseases, and represents a threat to global public health. Indeed, obesity is rising in incidence, prevalence, and economic burden. Once the number of released EVs and its cargo are altered during obesity, we hypothesize that EVs intercellular communication may play a role in obesity pathophysiology. In order to investigate the metabolic changes caused by EVs in obesity, C57BL/6 male mice fed a control balanced diet were injected (i.p.) with serum EVs derived from diet‐induced obese mice (400ug/mouse) every 3 days for 8 weeks. Body weight was assessed every week. Visceral and subcutaneous fat depots, liver and gastrocnemius and soleus muscles were dissected and weighed. Serum biochemical analysis were conducted, and the content of hepatic triglyceride and total cholesterol was measured. Glucose, insulin and pyruvate tolerance tests were performed, as well as body composition analysis and indirect calorimetry. Differences at p<0.05 were considered significant. Compared to the control group, EVs injection attenuated weight gain, reduced the weight of subcutaneous fat depot, gastrocnemius muscle and liver, decreased the respiratory exchange ratio and increased hepatic triglyceride content, oxygen consumption and insulin sensitivity. Our results suggest that EVs released in obesity can modulate lean mice metabolism. Altogether, these data provide initial insights on EVs function and signaling during obesity.

Support or Funding Information

This study was financed in part by grants #2018/05426‐0 and #2019/14999‐6, São Paulo Research Foundation (FAPESP) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001.
24‐Hour Total Sleep Deprivation Alters Cardiovagal Baroreflex Sensitivity and Blood Pressure Reactivity in Mid‐Life Adults
Authors: Colling Morgan E. , Greenlund Ian M. , Gonzalez Joshua E. , Carter Jason R.
Published: April 2020

Nearly 1 in 3 American adults fail to get recommended levels hours of sleep, and epidemiological studies report a significant relationship between chronic sleep deprivation and hypertension. A number of studies from our laboratory and others have focused on neural and cardiovascular responses to a 24‐hour total sleep deprivation (TSD) model. However, these prior studies have primarily been conducted in young or older populations, with a gap in mid‐life adults. This is relevant because mid‐life can often be when chronic sleep levels reach a lifetime nadir. The purpose of this study was to determine the cardiovascular effects of 24‐h TSD on mid‐life adults. Nine participants (6 men and 3 women, BMI: 28 ± 5 vs. 22 ± 3 kg/m2) between the ages of 40 and 55 yr were tested twice, once after 24‐h TSD and once after normal sleep (randomized, crossover design). In both tests, participants were equipped with an automated sphygmomanometer (Omron HEM‐907XL, Omron Health Care) to assess mean arterial pressure (MAP), finger plethysmography to measure beat‐to‐beat arterial pressure (NOVA, Finapres), and a three‐lead electrocardiogram to record heart rate. Following a 10‐minute supine baseline, participants performed three 15‐s Valsalva maneuvers (VM) with 1‐minute recovery periods. Following another 3‐minute post‐VM baseline, a 2‐minute cold pressor test (CPT) was performed by submersing each participant’s hand in ice water. Data were analyzed using WinCPRS software (Absolute Aliens, Turku, Finland). R‐waves were detected and marked in the time series. R‐R interval (ms) vs. systolic arterial pressure (SAP, mmHg) were plotted for Phase IV of each VM, which was defined as the interval between the first SAP that exceeded the mean supine resting baseline SAP to peak SAP during Phase IV “overshoot.” Cardiovagal baroreflex sensitivity (cBRS) was determined from the slope of the regression line. The cardiovascular responses to each VM were included if the correlation coefficient exceeded 0.7 and the SAP increase was at least 15 mmHg (n=7). Irregular “square waves” were excluded from analysis. Statistical analysis included repeated measures ANOVA (p<0.05). TSD did not alter baseline MAP (84±3 vs. 85±4 mmHg) or heart rate (62±4 vs. 58±3 beats/min, p>0.05 for both). Cardiovagal baroreflex sensitivity (cBRS) was significantly reduced after 24‐h TSD (21.9 ± 10.3 vs. 14.7 ± 6.47 ms/mmHg, p<0.05). SAP reactivity was augmented following TSD during CPT (Δ16±7 vs. Δ25±6 mmHg, p=0.05), and DAP reactivity trended higher following TSD during CPT (Δ14±3 vs. Δ19±4 mmHg, p=0.09). In summary, TSD impacts baroreflex sensitivity and blood pressure reactivity in mid‐life adults.

Support or Funding Information

Support was provided by the American Physiological Society’s (APS) Summer Undergraduate Research Fellowship, Michigan Space Grant Consortium, and the Portage Health Foundation.
Evolution and Adaptation in Malate Dehydrogenases
Authors: Blythe Hannah , Bell Ellis , Bell Jessica
Published: April 2020

What is the evolutionary relationship between structure, function and stability is a central concept in the molecular life sciences. Malate Dehydrogenase is an excellent model enzyme to ask questions about this concept since i) it exists throughout biology, playing a central role in energy metabolism and ii) has a number of isoforms including cytosolic, mitochondrial, and in plants, glyoxysomal: overall the organelle forms are thought to have evolved by symbiosis with bacterial forms and are quite different in sequence and properties than the cytosolic forms. Cytosolic forms tend to have higher affinity for NADH while organelle forms have higher affinity for oxaloacetate, broadly consistent with the metabolic roles of the isoforms. Initially, we compared sequence conservation in 22 mammaliam cytoplasmic forms with watermelon glyoxysomal malate dehydrogenase (WMgMDH). Of 40 conserved amino acids in the cytosolic forms, 36 were conserved in WMgMDH. From the structure of WMgMDH, we used HINT and PyMol to examine atomic interactions both within the protein and with substrate and cofactor of the four residues, L159, A221, I250 and K311. We changed each residue to that resembling the cytosolic form to explore which contributed most to cytosolic like properties. We hypothesized that by changing Leucine 159 to a Valine (L159V), the hydrophobic core and cofactor bonding network would be altered. Changing Alanine 221 to Serine (A221S) would create hydrogen bonding potential at this site. Changing Isoleucine 250 to Valine (I250V) would reduce the hydrophobic area and potential impact stability of the protein. Lastly, changing Lysine 311 to Proline (K311P) may affect an interaction with cofactor due to its close proximity. Each mutation was constructed using QuikChange mutagenesis. Sequence confirmed mutants were expressed and purified using NiNTA Affinity Chromatography. Purified proteins were screened using SDS PAGE to assess purity. Specific Activities and Michaelis Constants for NADH and Oxaloacetate were then determined. The K311P and L159V mutants had specific activities similar to wildtype. I250V and A221T had 2 fold and 8 fold lower specific activities, respectively. Within standard errors, the Km for NADH was similar to wildtype for K311P and I250V, while L159V had a two fold lower Km (at a greater than 95% confidence limit). For oxaloacetate binding (as judged by Km for Oxaloacetate), I250V had a significantly higher Km, while L159V had a significantly lower Km (both at greater than 95% confidence limits). The changes produced by the L159 and I250 mutations result in more “cytosolic like” properties, while K311 mutation produced no significant effect. These findings contribute to our understanding of the subtle changes that can contribute to evolution and adaptation of enzyme structure and function.
Smooth Muscle Cell – Extracellular Matrix Alterations in Genetic Vasculopathies
Authors: Buja Louis Maximilian , Zhao Bihong , Lelenwa Laura , Cecchi Alana , Safi Hazim , Estrera Anthony , Milewicz Dianna
Published: April 2020

Pathogenic variants in 11 genes predispose individuals to aneurysms and dissections of the thoracic aorta and in some cases, vascular complications throughout the arterial tree, termed heritable thoracic aortic disease (HTAD) (Renard M et al. JACC, 2018;72:605–615). These genes are involved in vascular smooth muscle cell (SMC) contraction and metabolism, extracellular matrix (ECM) integrity and transforming growth factor β (TGF β) signaling. A unifying hypothesis is that the mutated gene products contribute to altered mechano‐transduction leading to vasculopathy (Humphrey JD et al. Circ Res 2015;116:1448–1461; Pinard A, Jones GT, Milewicz DM. Circ Res 2019;124:588–606). The objective of this study was to employ light (LM) and electron (EM) microscopy to evaluate the thoracic aortic and other arterial tissue from individuals harboring pathogenic variants in the 11 established predisposition genes who underwent surgical aneurysm or dissection repair. These individuals (n) harbored pathogenic variants in the following genes: FBN1 (13), ACTA2 (6), TGFBR1 (7), TGFBR2 (3), TGFβ2 (3), SMAD3 (3), LOX (1), MYLK (1), PRKG1 (1), and COL3A1 (4); total 42. Arterial specimens were evaluated using LM consensus criteria (Halushka MK et al. Cardiovasc Pathol 2016;25:247–257). EM was performed on selected cases (Davis EC. Lab Invest 1993;68:89–99). By LM, specimens were graded (0 – 3+) for mucoid extracellular matrix accumulation (MEMA), elastic fiber fragmentation and/or loss (EFFL), SMC nuclear loss/laminar medial collapse (SMCNL/LMC), and overall medial degeneration (A 1–2, B 3–4, C 5–6, D 7–9); intimal lesions (0–3+) and medial dissection (+/−) also were noted. The 42 specimens included 17 with medial dissection and 25 non‐dissected aneurysms. By LM, cases showed a spectrum of extent and severity of medial degenerative changes: A mild 8 cases, B moderate 18 cases, C moderately severe 14 cases, and D severe 2 cases. By EM, COL3A1 specimens showed reduction of collagen fibrils and shape variations of residual collagen fibrils; elastin fiber connections to SMCs were intact. In contrast, specimens with mutations in the TGF β pathway exhibited multiple foci with deficiency of elastic fibers and loss of elastic fiber connections to SMC but had normal collagen ultrastructure. These results support the hypothesis that the pathogenesis of genetic vasculopathies involves structural defects in the interaction of ECM components and SMCs, including impairment of the elastin‐contractile unit directly or through loss of structural support provided by collagen.

Support or Funding Information

Local funds
Pharmacokinetic Analysis of Ionizable Zwitterionic Oximes: Antidotes to Organophosphate Exposure.
Authors: Samskey Nathan , Ho Kwok-Yiu , Momper Jeremiah , Radic Zoran , Sit Rakesh , Sharpless K. Barry , Taylor Palmer
Published: April 2020

Currently approved antidotes to organophosphate (OP) exposure rely on discoveries of Irwin Wilson in the 1950’s, employing site‐directed pyridinium aldoximes to reactivate OP‐conjugated acetyl‐cholinesterase (AChE). This led to approval of 2‐PAM in the US and other pyridinium aldoximes elsewhere as antidotes to exposures of OP pesticides and terrorism nerve agents. However, principal limitations of these quaternary agents reside in their inability to cross the blood‐brain barrier and reactivate CNS AChE rapidly and their inherently rapid overall body clearance. Moreover, certain pyridinium aldoximes carry risks of channel blocking side effects. Alternative compounds that are ionizable zwitterions (hydroxyiminoacetamido alkyl amines‐RS194B) show facile transfer across the blood‐brain barrier through its neutral species, limited reversible binding to OP conjugated AChE, oral bioavailability and minimal toxicity. However, high doses may be needed for acute treatment, since these agents are extruded by brain capillary transporters and are rapidly cleared as the parent compound by renal transporters. To understand and counter these actions, we employed two approaches: (a) examination of tissue disposition and pharmacokinetics in a P‐glycoprotein knock‐out strain of mice, and (b) employing small molecules to inhibit extrusion of RS194B by brain capillaries and limit rapid clearance by renal tubules. While gene knock‐out enhances brain levels in mice, this is not practical in humans, so antidote retention in the CNS and diminishing renal clearance require competing small molecules. To avoid an imposed requirement of approval of two new agents, the antidote and a clearance inhibitor, we examined current FDA‐approved drugs for repurposing in a formulation. We have achieved favorable outcomes with transport inhibitors already approved by the FDA for other therapeutic purposes. Our approach employs agents that are zwitterionic, block P‐glycoprotein transport in the CNS and kidney, and show limited liability or toxicity for short term use. We have compared various fluoroquinolones, biguanides, ion channel blockers and have achieved favorable ratios of enhanced CNS levels and diminished renal clearance of RS194B in rodents at doses not eliciting toxicity. These comparisons enable assessing efficacy of the combination in other animal species.

Support or Funding Information

(Supported in part by RO1 GM18360 and UO1 NS058046)
Mapping Key Active Site Residues of the PA3944 Gcn5‐related N‐acetyltransferase
Authors: Variot Cillian , Baumgartner Jackson , Houseman Charles , Zhang Brian , Joe Layton , Kuhn Misty L.
Published: April 2020

PA3944 is an acetyltransferase from Pseudomonas aeruginosa that acetylates polymyxin antibiotics in vitro. It belongs to the large Gcn5‐related N‐acetyltransferase (GNAT) superfamily. We previously determined the 3D structure of this enzyme, but were unable to obtain a complex in the presence of polymyxin. Since it is not know which residues in the active of this protein are critical for function and recognition of the polymyxin substrate, we created point mutations of residues in the active site and assayed their kinetic activity. We found two key residues that were critical for activity of the enyzme: one was important for the catalytic mechanism and the other played a structural role. Additionally, we found two mutants that improved the enzymatic activity, which could be used to further explore the possibility of using this enzyme for biotechnological applications to modify polymyxin antibiotics with reduced toxicity. Moreover, these results provide insight into GNAT evolution and how key residues can alter substrate specificity and structure.

Support or Funding Information

Research reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).
Overlooked and Underestimated: New Insight on Communicating Branches of The Facial Nerve
Authors: Freed Blair , Steele Robert , Coker Charles , Marolt Clayton , Dennis Jennifer
Published: April 2020

The facial nerve demonstrates wide anatomical variation and significant clinical relevance. Branching patterns of the facial nerve have been extensively studied, but communicating branches have comparatively received far less attention. Communicating branches of the facial nerve are hypothesized as an important means of motor recovery to facial nerve injury, and therefore must be carefully considered by physicians working in the region of innervation. Indeed, damage to branches of the nerve is associated with significant ramifications to patients, and facial nerve injury is the most common complication of facial surgery. Knowledge of communicating branches may contribute to preservation of facial nerve function, and therefore are important to describe and document. This study aims to clarify the frequency and branching patterns of communicating branches of the facial nerve. Cadaver specimens from the Gift Body Program at Kansas City University College of Osteopathic Medicine revealed the anatomy. Cadaveric dissections were performed bilaterally to identify and document communicating branches across the facial nerve extending from the temporal to cervical branches. Frequency and location of communicating branches were assessed among 15 body donors bilaterally, yielding 30 facial nerves for analysis. Due to the variability of facial nerve branches observed bilaterally on specimens, this study utilized right and left facial nerves of each donor as individual specimens. We document and describe the presence of communicating branches across the temporal, zygomatic, buccal, marginal mandibular, and cervical branches. This characterization has more accurately established the generalizable area(s) in which communicating branches are located. A higher frequency of communicating branches between the marginal mandibular and cervical branches was observed than has been previously reported. The locations of these branches are described in relation to nearby landmarks for use in clinical and surgical settings. Branch variations were carefully outlined and overlaid to demonstrate areas of density to support surgical and procedural awareness
DETERMINATION OF STRUCTURAL STABILITY AND CATALYTIC ACTIVITY OF G6PD VARIANTS USING MOLECULAR DYNAMIC SIMULATION (MDS) APPROACH
Authors: Hamza Muaawia Ahmed
Published: April 2020

Background

Glucose‐6‐phosphate dehydrogenase (G6PD) is a known housekeeping enzyme that is important in the pentose phosphate pathway (PPP) and essential for basic cellular function. G6PD also aids in producing compounds to prevent build‐up of reactive oxygen species (ROS) within the red blood cells. Glucose‐6‐phosphate dehydrogenase deficiency (G6PDD) is a known hereditary mutation and X‐linked recessive genetic disorder that have affected over 400 million people worldwide. More than 186 G6PD variants have been characterized that affect enzyme catalytic activities and biochemical reactions leading to various clinical phenotypes.

Objectives

The intention of this study was to investigate the genotype‐phenotype correlations of selected G6PD mutations via structural analysis and molecular dynamic simulation analysis (MDSA) tools.

Method

ESPript 3.0 software was used for multiple sequence alignments of G6PD from human and from other organisms. PyMOL molecular visualization tool was also used to shown and evaluate structural changes in ten selected variants of the protein.

Results

Multiple sequence alignment of G6PD from human and three other organisms, namely; Trypanasoma cruzi, Mycobacterium avium and Leuconostoc mesenteroides revealed three conserved regions. The findings of this study suggested that mutation to residues that are unable to adopt a cis conformation would lead to an extremely high activation energy barrier for the enzyme to adopt the correct transition state during the enzymatic reaction. The protein was subjected to several computational approaches as described in previous part. The simulation data from the molecular dynamic simulation helps to determine the protein dynamics through stability and flexibility of G6PD native and mutation.

The native G6PD have high RMSD average value than Class I, class II and class III mutation, thus, have low structure stability. The G163D has the lowest RMSD value thus have the most stable structure through simulation. From RMSF analysis, the residue at 310 until 320 (loop 1), 374 until 388 (loop2), 408 until 414 (loop 3) and 424 until 434 (loop 4) responsible for structure flexibility and stability. The loop regions produce larger fluctuation thus, providing the flexibility to the structure.

Findings from molecular dynamic analysis reported here will be more valuable if comparison to kinetic data of the mutants are made. Simulation of the enzyme dimer form could stipulate the relationship between structural changes and enzymatic activity of G6PD variants.

Support or Funding Information

Research Center, King Fahad Medical City, Riyadh, KSA
Exploring the Structural Link Between FOXP2 and ADHD in Human Adults
Authors: Savoy Kaitlyn , Nataraj Kruti , Olvera Sophia , Petursson Emma
Published: April 2020

The forkhead box protein P2 (FOXP2) gene, located on chromosome 7, codes for a forkhead/winged‐helix transcription factor which regulates transcript production, typically repressing expression of neutral targets. The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationship between this protein, FOXP2, and attention deficit hyperactivity disorder (ADHD) in human adults. An assessment of the relationship between the presence of each of 12 FOXP2 tagSNPs and the presence of ADHD in two European samples found 1 tagSNP to be significantly associated with combined ADHD in one sample in both a single‐ and multiple‐marker test. These encouraging results provided the foundation for further exploration of the structural link between FOXP2 and ADHD in adults. Current studies involve observation of a variety of samples and deep‐sequencing of the FOXP2 genomic region with the intent to identify the sequence variants directly involved in the genetic background of ADHD. Future studies may focus on genes regulating and regulated by FOXP2 to gain a better understanding of the mechanisms and functions of the protein.

Support or Funding Information

The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationship between this protein, FOXP2, and ADHD in human adults.

Sponsors: Chris Elniff and L.B. Fogt

Mentor: Brendan Mattingly, University of Kansas Edwards
Adiponectin Reduces Angiotensin II‐Induced Vascular Hypertrophy by Targeting ROS Formation and Actin Cytoskeleton Remodeling
Authors: Zeidan Asad , Radwan Zeina , Farht Rima , Ghantous Crystal
Published: April 2020

Background and aims

Adiponectin (APN), an adipocytokine, exerts cardioprotective effects, while angiotensin II (Ang II) plays a critical role in the pathogenesis of hypertrophy of vascular smooth muscle cells (VSMCs) induces hypertension and vascular remodeling. Ang II induces VSMC hypertrophy in part via activation of RhoA/ROCK pathway. In this study, we investigated the molecular mechanisms associated with Ang II‐induced RhoA activation and translocation and the ability of ANP to counteract Ang II‐hypertrophic effect through inhibition of RhoA activation, actin cytoskeleton remolding and reactive oxygen species (ROS) formation.

Methods

Studies were carried out using rat aortic tissues and VSMCs. The effect of APN on Ang II‐induced RhoA translocation was assessed by Western blotting (WB) and Immunohistochemistry. Leucine incorporation was used to examine NO effect on Ang II induced hypertrophy. The phosphorylation of cofilin‐2 and ERK1/2 was detected by WB. Moreover, immunohistochemistry was performed on aortic frozen sections to detect ROS, F‐actin and G‐actin levels, RhoA‐Cav 1 co‐localization and eNOS translocation.

Results

Ang II‐induced VSMCs hypertrophy was prevented by supplementing APN. The hypertrophic response to Ang II was associated with RhoA translocation to caveolae‐rich membrane, cofilin‐2 and ERK1/2 phosphorylation, in addition to a significant decrease in G/F actin ratio. Moreover, ROS production significantly increased with Ang II treatment as revealed by confocal microscopy. However, these effects were inhibited by APN and caveolae disruption. Taken together, these results suggest that Ang II hypertrophic effect is due, in part, to RhoA translocation to caveolae rich micro‐domains and its downstream effectors which is counteracted by APN.

Conclusion

These findings suggest the ability of APN to attenuate Angiotensin II‐induced vascular hypertrophy.