bluuebunny/crossref_metadata_2025 huggingface dataset:
Metformin down‐regulates TGF beta signal transduction and production of PAR2 N‐terminus cleaving protease activity in an NR4a1 dependent manner in a PC3 prostate cancer cell line
Authors: Mihara Koichiro , Saifeddine Mahmoud , Hollenberg Morley D.
Published: May 2022

OBJECTIVE

Our aim is to understand the mechanisms of metformin’s antitumor actions

HYPOTHESIS

Metformin is known to have antitumour effects in cancers like head & neck cancer, that may involve its regulation of TGF‐β signalling, but the metformin receptor mechanisms for this action are not known. We recently discovered that the action of metformin to preserve hypoglycaemia‐impaired endothelial cell function regulated by proteinase‐activated receptor‐2 (PAR2) requires its interaction with the orphan nuclear receptor, NR4A1 (PubMed ID 34452975). PAR2 also plays a role in regulating prostate cancer PC3 cell function whereby autocrine signaling occurs in PC3 cells which secrete a PAR2‐cleaving/activating matrix metalloproteinase, MMP2. We hypothesized that metformin can act in PC3 prostate cancer‐derived cells via NR4A1 to change TGF‐β signalling and to alter the production of PAR‐cleaving proteinases.

METHODS

The ability of TGF‐β to upregulate connective tissue growth factor (CTGF) mRNA in PC3 cells was measured for metformin treated cells (</=100 μM) that were transfected with NR4A1, C‐terminally tagged with red‐fluorescent protein (RFP): either wild‐type (WT) or mutant (PPGG) murine NR4A1 that does not interact with metformin. TGF‐β upregulated CTGF mRNA in PC3 cells was monitored after metformin treatment for 20 hours. In similarly transfected WT and mutant PPGG NR4A1‐expressing PC3 cells, treated or not with metformin (</=100 μM), the cleavage status of dually tagged transfected N‐terminal RFP/C‐terminal GFP‐PAR2 was monitored: (confocal imaging: yellow = intact receptor; green = cleaved receptor). Concurrently, the abundance of MMP activity of PC3 cell supernatants was measured by a fluorogenic MMP substrate, Mca‐RPKPVE‐Nval‐WRK(Dnp)‐NH2 (ES002).

RESULTS

The expected TGF‐β upregulated CTGF mRNA in PC3 cells was prevented by metformin treatment, but only for cells transfected with the WT and not the mutant PPGG NR4A1. Metformin acted at concentrations (</=100 μM) far lower than those that affect mitochondrial complex I (> mM). We also found that in WT, but not PPGG mutant‐transfected PC3 cells, metformin (</=100 μM) down‐regulated MMP PAR2‐cleaving proteinase activity.

CONCLUSIONS

Our results show that low concentrations of metformin (</=100 μM), in the presence of wild‐type but not PPGG mutant NR4A1 which does not enable metformin interactions at the NR4A1 alternative ligand binding site, can both block TGF‐ β signaling and reduce cell‐secreted MMP activity that in turn can drive tissue invasion and metastasis via PAR2 activation. Thus, NR4A1 may play a role in the antitumour actions of metformin.
Nitric Oxide Therapy Attenuates Heart Failure with Preserved Ejection Fraction
Authors: Koul Kashyap , LaPenna Kyle , Li Zhen , Doiron Jake , Wang John , Sharp Thomas E. , Lefer David J.
Published: May 2022

Introduction

Nitric oxide (NO) based therapeutics have proven to exert cardioprotective actions in the setting of heart failure with reduced ejection fraction (HFrEF). However, NO therapy has failed to exert beneficial effects in clinical studies of heart failure with preserved ejection fraction (HFpEF).

We investigated (1) the effects of the NO donor, sodium nitrite, as a monotherapy, (2) hydralazine monotherapy, a known vasodilator and antioxidant, and (3) combination therapy with both drugs in a murine model of HFpEF. We assessed left ventricular (LV) function, oxidative/nitrosative stress, exercise tolerance, and cardiac fibrosis.

Materials and Methods

Male, C57/BL6N (n=15 per group) were placed on a Western high fat diet (60% kCal from fat) and treated with L‐NG‐Nitro arginine methyl ester (L‐NAME, 0.5 g/L/day) in the drinking water beginning at 10 weeks of age. At 15 weeks of age, mice were randomly assigned to four separate groups for five additional weeks: HFpEF control, sodium nitrite in drinking water (75 mg/L), hydralazine (2 mg/kg/day, i.p., b.i.d.), or the combination of sodium nitrite and hydralazine.

Plasma was collected to measure circulating 8‐isoprostane and 3‐nitrotyrosine levels, biomarkers of oxidative and nitrosative stress respectively. In addition, cardiac tissue was harvested at 20 weeks for Masson’s Trichrome staining and to measure cardiac nitrite levels. Echocardiography and exercise tolerance testing was performed at 10, 15, and 20 weeks of age.

Results

There was a significant increase in circulating nitrite levels (p < 0.05) in the sodium nitrite + hydralazine group compared to the control and either monotherapy groups. There was no significant difference in the cardiac tissue nitrite levels between the combination therapy and the control group. Plasma 8‐isoprostane and 3‐nitrotyrosine levels were significantly (p < 0.05) reduced in the sodium nitrite + hydralazine group compared to the HFpEF control group, indicating a reduction in oxidative and nitrosative stress. Although there was no significant difference in the extent of interstitial cardiac fibrosis between groups, there was a significant decrease (p < 0.05) in myocardial perivascular fibrosis in the sodium nitrite + hydralazine group compared to control. Left ventricular end‐diastolic pressure (LVEDP) was significantly reduced in all treatment groups, and LVEDP was reduced to the greatest extent in the nitrite + hydralazine group vs. HFpEF control. Furthermore, we observed a significant (p < 0.01) improvement in LV diastolic function (i.e., E/e’) in the sodium nitrite and hydralazine group that was superior to either of the treatments alone. There was no significant difference in the exercise distance or duration among the various groups.

Conclusion

These data demonstrate that the combination of a powerful antioxidant with NO donor therapy significantly enhances the beneficial effects NO therapy in the setting of severe HFpEF.
Potential Synergistic Effects of the Bioflavonoid Quercetin with the Apoptosis‐Inducer, TRAIL
Authors: Bush Jycole E. M. , Byrd Sherell
Published: May 2022

Acute lymphoblastic leukemia (ALL) mutations in lymphocyte precursor cells cause continuous production of lymphocytes. TRAIL, TNF‐related apoptosis‐inducing ligand, stimulates apoptosis in a range of cancer cells. TRAIL is being used more frequently in cancer research because of its directed anti‐cancer abilities, however cancer cells often become resistant to TRAIL treatment. Plant flavonoids such as Kaempferol have been shown to boost the effects of TRAIL and reduce resistance. This study examines the use of both Kaempferol and Quercetin, a plant flavonoid found in several fruits, as co‐treatments with TRAIL in inducing apoptosis in ALL. While the flavonoids have been used in combination with other traditional cancer drugs, little has been done to examine how combinations of flavonoids and molecules like TRAIL affect apoptosis in ALL. We observed an effective concentration for quercetin of 2.5 uM which produced 80% apoptosis in ALL cells. Kaempferol’s effective concentration was 100 uM, resulting in 37% cell death. TRAIL, at concentrations of 50 and 100 nM, showed no effect on Molt‐4 cells, primarily due to Molt‐4’s known resistance to TRAIL. Co‐treatment with quercetin and TRAIL showed suppression of ALL viability as did a triple combination treatment. Co‐treatments also resulted in modification of anti‐ and pro‐apoptotic protein expression. These results indicate that quercetin and other flavonoids may improve the effectiveness of TRAIL, even in TRAIL‐resistant cancer cell lines.
The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase
Authors: Smith Cameryn , Rementeria Gabriel , Stamper Tatum , Mansfield Madison , Halsell Jonah , Duffy Michael , Blatt Alexander , Velasquez Danielle , Incaviglia Isabella , Balderas Max , Marquez Joshua , Zachariou Natalie , Daley Margaret , Bell Isaac , Bell Anthony , Bell Jessica , Bell Ellis
Published: May 2022

All malate dehydrogenases have a flexible loop that starts to close over the active site region of malate dehydrogenase when cofactor binds and locks down completing the active site when the dicarboxylic acid substrate binds. As such the active site loop appears to play a role in both substrate specificity and correct alignment of the substrate to allow hydride transfer and proton abstraction/donation depending on the direction of the reaction. Combined with available crystal structures, detailed analysis of the 1smk.pdb structure shows the loop in a series of microstates from fully open to a closed state induced by the inhibitor citrate binding (figure 1), sequence conservation suggests that the loop region contains a 19 amino acid stretch from P119 to N137 using the watermelon glyoxysomal enzyme as reference. Clustal Omega Analysis of malate dehydrogenases from prokaryotes to mammals shows 5 families of conservation of the loop region (figure 2) allowing a series of hypotheses about structure function relationships of specific residues within the sequence to be made which have been tested by a combination of computational and site directed mutagenesis wetlab approaches. In addition to the two canonical arginines involved in substrate binding, we have explored every other residue in the loop region. Overall, mutations in the first half of the loop had small impact on Specific Activity while mutations in the second half of the loop had larger impacts. R130 mutants to E or S had the most impact while mutation to A or Q had lesser effects. Three other residues in particular had striking effects on Oxaloacetate saturation, G121A, L133S and N137. L133 and N137, at the C terminal base of the loop, were hypothesized to play a role in the repulsive interactions between substrate and cofactor in the active site. Consistent with this hypothesis, creation of an L133S or D137A mutant showed that concomitant with a reduction in specific activity to 2% and 5% wildtype (respectively), Km for Oxaloacetate was dramatically increased while Km for NADH was decreased somewhat. Finally, a series of mutations of M128 (Q,I and A) were explored with varying effects on specific activity (Q>I>A) and significant effects on Km for Oxaloacetate but lesser or no effects on Km for NADH. The impact of M128 mutations in particular were further explored computationally using DeepDDG analysis and effects on stability correlated with impact on kinetic properties. Overall these studies provide a detailed picture of the complex interactions the flexible loop makes that contribute to both substrate binding and catalysis.
Dendritic Cells Direct Circadian Anti‐Tumor Immune Response
Authors: Wang Chen , Barnoud Coline , Kizil Burak , Scheiermann Christoph
Published: May 2022

Circadian time partitioning of the day is a ubiquitous feature of physiological processes. Recent observations demonstrate that the initial time of the day when an inflammatory stimulus is encountered by the host governs the strength of the ensuing immune response. This is the case for an acute setting, which is mainly driven by innate immunity but also for adaptive immunity, where the effect is observed even weeks later. Here, we demonstrate a clear effect of time‐of‐day of tumor engraftment on tumor growth. Using immunocompromised NSG as well as Rag2‐deficient mice we can trace this difference to the adaptive arm of the immune response. More specifically, rhythmicity in the anti‐tumor response is abrogated in mice exhibiting lineage‐specific loss of circadian rhythmicity in T cells or dendritic cells (DCs). Using chronotherapy and rhythmic targeting of tumors with DC vaccines reduces tumor burden in a time‐of‐day dependent manner. Together, these data indicate that a circadian immune system can control tumor growth.
Instructor Perceptions of Student Incivility in the Online Undergraduate Science Classroom
Authors: Abraham Anna , Busch Carly , Brownell Sara , Cooper Katelyn
Published: May 2022

Student incivility, defined as a behavior perceived by the instructor or other students to be disrespectful and/or disruptive to the overall learning environment, can negatively impact student learning and instructors. During the COVID‐19 pandemic, the transition to an online teaching format created a unique environment for undergraduate incivility to take place. Further, student incivility has historically been disproportionately experienced by instructors of particular demographic groups, including women and People of Color, but it is currently unknown whether these demographic differences exist in the online environment. In this study, we designed a survey based on previous literature to measure instructor perceptions of student incivility during the COVID‐19 pandemic. We surveyed 283 science instructors from a random subset of research‐intensive institutions, Masters‐granting institutions, primarily undergraduate institutions, and community colleges who taught undergraduate courses with synchronous online components in the fall 2020 semester about their experiences with student incivility. Using binary logistic regression and open‐coding to analyze our data, we found that over half of instructors surveyed reported experiencing student incivility during fall 2020, with women being significantly more likely to report student incivility than men (Figure 1). Compared to white instructors, Instructors of Color were significantly more likely to report an increase in student incivility in fall 2020 compared to teaching the same course in previous semesters (Figure 1). The most common uncivil behaviors reported by instructors were requests to change student grades, sleeping in class, and excessively communicating with the instructor outside of class. When discussing how they handled experiences of student incivility, over half of instructors reported being irritated or annoyed as a consequence and tolerating or ignoring the behavior as a coping strategy, which could have detrimental effects on the learning environment for all students. This work indicates that student incivility exists in the online synchronous learning environment and that the negative impacts of student incivility are not distributed equally among all instructors, presenting additional burdens for women and Instructors of Color.
The rostral brainstem's pivotal role in swallow‐breathing coordination
Authors: Dutschmann Mathias
Published: May 2022

Respiration and swallowing are motor behaviours that require the interaction of two brainstem central pattern generators (r‐CPG, sw‐CPG). Previously we studies the interaction of swallowing and breathing in the in situ perfused brainstem preparation that identified a pivotal role of the pontine Kolliker‐Fuse nuclei for the mediation of protective glottal closure. We recently developed a computational model to further aid and test network connectivity and neural interaction between sw‐ and r‐CPGs. The model is based on a stepwise integration of synaptic connectivity between central sensory relay, sw‐ and r‐CPG neuron populations. The model is validated by experimental data obtained in situ. In the model and experimental data, stimulation of the superior laryngeal nerve (SLN, 10s) reliably triggers sequential swallowing with concomitant swallowing‐related glottal closure and suppression of inspiratory activity. Short SLN stimulation (100ms) evokes single swallows and respiratory phase resetting in the model and in situ yielding comparable phase response curves. Consistent with experiments, numerical circuit‐busting simulations show that deletion of ponto‐medullary synaptic interactions triggers apneusis and eliminates swallowing‐related glottal closure during sequential swallowing. Additionally, systematic variations of the synaptic strengths of distinct network connections predict vulnerable network connections that can mediate clinically relevant breathing‐swallowing disorders observed in the elderly and patients with neurodegenerative disease. Thus, the present model provides novel insights that can guide future experiments and the development of efficient treatments for prevalent breathing‐swallowing disorders.
Differential Regulation of Lhx9 Expression in Mouse and Chicken Limbs
Authors: Kim Jean Young , Britton Jessica C. , Pira Charmaine U. , Oberg Kerby C.
Published: May 2022

The distal tip of the developing vertebrate limb is covered by a rim of thickened ectoderm called the apical ectodermal ridge (AER). The AER secretes fibroblast growth factors (Fgfs) into the underlying mesoderm to control outgrowth and proximal‐distal patterning. Patterning during outgrowth proceeds from proximal to distal, with the proximal segment or stylopod being laid down first, then the zeugopod, and the distal segment or autopod forming last. Secreted Fgfs from the AER maintain a sub‐AER population of undifferentiated mesoderm, called the progress zone, that provides cells for this progressive differentiation.

In mice, Lhx9and its homologue Lhx2are expressed in the sub‐AER mesoderm and appear to play redundant roles in maintaining the expression of other patterning molecules, such as Shh. Interestingly in the chicken, the expression of LHX9 is restricted to the anterior aspect of the sub‐AER mesoderm, while LHX2 retains the full distal expression seen in mice. Recent clinical reports of an LHX9 missense mutation in a patient with hypoplastic/missing thumbs and great toes suggest that LHX9 can play a critical role in normal limb development. We hypothesized that the chicken cis‐regulatory modules (CRMs) regulating Lhx9 might have subtle species‐specific sequence variations compared to the murine CRMs that correlate with this differential expression pattern.

To test this hypothesis, potential CRMs were identified near the Lhx9 locus by in silicoanalysis, using sequence conservation and regulatory chromatin markers from published ChIP‐seq data. The potential CRMs were isolated and inserted into CRM‐reporter constructs. The limb‐related activity of a potential CRM was then determined by targeted regional electroporation into the distal chicken limb mesoderm.

We identified a highly conserved, transcription‐factor‐rich 623 bp sequence within intron 12 of DENDD1B 132 kbp upstream of the chicken LHX9 transcription start site. The potential CRM was associated with H3k4me2, H3k27ac, p300 and H3k27me3. The associated CRM‐reporter construct displayed activity in both anterior and posterior sub‐AER limb mesoderm overlapping LHX9 and LHX2 expression. These findings are consistent with a distal sub‐AER LHX9enhancer, but do not account for the lack of posterior expression. This suggests that additional CRMs contribute to the final expression pattern of LHX9 in the limb. Twenty‐two additional potential CRMs were identified and will be further characterized to determine their role in restricting LHX9expression from the posterior mesoderm in chickens. Characterizing the differential regulation of Lhx9 during limb development may provide insight into the species‐specific variation of limb patterning.
Fascicular Organisation and Neuroanatomy of the Porcine and Human Vagus Nerves: Allowing for Spatially Selective Vagus Nerve Stimulation
Authors: Thompson Nicole , Ravagli Enrico , Mastitskaya Svetlana , Iacoviello Francesco , Perkins Justin , Shearing Paul , Aristovich Kirill , Holder David
Published: May 2022

Vagus nerve stimulation (VNS) often results in off‐target effects due to the unknown fascicular organisation of the cervical vagus nerve and thus nonspecific nature of stimulation. This currently limits its therapeutic efficacy in treating epilepsy and depression and restricts its extension to treating numerous other conditions, such as heart failure and lung injury, for which it could be a promising treatment. The purpose of this work was to determine the fascicular anatomical representation of the recurrent laryngeal, cardiac and pulmonary branches of the vagus nerve at the mid‐cervical level of VNS cuff placement in the vagus nerve in pigs using micro‐computed tomography (microCT) to trace anatomical connections to end‐organs with the intention of extension to humans.

The left vagus nerves were dissected from mid‐cervical level to the branches of the lungs, heart and larynx in four domestic female 60‐70 kg pigs. Nerves were fixed, sectioned into 4 cm lengths with registration landmarks, soaked in Lugol’s solution (1% total iodine) for five days for sufficient contrast staining and microCT‐scanned (Nikon XT H 225, Nikon Metrology) with a molybdenum target and 7 µm voxel size, 35 kVp energy, 114 mA current, 4 W power, 0.25 fps in 4 s exposure time, and 3176 projections. Subsequently, scans were reconstructed in CT Pro 3D (Nikon Metrology) and analysed in ImageJ. Fascicles of the three target organs/fascicle groups were segmented and traced with Neurolucida 360 (MBF Bioscience) from the point the organ‐specific branch enters the vagus nerve to mid‐cervical level (20‐25 cm total length per nerve).

The three identified fascicular groups remained discrete at the cervical level (26%, 23% and 4% of total vagus nerve area for pulmonary, recurrent laryngeal and cardiac fascicle groups, respectively, with significant separation of centres of mass (P<0.05)). These three distinct regions contained overlap only between a small number of pulmonary and recurrent laryngeal fascicles (<11%). The cardiac fascicles remained separate. This confirms organotopic organisation of the cervical vagus nerve according to organ/function for cardiac, pulmonary and recurrent laryngeal fascicles. Work is in progress to delineate similar fascicular organisation in excised cadaveric human vagus nerves. Knowledge of the fascicular organisation of the vagus nerve will enable accurate spatially selective VNS which may allow for avoidance of off‐target effects and so improve its therapeutic efficacy.
A Novel Zinc Binding Group for HDAC6 Inhibition
Authors: Cragin Abigail , Watson Paris R. , König Beate , Hansen Finn K. , Christianson David W.
Published: May 2022

Histone deacetylases (HDAC’s) are key regulatory enzymes in gene transcription and cellular motility through the deacetylation of lysine residues. These enzymes bear a distinct clinical significance, as the upregulation of HDACs has been associated with oncogenesis for many hematological malignancies and proliferation of other neurodegenerative or immune disorders. There are four different classes of HDACs, three of which require zinc for catalysis. Among the zinc dependent isozymes, HDAC6 is thought to be a particularly desirable therapeutic target, as its selective inhibition in malignant cells is accompanied by fewer associated toxicities in comparison to pan‐HDAC inhibition. Therefore, optimizing HDAC6 inhibitor selectivity has the potential to yield great clinical significance. This selectivity is often conferred by designing inhibitors with bulky capping groups and a hydrophobic linker region, which make favorable interactions in the hydrophobic region of the HDAC6 active site. Many inhibitors contain a hydroxamate zinc‐binding group, which can coordinate with bidentate or monodentate geometry. In efforts to optimize HDAC6 selectivity by exploring alternative zinc‐binding groups, a unique inhibitor containing an oxadiazole ring was discovered. Surprisingly, the crystal structure of its complex with HDAC6 reveals that the oxadiazole undergoes a ring opening reaction to yield an acylhydrazide that binds with an extended conformation in the active site.
Single‐cell spatial transcriptomics analysis of a regenerating liver
Authors: Monga Satdarshan P. , Ko Sungjin , Hu Shikai , Singh Sucha , Poddar Minakshi
Published: May 2022

Liver, the largest gland in the body performs more than 500 functions. It is comprised of lobules, which are the functional metabolic units formed by a central vein in the center, and multiple portal triads at the periphery. Each portal triad is composed of a portal vein, hepatic artery and bile duct. Blood enters the lobule through portal vein and hepatic artery and moves through sinusoids that are lined by the sinusoidal endothelial cells (LSECs) and eventually exits through the central vein. Hepatocytes line the other side of the endothelial cells along the porto‐central axis. Intriguingly, the hepatocytes located across the lobule express genes differently based on their proximity to portal triad (zone‐1) versus central vein (zone‐3). This process of metabolic zonation ensures division of labor making hepatocytes more efficient in performing their endowed functions of synthesis, metabolism and detoxification based on their location in specific zones. Recent studies have begun to show that zonation of gene expression exists not only in hepatocytes but also in other minority cell types of the liver including cholangiocytes, endothelial cells, macrophages and stellate cells. Liver is also a unique organ with an innate capability to regenerate following toxicant‐induced injury or surgical resection. The process of regeneration is ensured by replication of all surviving cells in the liver. Very little is known about how metabolic zonation changes during the process of regeneration and how the various functions performed by the liver are maintained while the hepatocytes and other cells undergo replication towards attaining the pre‐hepatectomy mass. Here, we perform Molecular CartographyTM, a single cell spatial transcriptomics technique, to address changes in the both the expression and location of both zonated and non‐zonated genes in various cell types of the liver following partial hepatectomy. We address how these one hundred carefully selected genes undergo dynamic regulation as cells in the remnant liver attempt to maintain both function and proliferate to restore liver mass. In our current study we identify dynamic changes in spatiotemporal regulation of multiple important genes associated with most proliferation and function of cells during the process of liver regeneration.
Investigating the Status of Whole‐Body Donation Across the United States of America
Authors: Bagian Lauren K. , Wyatt Taylor B. , Balta Joy Y.
Published: May 2022

Cadaveric dissection has been used throughout history to gain a better understanding of human anatomy and to train medical practitioners. Today, these bodies are predominantly sourced through whole‐body donation programs housed by academic institutions (Champney et al., 2018). Universities rely on these programs to supply students, researchers, and clinicians with valuable, hands‐on learning experiences (Balta et al., 2016). The aim of this study was to gather information about the process of whole‐body donation at universities across the United States in order to better understand how body donors are procured for educational and research purposes. A Qualtrics survey was administered via email to 125 body donation programs in the United States. Results were gathered from the 50 programs that responded to the questionnaire. All but one of the respondents were accepting donations into their programs at the time of data collection. Collectively, these programs receive approximately 9,855 whole‐body donations annually. Our findings show that 82% of the donation programs receive enough donations to fit or exceed the needs of their institutions, while the remaining 18% receive too few donations to fit their institutional needs. Results showed that 37 programs permit next of kin body donation regularly or in times of need, and only 3 programs accept donations of fetal anatomical materials for educational and research purposes. Of the programs surveyed, 44% reported that other departments from their institution procure human anatomical materials from sources outside of the university’s donation program. Forty‐four programs reported that on average, over 80% of their registered donor population is composed of white individuals, yet only 5 institutions have methods in place to promote diversity among their donor population. Overall, we conclude that there is considerable variability in the operation of body donation programs across the United States. This is likely due to the lack of national policies to regulate these programs. These findings can be used to make recommendations about donor enrollment and program operations to build trust with prospective donors. By sharing information and best practices, anatomical gift programs across the United States can improve the donation process overall so that American universities may continue to reap the benefits of the immense gift that only whole‐body donors can provide.
Methods for studying the ductus venosus in animals
Authors: Melnikov Sergey , Bylinskaya Darya , Zelenevskiy Nikolay , Shchipakin Mikhail , Khvatov Victor , Glushonok Sofia
Published: May 2022

The ductus venosus is a physiological vascular shunt to the umbilical and caudal vena cava in the fetus. The umbilical vein (vena umbilicalis) leaves the placenta, enters the fetus through the foramen umbilical, and goes to the liver hilum. It carries oxygenated and nutrient‐rich blood from the placenta to the fetus. Part of the blood enters the liver through the umbilical vein, through the shunt ‐ the ductus vein enters the central venous line ‐ the caudal vena cava. This mechanism of blood redistribution is necessary for feeding the fetal brain with blood that is more saturated with oxygen. Given the scarce data on the topography, shape, and morphometric parameters of the venous duct in animals, we set the goal of revealing the methods used for its postmortem study. For a complete and comprehensive study of the topography of the venous duct, it is necessary to use a set of anatomical methods: thin anatomical preparation, vasoradiography, production of corrosive preparations, and morphometry. When conducting vasoradiography, a mass for injection prepared according to the recipe was used as a radio‐opaque mass: 45% ‐ lead white, 45% ‐ gum turpentine, 10% ‐ medical gypsum powder (M.V. Shchipakina, A.V. Prusakova, D.S. Bylinskaya, S.A. Kuga, 2013). In the manufacture of corrosive preparations, self‐hardening dental plastic was used. It belongs to the acrylic group of cold curing plastics and consists of powder and solvent components. In both cases, the injection of these substances was carried out through the umbilical vein, without opening the abdominal cavity, by accessing through the umbilical cord. After introducing the X‐ray contrast mass, the objects of study were placed in a fixing solution (10% formalin solution) and left in it for up to 5 days. Subsequently, thin anatomical preparation and photography, as well as radiography, were performed. After introducing a plastic solution, the objects were placed in chambers with a temperature of +4°C for two days. Subsequently, corrosion treatment was carried out in an aqueous potassium hydroxide solution for 4 ‐ 5 days. The morphometry of the venous duct in the study of vaso‐radiographs was carried out in the RadiAnt computer program, in the study of a corrosive preparation ‐ using an electronic caliper (Stainless hardened). On vaso‐radiographs, the ductus venous is identified as a sizeable cone‐shaped vessel located in the area of the liver hilum. The ductus venosus is identified as a craniodorsal blood vessel that flows into the caudal vena cava on the casts of the vascular bed. The proposed methods for studying the ductus venosus make it possible to accurately anatomically describe the position, shape, and size of the ductus venosus in fetuses.
Changes to the Skeletal Muscle Gene Expression Signature in Response to Nutrient and/or Mechanical Stimuli
Authors: Laskin Grant R. , Gordon Bradley S.
Published: May 2022

Nutrient consumption and mechanical overload influence skeletal muscle metabolism and function. These metabolic and functional outcomes are mediated at least in part by changes to the types of genes that are expressed in skeletal muscle. The extent to which nutrient consumption and mechanical overload act synergistically or independent of each other to change the skeletal muscle gene expression signature remains ill defined. Therefore, the purpose of this study was to define the differential and conserved gene expression responses to either nutrient consumption or mechanical overload as well as the combination of these stimuli. Published RNA‐Seq data sets generated from the muscle of humans subjected to acute resistance exercise were uploaded into the Database for Annotation Visualization and Integrated Discovery (DAVID) to define important functional gene categories related to muscle metabolism and/or function. Genes from the functional categories of ‘cell cycle’, ‘FoxO pathway’, and ‘transcription’ were marked for analysis in a mouse model of acute feeding and mechanical overload. For this model, C57BL/6 mice were initially fasted beginning at 1700 hr. The next morning (0800‐1000 hr), mice were randomized to remain fasted or allowed access to food for 30 min. Then, a mechanical overload was placed upon the left tibialis anterior (TA) muscle of all mice by subjecting the muscle to 10 sets of 6 eccentric muscle contractions by unilateral electrical stimulation of the sciatic nerve. The contralateral TA served as the non‐overloaded control muscle. All mice were deprived of food following the contractions protocol until euthanasia 4 hr later. The relative mRNA expression of Gadd45a, Usp2, Foxo1, Foxo3, and Myod1 were determined by RT‐PCR. Feeding and contractions each decreased the mRNA expression of Gadd45a (cell cycle), Foxo1, and Foxo3 (FoxO pathway), with the combination of feeding and contractions acting to synergistically decrease the expression of each gene. Conversely, the mRNA expression of Usp2 (cell cycle) was decreased in response to muscle contractions, with no effect of feeding noted. Finally, the mRNA expression of Myod1 (transcription) was significantly increased in response to feeding, with contractions causing a differential change in direction of Myod1 mRNA expression depending upon the fasting/feeding state. These data show that nutrient consumption and mechanical overload have shared and differential effects on the skeletal muscle gene expression signature. A more thorough understanding of the genetic changes regulated by each stimulus alone, as well as the combination of stimuli, will provide novel insight for how nutrients and mechanical overload alter metabolism and function in muscle.
The Impact of HIV Infection in Cocaine Seeking Behaviors
Authors: Xie Qiaowei , Jackson Joshua , Barker Jacqueline
Published: May 2022

Background

Substance use disorders (SUDs) are characterized by high propensity to relapse and are highly comorbid with HIV infection. The underlying neurobiology of this relationship is poorly understood. Preclinical research on the neurobiobehavioral outcomes of progressive HIV infections may yield crucial information to improve SUD prognosis and reduce risk of relapse in people living with HIV.

Methods

To model progressive HIV, adult male and female C57BL6J mice were inoculated with EcoHIV, a chimeric HIV‐1 which infects murine cells by replacing envelope glycoprotein gp120 with gp80. Five weeks after inoculation mice were trained in a cocaine conditioned place preference (CPP) paradigm (10mg/kg, i.p.) followed by extinction training. Stress‐induced reinstatement was assessed following administration of yohimbine, an α2 adrenergic antagonist, to determine EcoHIV effects. Neuronal activation in the prefrontal cortex (PFC) and nucleus accumbens (NAc) by cocaine exposure was assessed by quantitative analysis the expression of the immediate early gene, cFos.

Results

EcoHIV and sham‐inoculated mice exhibited similar cocaine CPP and extinction. However, EcoHIV mice showed significantly increased yohimbine‐induced reinstatement rate (p<0.05). cFos analysis revealed an elevated neural activation in PFC and NAc in response to cocaine. However, only neurons within infralimbic PFC (IL) and NAc shell subregions showed further increases in activity in cocaine exposed mice infected with EcoHIV vs shams.

Conclusion

EcoHIV infected mice escalated stress‐induced reinstatement of cocaine seeking compared to controls, suggesting that people living with HIV may be at elevated risk for stress‐induced relapse. Progressive EcoHIV infection may dysregulate IL and NAcShell activity to facilitate the cocaine seeking and reinstatement. Ongoing research will characterize how EcoHIV infection dysregulate IL‐NAcShell pathway specific activity during cocaine reward learning and stress‐induced relapse.
Investigating the Effect of the Atg8 Conjugation System on Autophagosome Size and Number
Authors: Silvia S.K. Nadia Rahman , Roberts Jacquelyn R. , Backues Steven K.
Published: May 2022

Autophagy is the cellular degradation process in which cellular contents are encapsulated by double‐membrane vesicles, autophagosomes, and delivered to the vacuole to be degraded and recycled. This process is important for cell health and homeostasis. It involves the upregulation of many autophagy‐related proteins, including Atg3 and Atg10. We are investigating the role of these proteins in the model system S. Cerevisiae is to measure the size and number of autophagosomes formed when insufficient amounts of that protein are available. Atg8 levels are known to affect only autophagosome size. However, we have found that levels of Atg7, the most upstream protein in the Atg8‐PE conjugation pathway, affect both autophagosome size and number. Atg3 and Atg10 are intermediate proteins involved downstream of Atg7 and upstream of Atg8. Atg3 directly conjugates Atg8 to PE, while Atg10 conjugates Atg12 to Atg5, and the resulting Atg12‐5 complex stimulates the activity of Atg3. It is possible that the Atg12‐5 complex has additional roles independent of Atg8 that explain the effect of Atg7 levels on autophagosome number. If so, we would expect that Atg3 activity would correlate only with autophagosome size, while Atg10 might control size and number. We are generating Atg3 and Atg10 active site mutants and testing the functionality of these mutants by performing western blots and enzymatic assays. Mutants that give a partial loss of autophagic activity will be selected for later measurement of autophagosome size and number.
SPAK‐Independent NKCC2 Phosphorylation in Dahl SS Rats: A Role for TNIK in NKCC2 Hyperphosphorylation
Authors: Granados Pineda Jessica , Haque Mohamed , Ares‐Sarmiento Gustavo , Ortiz Pablo A.
Published: May 2022

Increased NaCl reabsorption in the Thick Ascending Limb (TAL) is implicated in salt sensitive hypertension. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. We reported that NKCC2 phosphorylation at Tre 96,101 and surface expression are enhanced in Dahl Salt sensitive rats (Dahl SS) on normal or high salt diets. The phosphorylation of SPS1‐related proline/alanine‐rich kinase (SPAK), an upstream kinase for Thre 96,101 in NKCC2, was found to be higher in Dahl SS TALs. Deletion of SPAK in Dahl SS, lowers NKCC2 phosphorylation and expression and blunts salt‐sensitive hypertension, but does not completely prevent this. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thre 96,101 in NKCC2. We hypothesize that TNIK is in part responsible for enhanced NKCC2 phosphorylation in Dahl SS rats. We first measured total expression of SPAK, OSR1 and TNIK by Western blot in TALs isolated form Dahl SS and control rats. SPAK and OSR1 expression (normalized to GAPDH) was similar between strains whereas TNIK expression was 3‐fold higher in Dahl SS TALs (control: 100, Dahl SS: 396±80%, n =5, p<.025). We then tested whether a novel TNIK inhibitor (KY‐05009) decreased NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with KY‐05009 (1 µM) for 25 min decreased NKCC2 Thre 96,101 phosphorylation by 40±4% (p<0.05, n=5). To study whether NKCC2 phosphorylation can be stimulated in the absence of SPAK, we used SPAK knockout Dahl SS rats. Baseline NKCC2 expression and phosphorylation in TALs was 50% lower in Dahl SS‐SPAK KO TALs, however, stimulation of Beta‐adrenergic receptors with Isoproterenol increased NKCC2 phosphorylation by 192±49% (p<0.01, n =4) in SPAK‐KO. Similarly, the cAMP analogue db‐cAMP (500 µM) increased NKCC2 phosphorylation by 939±183% in SPAK KO (p<0.01 n=4) and by 497±105% (p<0.01 n=4) in control rat TALs, indicating that a kinase other than SPAK is essential for baseline and cAMP‐stimulated NKCC2 phosphorylation in Dahl SS rats. We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK, which show 3‐fold higher expression in this strain. SPAK also contributes to higher baseline NKCC2 phosphorylation, whereas a kinase other than SPAK (either TNIK or OSR1) is involved in cAMP‐stimulated NKCC2 Thre 96,101 phosphorylation in Dahl SS TALs.
Revealing Sex and Alzheimer’s Disease‐related Changes in the Spatial Localization of Brain Lipids in Mice using Mass Spectrometry Imaging
Authors: Seneviratne Herana K. , Wheeler Abigail , Eberhard Colten , Paul Bindu , Bumpus Namandjé
Published: May 2022

Lipids are essential biomolecules in the brain, as they have important roles in signaling and structural integrity. Of note, the central nervous system has a high concentration of lipids, second only to adipose tissue. Recently, it has been shown that abnormalities in lipid levels play a critical role in the onset of neurodegenerative diseases including Alzheimer’s disease (AD). AD is a progressive and severe neurodegenerative disease and the leading cause for dementia, affecting millions worldwide. However, the spatial localization of individual lipid molecules in normal brain tissues, as well as region‐specific lipid alterations during AD are yet to be explored. Additionally, current understanding of potential sexual dimorphisms in lipid localization in brain is incomplete. With these in mind, we employed high‐resolution matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) coupled to ion mobility mass spectrometry to characterize region‐specific lipid distribution in brain obtained from age‐ and sex‐matched wild type and AD mice (18‐month‐old female C57BL/6J and 3xTg‐AD mice, which exhibits both amyloid and tau pathology, n=3 per group). Additionally, the spatial distributions of lipids were investigated in male and female mice within two different age groups (4 and 12‐week‐old, n=3 per group). From these experiments, we were able to detect more than one thousand ions in brain tissue sections. Following detection, we annotated an approximate 100 lipid molecules with high confidence. Tissue imaging results revealed region‐specific distributions of specific lipids representing a range of classes including phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, ceramide phosphates, sphingomyelins, sulfated hexosyl ceramides, and ceramide phosphoinositols across male and female mice. Interestingly, ceramide phosphate (30:7), lysophosphatidylcholine (16:0), and phosphatidylcholine (36:5) molecules exhibited sex‐specific localization patterns restricted to the cerebellum in 4‐week‐old male mice. Further, ceramide phosphoinositol (42:6) was localized to the cerebellum region of 12‐week‐old male and female mice whereas 4‐week‐old male and female mice showed relative homogeneous spatial distribution. Notably, we observed region‐specific alterations in the spatial localization of several lipid molecules including phosphatidylcholine [(40:6), and (40:7)] and sulfated hexosyl ceramide [(16:1;2O/22:2;O), (24:2;2O/12:1;O), and (19:0;2O/15:0;O)] in AD mice as compared to age‐ and sex‐matched controls. These observed changes occurred in the hippocampus region and were further confirmed using receiver operating characteristic analysis. Taken together, the above results reveal highly localized patterns of alterations in lipid molecules across male and female mice, as well as during AD.
Inflammation at High Altitude and Potential Links to Physiological Responses to Hypoxia
Authors: Pham Kathy , Frost Shyleen , Oeung Britney , Puvvula Nikhil , Heinrich Erica
Published: May 2022

Inflammation plays a critical role in the physiological response to hypoxic stress. Evolutionary conserved inflammatory responses to oxygen limitation promote adaptation to the hypoxic environment. However, these responses may also become maladaptive during chronic exposure if not properly resolved, possibly contributing to pathologies such as Acute or Chronic Mountain Sickness. We hypothesize that pro‐inflammatory gene expression increases upon acute high‐altitude exposure, followed by a subsequent blunting of the inflammatory profile with acclimatization. We compared the inflammatory profiles in whole blood samples collected in the morning during fasting at sea level and after one and three nights at high altitude (3800 m elevation) in 15 healthy sojourners (5 women, 10 men). RNA sequencing was coupled with a nanoString Human Inflammatory Panel. Previously, we found key components of the TLR4 signaling pathway upregulated under acute high‐altitude exposure. To complement this, we found that IL8, a chemotactic cytokine that induces neutrophil recruitment, was significantly upregulated upon the first and third day at high altitude, and larger increases in IL8 may be associated with lower oxygen saturation (SpO2) at high altitude (R= ‐0.51; p=0.05). Additionally, FASLG, which is essential for immune system regulation, was associated with both SpO2 (R=0.68; p<0.01) and AMS score (R= ‐0.71; p<0.01) at high altitude. The upregulation of TLR4 signaling pathway genes observed during high‐altitude exposure in vivo suggested that hypoxia may exacerbate responses to inflammatory stimuli. To test this, we cultured whole blood in the presence of normoxia or hypoxia with or without a lipopolysaccharide (LPS) stimulus for 24 hours. Cultures were further treated with DMOG (HIF prolyl hydroxylase inhibitor), PX‐478 (HIF inhibitor), or media (control) to determine if any of these outcomes were HIF‐dependent. Preliminary data suggests that, in the presence of both inflammatory stimuli (LPS and hypoxia), TNF production was exacerbated (p=0.03). However, when HIF was inhibited with PX‐478, this exacerbated response was mitigated. Additionally, we found that DMOG abrogated TNF, IL6, and IL8 production in all treatments, suggesting a key role of prolyl hydroxylase activity in inflammation. In conclusion, our results indicate that acute high‐altitude exposure may cause an initial priming of the innate immune system with a synergistic TLR4‐induced sensitization to subsequent inflammatory stimuli and neutrophil activation and recruitment. In the case that the inflammatory response is not properly blunted during acclimatization, this may lead to the development of hypoxia‐induced pathologies. This may be particularly important in the context of pulmonary diseases if hypoxemia initiates an exacerbated inflammatory response to infection.