bluuebunny/crossref_metadata_2025 huggingface dataset:
SGLT2 Inhibitor Effects on Proximal Tubule Cell Function
Authors: Youm Elynna B. , Albalawy Wafaa N. , Long Kimberly R. , Rbaibi Youssef , Shipman Katherine E. , Baty Catherine J. , Weisz Ora A.
Published: May 2022

Sodium glucose transporter inhibitors (SGLTis) have been shown to improve cardiac and kidney health outcomes in patients with type 2 diabetes mellitus as well as in nondiabetic individuals. Both renal and extrarenal targets of SGLTis have been implicated in these protective effects, and it is unclear whether SGLTi protection against kidney disease occurs via the same mechanisms under diabetic vs nondiabetic conditions. To begin to address these questions, we examined the effects of SGLTis on proximal tubule (PT) transport and endocytosis in a well‐differentiated opossum kidney (OK) cell model that recapitulates essential morphological, metabolic, and functional characteristics of the PT in vivo. Treatment with canagliflozin, empagliflozin, or tofogliflozin reduced endocytic uptake of albumin by these cells in a dose‐dependent manner. Similarly, treatment with SGLTis inhibited fluid transport across OK cell monolayers. SGLTis have been shown to impair NHE3 transport, and as predicted, inhibition of NHE3 had similar effects on endocytic uptake and fluid transport. We recently discovered that knockout (KO) of megalin in OK cells results in a dramatic reduction in SGLT2 transcription with no change in NHE3 expression. Megalin KO cells had considerably lower rates of fluid transport that were not further affected by SGLT2i treatment. Current studies are directed towards understanding the interplay between megalin, SGLTs, and NHE3 expression and activity at the plasma membrane and in endosomal compartments in the modulation of PT endocytic function and transport.
The Impact of Profilin‐1 Mutations on Protein Homeostasis in Amyotrophic Lateral Sclerosis
Authors: Castle Julianna , Ferrara Jay , Gau David , Gleixner Amanda , Roy Partha , Kolarcik Christi L.
Published: May 2022

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized by the dysfunction and degeneration of motor neurons in the central nervous system. ALS has been linked to a number of genetic mutations, including point mutations in the actin‐binding protein profilin‐1 (Pfn1). While the prevalence of Pfn1 mutations is low, around 1% of inherited forms of the disease, an understanding of their impact on motor neuron cell death can provide insight into the mechanisms underlying ALS onset and progression. As Pfn1 is an important regulator of the actin cytoskeleton, we hypothesize that ALS‐linked mutations of Pfn1 promote defects in protein homeostasis and synaptic communication. To test this hypothesis, neuronal cells were transfected with Pfn1 constructs (including wild‐type, C71G, and M118V) and subjected to cellular stressors (including heat shock and sodium arsenite treatments). Immunostaining was then used to visualize and quantify differences in protein aggregation and stress granule formation. Our results have established the baseline response with the wild‐type construct and indicate that there is an increase in protein aggregation as well as in the number and size of stress granules with the mutant constructs. Our ongoing studies are aimed at validating these preliminary results, and our goal is to provide mechanistic insight pertaining to Pfn1’s role in ALS disease pathogenesis.
Neonatal Opioid Withdrawal Syndrome in Mice: Acute Molecular and Long‐Term Behavioral Consequences
Authors: Ferrante Julia R. , Dunn Amelia D. , Mengaziol Juliet , Noreck Julia , Lemchi Crystal O. , Creus‐Muncunill Jordi , Ehrlich Michelle E. , Blendy Julie A.
Published: May 2022

Populations affected by the ongoing opioid epidemic include pregnant women and their offspring. Infants exposed to opioids in utero are at risk of developing Neonatal Opioid Withdrawal Syndrome (NOWS), a combination of somatic withdrawal symptoms including high pitched crying, sleeplessness, irritability, and potentially seizures. Confounding environmental factors and difficulty in conducting longitudinal clinical studies have made it challenging to elucidate the long‐term consequences of NOWS. In order to investigate the molecular mechanisms and long‐term effects of NOWS, we have developed a mouse model of opioid exposure and withdrawal that encompasses the developmental equivalent of all three trimesters of the human pregnancy. Female mice are implanted with morphine minipumps (10 mg/kg/day) before mating, ensuring that pups are exposed to morphine in utero for the entirety of embryonic development. Mice are then given twice daily morphine injections (10 mg/kg/day, SC) for post‐natal days 1‐14. Mice are tested for developmental milestones during this period, including eye opening, extinguishing pivoting, surface righting, and forelimb grasp. Following the final morphine injection withdrawal behaviors were measured by ultrasonic vocalizations, 55°C hotplate analgesia latency, and somatic withdrawal signs. After reaching adulthood, mice were tested for changes in affective behavior (elevated zero maze, tail suspension test and learned helplessness), opioid response (conditioned place preference and intravenous self‐administration), social behavior (three‐chamber social behavior test), and sleep patterns. Mice exposed to this paradigm show delayed latency to reach developmental milestones and acute withdrawal phenotypes. In adulthood, social behavior and sleep patterns were affected in a sex‐dependent manner, while affective behavior and opioid response was unchanged. Bulk RNA sequencing of mice at post‐natal day 1 revealed changes in gene expression related to synapse development and function, the GABAergic system, and mitochondrial function. These transcriptome changes may underlie the behavioral long‐term effects of NOWS.
Morning Cardiovascular and Sympathetic Reactivity to Cold Pressor Test After Evening Binge Drinking: Sex Differences
Authors: Greenlund Ian M. , Bigalke Jeremy A. , Tikkanen Anne L. , Nicevski Jennifer R. , Smoot Carl A. , Durocher John J. , Carter Jason R.
Published: May 2022

Heavy alcohol consumption, including binge drinking, is associated with increased cardiovascular risk There is consistent evidence for increased muscle sympathetic nerve activity (MSNA) after acute alcohol consumption, and recent evidence for augmented sympathetic reactivity the morning after evening binge drinking. Given well documented differences in neural cardiovascular control between men and women, we hypothesized that women would exhibit augmented morning cardiovascular and sympathetic neural reactivity to cold stress following evening binge alcohol consumption. Thirty volunteers (15 women, 15 men; 25±1 years; 27±1 kg/m2) participated in the present study. Utilizing a randomized, cross‐over design, participants visited the laboratory for fluid control and alcohol sessions separated by ~1 month. The alcohol dose was based upon biological sex and body weight (1 g/kg dose in men and 0.85 g/kg dose in women). Beverage consumption occurred at 8:00pm and 9:00pm to simulate binge alcohol consumption (i.e., 4‐5 drink equivalent), with lights out at 11:00pm. An 8‐hour sleep opportunity was allotted from 11:00pm to 7:00am for overnight polysomnography (NATUS, Middleton, WI). Following lights on, twenty‐two (10 women, 12 men) participants were instrumented for a morning autonomic study with measurements of muscle sympathetic nerve activity (MSNA; microneurography), beat‐to‐beat blood pressure (finger plethysmography), and heart rate (electrocardiogram). MSNA, blood pressure, and heart rate were measured during a 3‐minute baseline, 2‐minute cold pressor test (CPT), and 3‐minute recovery. MSNA was scored and confirmed by one trained investigator (JR Carter). Statistical analysis included repeated measures ANOVA with condition and time as within subject factors and sex as a between subject factor (α = 0.05). Total sleep time and sleep efficiency were reduced following binge alcohol consumption in both sexes (condition: p < 0.05), but women had more frequent arousals following alcohol consumption (condition × sex: p = 0.008). During CPT, women exhibited an augmented pressor response via mean arterial pressure following evening binge alcohol consumption compared to fluid control, while men did not (condition × time × sex: p = 0.010). MSNA burst frequency was elevated during CPT following evening binge alcohol consumption compared to fluid control (condition × time: p = 0.033), but this was not different between sexes. Heart rate reactivity was not different across conditions (condition × time: p = 0.405) or between sexes (condition × time × sex: p = 0.818). In conclusion, our results indicate that evening binge drinking results in augmented cardiovascular reactivity in women compared to men, but these differences do not appear to be mediated via sympathetic neural reactivity.
Uncovering the Effects of Estrogen in an iPSC Derived‐Vascular Smooth Muscle Model of Marfan Syndrome
Authors: Escopete Sean S. , Ariyasinghe Nethika R. , De Souza Santos Roberta , Gross Andrew , Sareen Dhruv , Parker Sarah J.
Published: May 2022

The phenotype and severity of cardiovascular diseases, such as a thoracic aortic aneurysm and dissection and atherosclerosis, differ between females and males. Notably, coronary artery dissection risk increases four‐fold in women during late pregnancy and postpartum, suggesting that female sex hormones such as estrogen may play a role in vascular pathophysiology, yet are considered protective under most other circumstances. This paradox underscores the fact that the effect of estrogen on the vasculature is not fully understood at a molecular level, and most previous work in this area has been done using immortalized cell lines or animal models, which may not accurately model human responses. Furthermore, examining these effects in genetic diseases such as Marfan syndrome (MFS) allows us to view these effects in an aneurysm phenotypic model. Marfan syndrome is a connective tissue disorder caused by a mutation in the fibrillin‐1 gene. Individuals typically develop aortic root aneurysms with aortic disruption and rupture that remains a leading cause of death. Here, we used Healthy and MFS iPSC‐derived vascular smooth muscle cells (iVSMCs) to examine the effects of estrogen. Healthy and MFS IPSC’s were differentiated into iVSMCs for 21 days then treated with estrogen for 72hrs. Immunofluorescence of smooth muscle markers and contractility assays were conducted to confirm a contractile vascular smooth muscle phenotype. A proteomic profile was established to evaluate molecular differences. Peptide data was collected using a Thermo Fusion Lumos Orbitrap mass spectrometer. Data were analyzed using the DIA‐Neural networks software. We showed expression of smooth muscle markers and contractile response to stimuli. MFS iVSMCs showed decreased contractility response and a decrease in FBN1 expression compared to healthy control (MFS iVSMC vs Control iVMSC of‐1.4). The proteomic profile revealed an upregulation in proteins related to elastin fiber formation and cell adhesion in both Healthy and Marfan iVSMCs following estrogen treatment. FBN1 expression increased in response to estrogen in both Healthy and Marfan iVSMC. As well as increases in Lysyl Oxidase and TGFB2 expression in MFS iVSMC following treatment. We also see a downregulation of collagen expression and RNA synthesis in healthy iVSMCs. These findings will allow us to gain a greater understanding of the complex interaction estrogen has on the vasculature and to determine their role in cardiovascular disease risk.
DNA Polymerase‐Parental DNA Interaction is Essential for Helicase‐Polymerase Coupling in Bacteriophage T7 DNA Replication
Authors: Lo Chen‐Yu
Published: May 2022

DNA helicase and polymerase work cooperatively at the replication fork to unwind the parental DNA and perform leading strand DNA synthesis. It was believed that the helicase migrates at the forefront of the replication fork and unwinds DNA to provide a template for DNA polymerase. Although extensively investigated, the molecular basis of the helicase‐polymerase coupling is not fully understood. Replisome from bacteriophage T7 has been a model system for estimating mechanisms of DNA replication. Recently, the first structure of the helicase‐polymerase complex at a replication fork was reported with the T7 system. The T7 replisome structure suggested that the helicase and polymerase sandwich the parental DNA, with each enzyme pulling one daughter strand in opposite directions. Interestingly, it is the T7 polymerase but not helicase that carries the parental DNA with a β‐hairpin loop stacking at the fork opening and a positively charged cleft that holds the parental DNA. However, the role of the polymerase‐parental DNA interaction in helicase‐polymerase coupling has not been investigated. Here, we created and characterized T7 polymerases with perturbed β‐hairpin loop and the positively charged cleft. Mutations on both structural elements significantly reduced the strand displacement synthesis by T7 polymerase but had a minor effect on DNA synthesis against linear DNA substrate. Moreover, the mutations eliminate synergistic helicase polymerase binding to the DNA fork substrate and replication fork progression in mobility shift assay and rolling‐circle replication assays. Our biochemical data suggested that polymerase plays a dominant role in helicase‐polymerase coupling and replisome progression.
The Effects of Pyrene on Complex I and Complex II‐mediated Respiration in Isolated Skeletal Muscle Mitochondria in the Leopard frog, Rana pipiens
Authors: Alscher Lianna , Stabenau Erich K.
Published: May 2022

We have previously shown that in‐water exposure of the Northern Leopard frog, Rana pipiens, to pyrene significantly reduces many ATP‐dependent processes including hepatocyte cell volume regulation, skin Na+ transport, and skeletal muscle contractility. Furthermore, skin, liver, and muscle mitochondrial O2 consumption is significantly less in pyrene‐exposed frogs when compared to frogs under control conditions. One plausible explanation for the pyrene‐induced reduction in mitochondrial performance is through inhibition of mitochondrial complex activity. Therefore, the purpose of the present study was to isolate skeletal muscle mitochondria from Northern Leopard frogs and to examine Complex I and Complex II‐mediated respiration following direct exposure to pyrene or control solutions. After isolation of skeletal muscle mitochondria, Complex I and Complex II‐mediated respiration was examined using glutamate/malate and succinate, respectively, in an Oroboros O2k‐fluorespirometer equipped with a polarographic O2 sensor. To verify that decreased mitochondrial functionality could be detected, Complex I enzyme activity was inhibited with rotenone, whereas Complex II enzyme activity was inhibited with malonic acid. In order to assess the direct effects of exposure on mitochondrial function, 1.48 mM pyrene was added to extraction and suspension media. Isolated mitochondria were then incubated in suspension media with 1.48 mM pyrene for 1‐1.5 h. The Complex I and II enzyme activity was assessed and compared with mitochondria exposed to control solutions. There were no significant differences in mitochondrial Complex I and II‐mediated respiration following incubation with pyrene. These data suggest that the previously measured decrease in skeletal muscle mitochondrial performance following in‐water exposure of Northern Leopard frogs to pyrene is the result of effects on the proteins downstream of Complex I and II. Deficiencies in CoQ, Complex III, CytoC, and Complex IV activities could lead to the observed reduced mitochondrial respiration.
Reduced Hippocampal Neuronal Activity and Impaired Cognitive Function in a Mouse Model of Chronic Kidney Disease
Authors: Suzaki Aoi , Sul Julian , Day Helen , Goba Lihle , Huang Ting‐Ting
Published: May 2022

Chronic kidney disease (CKD) is characterized by a progressive loss of renal function that leads to an accumulation of uremic toxins in the circulation, despite regular dialysis. In CKD, uremic toxins can cross the blood‐brain barrier and cause neuroinflammation. Consequently, patients with CKD experience various symptoms, including fatigue and impaired cognitive function. A significant barrier to investigation of uremic toxicity and its impact on cognitive function has been the difficulty of performing studies in humans, due in part to the lack of access to relevant tissues. To examine the pathogenic mechanism of uremic toxicity in the development of cognitive impairments, a partial nephrectomy (PNx) mouse model was used as the experimental system. Sham and PNx mice were maintained for 12‐14 weeks after 5/6 nephrectomy and then assessed for motor function, cognitive function, and neuronal activity. Kidney function, as measured by glomerular filtration rate, was reduced by 40% in PNx mice, which corresponded to a 65% reduction in kidney mass. Blood urea nitrogen levels were 2.3 times higher in PNx mice, but no significant changes in plasma creatinine levels were observed. Despite reduced kidney function, PNx mice performed at the same level as sham controls in ambulatory activity, working spatial memory, and object recognition. There was also no evidence of increased anxiety. However, in the Puzzle Box paradigm, in which mice were tested for problem solving skills over multiple days with increasingly difficult tasks, PNx mice on average took three times longer to overcome the obstacle in the final two tasks. Ex vivo examination of neuronal activity following exposure to a novel environment revealed a 30% reduction in c‐Fos positive neurons in the hippocampal dentate gyrus in PNx mice. Similarly, the population of fast‐spiking parvalbumin positive interneurons, which are essential for coordinating neuronal activities, were also reduced in PNx mice. Collectively, the data support a correlation between reduced neuronal activity and impaired cognitive performance in PNx mice. These impairments are likely a consequence of exposure to elevated levels of uremic toxins in the brain due to reduced kidney function.
Calcium‐dependent signaling via gap junctions drives RhoA/ROCK activation during collective cell migration
Authors: Mancini Allison E. , Rizzo Megan A.
Published: May 2022

Collective cell migration (CCM) plays an important role in embryogenesis, vascular sprouting, and wound healing, but is also a significant driver of cancer metastasis. Ras homolog family member A (RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the precise spatiotemporal regulation of RhoA during CCM, we wanted to investigate the activity of downstream effector Rho‐associated kinase (ROCK) in collectively migrating fibroblasts. Using a new FRET‐based ROCK biosensor, we have observed calcium‐dependent activation of ROCK. We have previously demonstrated blunting of CCM in response to pharmacological inhibition of ROCK or depletion of intracellular calcium with 100 µM EGTA. To study the role of gap junction signaling in calcium‐dependent activation of ROCK during CCM, carbenoxolone, a gap junction blocker, was applied to collectively migrating cells following scratch wounding. Carbenoxolone treatment blunted CCM, as well as decreased ROCK activity and intracellular calcium levels at corresponding time points. Treatment with PQ7, a gap junction activator, caused modest increases in CCM. These results indicate that calcium signaling through gap junctions drives ROCK activity in the context of collective cell migration.
Microbial Community Composition Dependence on Season, Site, and Flow Rate in the Macatawa Watershed
Authors: Currier Jenna K. , Dawson Nicholas J. , Horsburgh Luke , Cooper Carolyn E. A. , Slater Adam D. , Brokus Sarah A. , Wade Randall D. , Best Aaron A. , Pikaart Michael J. , Krueger Brent P.
Published: May 2022

The poor condition of the Macatawa watershed has been an area of focus for the greater Holland community for decades. Urbanization and agricultural development are key reasons for the poor water quality as they introduce excess nutrients. These nutrients, such as nitrates and orthophosphates, lead to hypereutrophication of the lake and catalyze the growth of algal blooms and the spread of potentially dangerous bacteria. Hope College has been collecting data to better understand trends and patterns within the lake that affect both levels of nutrients and the bacterial community. We continuously monitor levels of Escherichia coli, orthophosphates, nitrates, total suspended solids (TSS), and other biological and chemical attributes. This poster focuses on investigating the composition of microbial communities under certain conditions such as changing seasons (e.g. winter vs. summer) and different types of sites (e.g. lake vs. stream). These data showed stark differences in the relative abundance of some bacteria when correlated with season and site type. For instance, Cyanobacteria and Cryomorphaceae showed higher abundance in the summer at lake sites. We also analyzed the abundance of microbes during standard flow rates compared with high flow rates. Details of these changes in microbial community structure are presented in this poster. These results ultimately tell us that there are seasonal and flow rate influences on bacterial community composition within the Macatawa watershed.
Mind and Body Donors: Can Meditation Help Students Maintain Empathy During Cadaveric Dissection?
Authors: Chakoumakos Madison , Eyler Lisa , Chun Jane , Tutjer Jenna , Noel Geoffroy
Published: May 2022

The adoption of humanistic practices in anatomical education is increasing with studies identifying educational practices that foster empathetic responses as a crucial aspect of pre‐clinical education. However, there has been little research evaluating the implementation of novel curriculum in pre‐clinical anatomy courses that help students maintain empathy during cadaveric dissections. Specifically, we hypothesize that implementing compassion training (CT) offered by trained professionals during a pre‐clinical anatomy course will help students maintain their empathy towards body donors. We implemented five training sessions run by the Sanford Institute of Empathy and Compassion in the pre‐clinical anatomy thread at the University of California, San Diego in this IRB‐approved study. We sought to assess baseline empathy and prior experience with meditation/mental training among both first and second‐year medical students. We specifically used the Jefferson Scale of Empathy (JSE) to evaluate empathy, which is broadly used in medical professions to assess this attribute. Interestingly, prior to the start of the course, the mean experience with contemplative compassion practices was 0.840 ± 1.042 (n=81) with zero being no experience, but the mean interest in participating in compassion exercises was 2.954 ± 1.422 (n=87) with five being highly interested. Among the 137 students that initially responded to the pre‐survey, 47 students chose to participate in a longitudinal study where students’ connection to the body donor was assessed with the Inclusion of Other in Self (IOS) scale. The emotional state of students was assessed using self‐reported scales of both pleasure and arousal. Students in the longitudinal study completed 10 anatomy sessions with five including CT. There was no significant difference in IOS scores between anatomy labs with CT and those without intervention. However, there was a significant difference between total mean levels of engagement during compassion practices with students showing higher levels of engagement in laboratory sessions with CT (With CT: 2.509 ± 1.089 n=106; Without CT: 1.500 ± 0.927 n=54, p<0.001). In conclusion, students are more likely to engage in compassion practices when training is incorporated into laboratory sessions. The question remains how engaging in these exercises affects students’ ability to navigate complex emotions during cadaveric dissection. Further investigation is needed to determine if higher levels of engagement are associated with increased empathy towards body donors. In the future, we plan to evaluate whether increased engagement with compassion practices correlates to increased empathy after completion of pre‐clinical anatomy. We ultimately hope that this research will help educators design educational initiatives that help students navigate between empathy and clinical detachment during cadaveric dissection.
microRNA‐34a modulates the senescence of activated hepatic stellate cells in alcohol‐associated liver injury
Authors: Slevin Elise , Wan Ying , Harrison Kelly , Zhang Yudian , Li Xuedong , Li Tian , Lorenzo Sugeily R. , Xu Wenjuan , Klaunig James E. , Shetty Ashok K. , Meng Fanyin
Published: May 2022

Background

Reversion of activated hepatic stellate cells (HSCs) to quiescence is likely to be a promising pathway in liver fibrosis regression. The development of senescence in HSCs during alcoholic liver injury may lead to liver compensation against fibrosis. The current study aims to characterize the functional role of miR‐34a regulated cellular senescence during alcohol‐associated hepatitis.

Methods

Senescence related gene and miR‐34a expression was assessed using a PCR Array and/or real‐time PCR analysis. Cellular senescence in cultured HSCs were measured by SA‐β‐gal staining. Senescence mediators were defined in LPS treated hepatic stellate cells in vitro, and in TLR‐4 knockout mice or morpholino antisense oligomer against miR‐34a (miR‐34a Morpho/AS) treated mice with chronic ethanol feeding in vivo.

Results

We identified that 5 weeks of ethanol feeding significantly increased the total liver histopathology score and miR‐34a expression, along with the activation of hepatic stellate cells marked by enhanced α‐SMA staining. Treatment of HSCs with LPS (20 ng/ml) for 24 hr significantly increased miR‐34a expression, along with the enhanced SA‐β‐gal activity, up‐regulated α‐SMA/Collagen A1 expression and increased cellular viability. Silencing of miR‐34a decreased LPS‐induced activation in HSCs by downregulation of fibrosis markers α‐SMA, Col1a1 and TIMP‐1, and increased SA‐β‐gal activity as well as the cellular senescence marker CCl2, whereas silencing of the LPS receptor, TLR4, decreased this marker in the same group of HSCs, suggesting anti‐miR‐34a reversed LPS‐mediated HSC activation through inducing cellular senescence. Furthermore, the expression of miR‐34a and verified miR‐34a related senescence marker CCl2 were significantly altered in hepatic stellate cells from ethanol‐fed mouse liver specimens compared to controls. TLR4 knockout mice and miR‐34a Morpho/AS treated mice displayed less sensitivity to alcoholic injury, along with enhanced SA‐β‐gal activity and CCl2 level in HSCs, and recovered expressions of α‐SMA, Col1a1 and TIMP‐1. Summary and

Conclusion

Our results show that miR‐34a mediated cellular senescence is essential for the potential reversion from activated to quiescent hepatic stellate cells during alcohol‐associated hepatitis. These findings provide new insight into the function of microRNA regulated

cellular senescence in human HSCs and increase opportunities for the development of novel treatment paradigms for the management of alcohol‐associated liver diseases.
Reduction of Postoperative Cognitive Deficits in Aged Mice by Chronic Intermittent Propofol
Authors: Nagarajan Rajasekar , Lyu Jinrui , Kambali Maltesh , Wang Muxiao , Pearce Robert A. , Rudolph Uwe
Published: May 2022

Postoperative cognitive dysfunction (POCD) can increase morbidity and mortality after surgery, especially in the elderly. Current evidence from both clinical/epidemiological studies and animal studies suggest that POCD is likely due to factors related to the surgery itself and at least not primarily due to the effects of general anesthetic agents. In contrast, propofol, the most commonly used intravenous general anesthetic agent which is acting mainly via GABAA receptors, has been shown to have neuroprotective effects by attenuating caspase activation, apoptosis, and mitochondrial dysfunction. We therefore set out to assess the effects of chronic intermittent propofol on postoperative cognitive deficits in aged mice. Abdominal surgery (exploratory laparotomy) under isoflurane anesthesia was performed in 21‐24 months‐old mice. Animals received either chronic intermittent propofol (75 mg/kg i.p.) or vehicle (IntralipidR) every 5th day throughout the experiment. The laparotomy was performed on day 17. Two days later, the mice were employed in the open field test. There was no difference in postoperative locomotor activity among groups (Surgery+Vehicle, Surgery+Propofol, No surgery+Vehicle, No surgery+Propofol). Then, cognitive functions were assessed using the Y maze, novel object recognition, contextual fear conditioning and Morris water maze tests. We found that laparotomy resulted in impaired learning and memory as determined in the behavioral test battery compared to no surgery controls. However, perioperative chronic intermittent propofol strongly attenuated the surgery‐induced memory impairments. Moreover, propofol per se improved the (likely age‐related) cognitive dysfunction in the no surgery controls. Our findings suggest that perioperative intermittent propofol administration might prevent or attenuate surgery‐induced cognitive dysfunction, and thus potentially reduce the liability for undesired long‐term outcomes. Thus, chronic intermittent administration of propofol may represent a novel therapeutic strategy with translational potential.
Role of Cortical Dopamine circuits in regulating Striatal Dopamine dynamics during Reversal Learning
Authors: Kumar Mayank , Green Sara M. , Urs Nikhil M.
Published: May 2022

Dopamine is a catecholamine neuromodulator implicated in locomotion, motivation, learning and cognitive behaviors. Although striatal dopamine signaling and circuitry are well established, the role of cortical dopamine projection circuitry in regulating striatal dopamine dynamics and behavior is not clear. Glutamatergic pyramidal neurons in the prefrontal cortex (PFC) are topographically organized and dopamine D1 and D2 receptors are expressed on glutamatergic pyramidal neurons in the PFC. Using a retrograde adeno‐associated virus (AAVRG)‐based approach we show that D1R+ subpopulations in medial orbitofrontal or prelimbic regions project to nucleus accumbens core (NAcc) or dorsal striatum (dSTR), respectively. However, D2R+ subpopulations in medial orbitofrontal, or medial prelimbic PFC, project to NAcc or the midbrain (SNpc/VTA) but not dSTR, respectively. Additionally, D1R+ and D2R+ medial orbitofrontal subpopulations have indirect connections to the medial SNpc through the NAcc core. We next wanted to test which of these topographically organized circuits regulate striatal dopamine dynamics during reversal learning. We used fiber photometry to measure striatal dopamine dynamics in the dorsomedial striatum (DMS) during reversal learning. We were able to measure reward prediction error (RPE) like responses in the DMS during reversal learning. We will next use an intersectional genetic approach to specifically activate or inhibit dopamine receptor cortical projection circuits and test their effect on striatal dopamine dynamics and reversal learning. Our studies will identify previously unappreciated roles for cortical dopamine projection circuits and their regulation of striatal dopamine dynamics.
Mitochondrial calcium retention capacity correlates with markers of muscle mass, strength, power and functional capacities in young and old adults
Authors: Cefis Marina , Marcangeli Vincent , Granet Jordan , Hammad Rami , Leduc‐Gaudet Jean‐Philippe , Gaudreau Pierrette , Robitaille Richard , Belanger Marc , Aubertin‐Leheudre Mylène , Morais José , Gouspillou Gilles
Published: May 2022

Rationale

The aging‐related loss of muscle mass and function is a major risk factor for falls, fractures, frailty, loss of independence, institutionalization and even death. Although the underlying cellular and molecular mechanisms remain partly understood, impaired mitochondrial permeability transition pore (mPTP) function has been proposed as a potential mechanism. Indeed, altered mitochondrial calcium retention capacity (mCRC), a marker of mPTP function, has been documented with aging in both rodent and human skeletal muscles and mPTP opening was shown to cause muscle atrophy. However, studies on the impact of aging on mPTP function in humans remain scarce, and whether impaired mPTP function correlates with markers of muscle mass, performance and functional capacities remain unknown.

Objective

The present study aimed at investigating whether mCRC is associated with markers of muscle mass, muscle function and functional capacities in humans.

Methods

Twelve young (29.4 ± 4.4y) and fourteen old (80 ± 7.4y) healthy men were enrolled in the present study. Leg lean mass (leg LM) was assessed using DEXA. Maximal isometric knee extension strength (iKES) and power were measured using a strain gauge system attached to a chair and the Nottingham Leg Extensor Power rig, respectively. To assess functional capacities, the performance at the 6‐min walk test and the step test (maximum number of steps in 20s) were evaluated. To assess mPTP function, mCRC was measured in permeabilized myofibers prepared from muscle biopsies obtained in the vastus lateralis.

Results

As compared to young adults, old individuals displayed significantly lower leg LM (p<0.01), iKES (p<0.01), and power (p<0.01). The performance at the 6‐min walk test (p<0.01) and the step test (p<0.01) were also lower in old individuals. Old individuals also displayed a significantly lower mCRC (p<0.01). mCRC was significantly correlated with leg LM (r=0.34; p=0.045), iKES (r=0.35; p=0.04) and muscle power (r=0.41; p=0.02). In addition, mCRC was significantly correlated with the performance at the 6‐min walking test (r=0.42;p=0.01) and step test (r=0.46; p<0.01).

Conclusions

These results indicate that mPTP function is altered with aging in humans and highlight positive associations between mPTP, muscle mass, strength, power, and functional capacities. These findings therefore strengthen the emerging view that altered mPTP function contributes to the aging‐related loss of muscle mass and function in humans.
GPR75 Deficiency Protects Against High‐Fat Diet Induced Obesity
Authors: Hossain Sakib , Gilani Ankit , Villegas Elizabeth , Agostinucci Kevin , Diegisser Danielle , Dirice Ercument , Garcia Victor , Schwartzman Michal
Published: May 2022

The CDC estimates the prevalence of obesity in the United States to be over 42.5% in 2021. With obesity related conditions including hypertension, heart disease, and type 2 diabetes, therapeutic approaches to treat obesity are necessary to prevent premature death. Recent studies have identified the orphan G‐protein coupled receptor, GPR75, as a possible target. For example, Akbari et. al., (Science, 2021) found that truncated loss of function variants of GPR75 were associated with 5.3 kg lower bodyweight and 54% lower odds of obesity in heterozygous individuals. Based on these findings, we hypothesized that global deletion of Gpr75 protects against diet‐induced obesity (DIO) and insulin resistance. Wild‐type and Gpr75null mice were subjected to 14 weeks of regular chow or high‐fat diet (HFD) feeding. Body composition, intraperitoneal glucose and insulin tolerance tests, and oxygen consumption were measured initially and at week 14. There were no significant differences in body composition, glucose homeostasis, and oxygen consumption between wild‐type (WT), Gpr75‐/‐ (KO) and Gpr75+/‐ (HET) mice initially. However, WT mice obtained a diabetogenic phenotype after HFD‐feeding while KO and HET counterparts were robustly protected as indicated by reduced body weight, (45.32 ± 1.387 grams, 28.29 ± 1.47 grams, and 36.60 ± 1.8 grams respectively) and increased sensitivity to insulin (blood glucose 30 min after insulin injection as percent change, ‐28.5 ± 4.41%, ‐50.68 ± 4.15% and ‐39.07 ± 1.79% respectively, p<0.0001). Weekly energy intake calculated from food consumption showed no significant differences between all three groups. However, oxygen consumption measured over 60 minutes indicated an increase in energy expenditure in KO compared to WT mice (73.76 ml/min/kg vs. 44.61 ml/min/kg respectively, p<0.0001). This correlated with a 2.63‐fold increase in brown adipose tissue (BAT) UCP1 mRNA expression in KO compared to WT mice. While the abundance of BAT is low in humans compared to mice, our findings suggest that improved glucose homeostasis as a result of GPR75 deficiency is linked to increased BAT thermogenesis. This may provide a potential novel route for the control of metabolism to combat obesity‐driven metabolic disorders.
Red Blood Cells Function as Nucleic Acid Sensors
Authors: Mangalmurti Nilam
Published: May 2022

Red blood cells are the most abundant cell in the human body and have tremendous potential to modulate immune function. While other enucleate cells such as platelets are known to play a vital role in innate immunity and immunothrombosis, astonishingly little is known about RBC immune function. My lab's primary, overarching long‐term objective is to define RBC immunomodulatory functions that may alter the immune response to pathogens and sterile injury. We recently found that RBCs function as DNA scavengers by expressing the nucleic acid sensing‐toll‐like receptor, TLR9. In addition, our new data demonstrate the presence of another nucleic acid‐sensing TLR, TLR7, on mammalian RBCs. Collectively our data indicate that RBCs function as nucleic acid sensors. These findings challenge the long‐standing belief that RBCs lack immune function and may elucidate novel diagnostic approaches and therapeutic targets for inflammatory syndromes.
Paper‐MAP: A Novel Tool for Super‐resolution Imaging of Pathological Tissues by Rapidly Tissue Expansion and Clearing
Authors: Lee Mirae , Woo Jiwon , Chang Jin Woo , Park Jeong‐Yoon
Published: May 2022

Super‐resolution mapping of three‐dimensional (3D) structures in intact tissues is essential in many biological studies. Biological tissues has very complex structure of functional networking with various cell population/organization. Since most biological tissues are not transparent, and imaging processes are limited by light scatter, imaging deep within the tissues is problematic. In this work, we developed the novel method as call Paper‐MAP for rapidly and simple clearing/expansion of based on MAP (Magnified Analysis of Proteome) using very thin tissue. The Paper‐MAP can be analyzed to super‐resolution 3D imaging of hybrid tissue with rapidly 4‐fold clearing/expansion in under 14 hours, while preserving their overall architecture and protein content of subcellular details after hydrogel embedding. Using a Paper‐MAP, we performed the super‐resolution imaging of pathological samples, such as injured spinal cord, brain hippocampus, salivary gland organoid, tumor sphere, and glioblastoma tumor of human patients. This technique provides an ideal way for researchers to examine the pathologic patterns as well as various intact biological samples.
Pharmacological inhibition of ER stress mitigates testicular pathology in hind‐limb unloaded mice
Authors: Ranade Anu V. , Qaisar Rizwan , Khan Amir A. , Karim Asima , Gul Muhammed T. , Azeem Muhammad , Jose Josemin , Ramachandran Gopika , Ibrahim Zainab , Nair Vidhya , Elmoselhi Adel , Rahman Hassan Wael A.
Published: May 2022

Reduced physical activity and cephalic fluid shift are common occurrences in prolonged bed rest and space flight, and can lead to testicular dysfunction. Experimental mouse model of hind‐limbs‐unloading (HU) mimics spaceflight and bed rest and recapitulates several features of testicular anomalies in such conditions including disruption of tubular architecture and associated histological changes that together contribute to male infertility. However, an effective pharmacological intervention to these changes remain elusive, partly because molecular mechanisms are poorly understood. Disruption of protein folding by endoplasmic reticulum (ER) or ER stress is a common etiology in several diseases but its contribution to testicular pathology is not known. We hypothesize that HU is associated with activation of ER stress and subsequent disruption of normal testicular histology in a time‐dependent manner, while treatment with 4‐Phenylburtyrate (4‐PBA; an ER‐stress inhibitor) will reverse these changes to normal levels. To test this hypothesis, 4‐months‐old male c57B16/j mice were subjected to 14 and 28‐days of HU. A significant reduction in thickness and organization of seminiferous tubular epithelium and discontinuation of germ cell layers was seen after 14 days of HU. We also observed a significant increase in the tubular and luminal diameter along with a reduction in the density of spermatozoa at 14 days. These changes were further intensified at 28 days of HU and were associated with signature alterations in the Raman spectroscopic analysis of the HU testis. Importantly, treatment with 4‐PBA for 3‐4 weeks (100mg/kg BW/d via intraperitoneal injections) partially reversed most of the pathological changes in mouse testis.

Collectively, our findings reveal the pivotal role of ER stress in triggering testicular pathology in conditions mimicking spaceflight and present 4‐PBA as a potential therapeutic agent to prevent and/or reduce testicular disruption in such cases.

Currently, we are characterizing the associated molecular phenotype in testicular disruption by mapping the global transcriptome and detailed Raman spectroscopic alterations in mouse testes under HU with and without 4‐PBA treatment.
Phosphorylation of the electrogenic sodium/bicarbonate co‐transporter 1 (NBCe1) at Ser245 and Ser255‐257is linked to mTOR‐mediated transporter activity
Authors: Giannaki Marina , Heermann Stephan , Roussa Eleni
Published: May 2022

The electrogenic sodium bicarbonate co‐transporter 1 (NBCe1, Slc4a4) is a key pH‐regulating membrane protein and is widely expressed in kidney, pancreas, cornea, heart and brain. Phosphorylation in several residues can affect the surface expression and the activity of the transporter. NBCe1 has been found to be phosphorylated in several residues among them in Ser65 where SPAK inhibits its activity and in Thr49required for regulation of NBCe1 by IRBIT and SPAK. Moreover, IRBIT regulates the activity of NBCe1 by controlling the phosphorylation status of Ser232, Ser233 and Ser235. The aim of this study is to identify novel phosphorylation sites and examine their putative contribution in the transport activity of NBCe1.

Phosphoproteomic analysis in mouse cortical astrocytes revealed constitutively phosphorylated NBCe1 at Ser245 and Ser255‐257. Mutational analysis in Hela cells and intracellular H+ recordings using pH‐sensitive fluorescent dye (BCECF) showed that the activity of NBCe1 is regulated by the phosphorylation of these residues, phospho‐Ser245 significantly reduced the activity of NBCe1 whereas phospho‐Ser255‐257led to increased NBCe1 activity. Additionally, in the presence of the mTOR inhibitor rapamycin or the mTOR activator 3BDO our data demonstrated that mTOR signaling pathway regulates the phosphorylation of the Ser245 and Ser255‐257.

Our findings reveal phosphorylation at Ser245 and Ser255‐257as novel regulators of NBCe1 transport activity. We suggest that mTOR pathway is capable to fine tune NBCe1 activity, an event with putative implications during pathophysiological conditions.
Pharmacological evaluation of a complex drug against anthrax and parasitosis of Rangifer tarandus
Authors: Nikolaev Semen
Published: May 2022

Reindeer husbandry is one of the leading areas of animal husbandry in the regions of the Far North of the Russian Federation. It serves as the primary source of life and employment for the local population. The most common pathologies in reindeer are gadfly infestations and anthrax. Carrying out mass treatment and preventive measures in reindeer husbandry is a rather laborious process and is associated with high economic costs. The research aims to develop and study the possibility of using the composition of aversectins with an anthrax vaccine in reindeer husbandry. The studies were carried out in the Bolshezemelskaya tundra of the Komi Republic. The studied drug had the following composition: 10 mg of aversectin C, 10 million live spores of the attenuated capsule‐free anthrax bacillus strain 55‐VNIIVViM, excipients (polyethylene oxide, water) up to 1 ml. For the experiment, 3 groups of full‐aged animals were formed. The first group of deer (n = 10) was injected intramuscularly with the developed composition at the rate of 1 ml of the drug per 50 kg of body weight, the second group (n = 10) received only aversesectin, the third group (n = 10) received only the anthrax vaccine. Deer were processed in September. For 14 days, the animals were measured for body temperature, pulse rate, respiration, tissue reaction at the injection site, and blood was obtained every three days for hematological and biochemical studies. The effectiveness of the prophylactic action of the investigated agent was taken into account during the planned slaughter of deer in December, by the method of counting gadfly larvae on the skins of deer of different groups, as well as by determining the titer of anthrax antibodies by the precipitation reaction of two‐fold dilutions of blood serum with anthrax antigen. The clinical picture and morpho‐biochemical composition of the blood of the animals treated with the test agent did not differ significantly from those of the deer of other groups. Furthermore, a response of the body to the drug at the injection site was also not established. The average level of antibodies to the anthrax bacillus in the first group was 1: 288 ± 46, in the second 1: 56 ± 7, in the third 1: 256 ± 26. Thus, the developed composition provides for the formation of intense immunity to the pathogen. A post‐mortem examination of the skins of animals treated with the study drug and native aversectin indicates that the use of the agent is 100% effective against edemagenosis of reindeer. In comparison, the use of one vaccine was characterized by 100% gadfly invasion of animals with an average intensity of 86 ± 18 larvae per animal. Thus, the developed drug shows high efficacy against edemogenosis and anthrax and makes it possible to reduce the number of veterinary manipulations during preventive treatments of reindeer.
Iatrogenic dehydration drives organic osmolyte production in critical COVID‐19
Authors: Hultström Michael , Nihlén Sandra , Titze Jens M. , Lipcsey Miklos , Frithiof Robert
Published: May 2022

Fluid removal with concomitant dehydration is a common treatment strategy in acute respiratory distress syndrome (ARDS). We have previously shown that iatrogenic dehydration during treatment in the intensive care unit (ICU) is associated with a shift to organic osmolyte production in the general ICU population. In this study we investigate the development of estimated osmolality (eOSM = 2Na+2K+glucose+urea) over time in ICU, as well as the relationship between the major osmolytes and eOSM. The study includes 370 COVID‐19 patients admitted to the ICU at Uppsala University Hospital between March 13. 2020 and June 11. 2021. Complete data was available for 176 of these patients, which were used for the analysis. The results show that eOSM increases with time spent in ICU (R2 = 0.75, P<0.001), which indicates iatrogenic dehydration. Further, increasing eOSM is correlated with increasing role of urea as an osmolyte (R2 = 0.6, P<0.001), while the role of sodium and potassium are inversely correlated to eOSM. This is consistent with the aestivation response where dehydration with increased osmolality drives production of organic osmolytes through gluconeogenesis and urea synthesis. Interestingly, glucose shows a weak negative correlation to eOSM (R2 = 0.01, P<0.01), which we interpret as a sign of iatrogenic control using insulin treatment. Acute Kidney Injury with reduced kidney function is an alternative explanation for increasing urea concentration. However, although there is a correlation between urea and the GFR‐markers creatinine and cystatin‐C the effect size cannot explain more than part of the association. In conclusion, iatrogenic dehydration causes an aestivation response in critical COVID‐19 where urea plays an increasing role as an osmolyte with increasing total osmolality.