bluuebunny/crossref_metadata_2025 huggingface dataset:
Sigmar1 Deficiency Results in Pulmonary Fibrosis Associated with Increased Pulmonary Inflammation and Surfactant Protein Levels in Mice Lung
Authors: Remex Naznin S. , Abdullah Chowdhury S. , Aishwarya Richa , Morshed Mohammed M. , Nitu Sadia , Traylor James , Hartman Brandon , King Judy , Kevil Christopher , Orr Anthony W. , Bhuiyan Md. S.
Published: May 2022

Background

Sigmar1 is a ubiquitously expressed multifunctional molecular chaperone protein. Extensive studies demonstrated Sigmar1’s molecular functions in the central nervous system, and mutations in the Sigmar1 gene showed association with different neurodegenerative diseases, including amyotrophic lateral sclerosis and distal hereditary motor neuropathy. Studies also showed the protective roles of Sigmar1 in cardiovascular diseases in animal models of cardiac hypertrophy and heart failure. Recent studies focused on Sigmar1 in COVID‐19 drug repurposing as Sigmar1 was identified to interact with SARS‐Cov‐2 replication proteins using proteomic screening and Sigmar1 ligands showed promising protective effects in clinical trials. Despite all these studies showing a function of Sigmar1 in the respiratory system, the Sigmar1’s expression, localization, molecular and physiological functions in the lung remained unknown.

Objectives

The main objective of this study was to characterize the expression and localization of Sigmar1 in human and mouse lungs. We also investigated the pathophysiological role of Sigmar1 in the lung using Sigmar1 null mice.

Results

Using a combination of immunohistochemistry, immunofluorescence, and biochemical experiments, we confirmed the expression of Sigmar1 in rodent and human lungs. Hematoxylin and Eosin staining showed that Sigmar1 global knockout (Sigmar1‐/‐) mice lungs develop altered alveolar structure and organization and higher immune‐cells infiltration compared to Wildtype (Wt) littermate controls. This was associated with an upregulated protein level of phospho‐NFκB in Sigmar1‐/‐ mice lungs. Sirius red and Masson’s trichrome staining of lung tissue sections showed increased collagen deposition, pulmonary fibrotic remodeling associated with elevated fibronectin and phospho‐SMAD2 protein levels in Sigmar1‐/‐ mice lungs. Ultrastructural analysis by transmission electron microscopy (TEM) showed structural alteration in the multilamellar bodies with partial loss of lipid lamellae in alveolar‐type II epithelial cells (AV‐II) in Sigmar1‐/‐ mice lungs. Multilamellar bodies in AV‐II cells serve as secretory organelles releasing pulmonary surfactants into the extracellular space. Elevated levels of surfactant proteins (SP), SP‐A and SP–D, were reported to serve as biomarkers for acute respiratory failure and lung inflammation. Consistent with the TEM data, we also found increased SP (A, B, C, and D) protein levels in the lungs indicating the AV‐II pneumocytes dysfunction in Sigmar1‐/‐ mice.

Conclusion

Our findings suggested that Sigmar1’s lack of function leads to increased pulmonary inflammation, pulmonary fibrotic remodeling, altered structure and functions of multilamellar bodies, and AV‐II pneumocytes dysfunction in Sigmar1‐/‐mice.
Keratin 17 is a Negative Prognostic Biomarker in Non‐Small Cell Lung Cancer
Authors: Babu Sruthi , Roa‐Peña Lucia , Akalin Ali , Escobar‐Hoyos Luisa , Shroyer Kenneth
Published: May 2022

Although the overall prognosis for patients with non‐small cell lung cancer (NSCLC) has improved over the past several decades, there are still survival differences that are not accurately defined by clinicopathological factors. Thus, there is an unmet clinical need to develop novel approaches to enhance prognostic accuracy for the assessment of these patients. Prior studies have established that keratin 17 (K17) is a negative prognostic biomarker in a wide range of cancer types, including pulmonary adenocarcinoma (LUAD). Although keratin expression profiles have been widely utilized as markers to distinguish between metastatic versus primary squamous cell carcinomas (SCC), K17 has not been previously investigated for its potential accuracy as a prognostic biomarker in lung squamous cell carcinoma (LSCC). Here, we set out to determine if K17 is a prognostic biomarker that drives poor survival in both LSCC and LUAD. Data mining was performed to analyze K17 mRNA in independent cohorts of LSCC (n=266) and LUAD (n=271) and immunohistochemistry (IHC) for K17 was performed in separate cohorts of LSCC (n=84) and LUAD (n=107). Kaplan‐Meier and Cox proportional‐hazard regression models were used to determine overall survival differences between low vs. high K17 expressing cases. High K17 mRNA correlated with decreased overall survival in both LSCC (HR: 1.789, p=0.0646) and LUAD (HR: 1.894, p=0.0142). Similarly, by IHC, high K17 correlated with decreased overall survival in LSCC (HR: 1.894, p=0.0375) and LUAD (HR: 2.105, p=0.0257). Thus, K17, defined either by mRNA expression or immunohistochemical staining, is a negative prognostic biomarker for non‐small cell lung cancer.
RNA Interference Via shRNA Decreases the Number of Transcripts of Scn8a in Glutamatergic Interneurons in the preBötC
Authors: da Silva Carlos Aparecido , Picardo Maria Cristina D. , Del Negro Christopher A.
Published: May 2022

Rhythmic neural activity that drives the inspiratory phase of the breathing cycle in mammals emanates from glutamatergic interneurons of the pre‐Bötzinger Complex (preBötC). A longstanding question in neuroscience is: what are the underlying mechanisms for inspiratory rhythmogenesis? One proposed mechanism involves the persistent sodium current (INaP), which is the ionic mechanism that underlies the voltage‐dependent bursting‐pacemaker activity of neurons whose rhythmic burst discharges are putatively obligatory for inspiratory rhythm generation. Specifically in brain tissues, the Scn8a gene codes for Nav1.6 ion channels, and those ion channels give rise to INaP.

Different genetic tools can help us to understand the role of specific molecules in the generation of motor behaviors, such as breathing. The short hairpin RNA (shRNA) is an artificial RNA molecule, delivered through adeno‐associated virus (AAV), that can inhibit gene expression and promote long‐term knock‐down of the targeted gene.

Here we administered Cre‐dependent shRNA to knock‐down Scn8a (thus Nav1.6) in Cre‐expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10‐14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used in situ hybridization (RNAscope® Fluorescent Assay) to label mRNA of Scn8a and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four‐Channel, Upright Confocal Microscope Body Based on Cerna® System (ThorLabs), and images were acquired using the ThorImage®LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open‐source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t‐test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non‐targeting shRNA sequence).

Our results show that at 2 weeks, the control group had 27.5±9.5 RNA/neuron, whereas the shRNA‐injected had 12.8±3.5, which represents a 53% of decrease in the number of transcripts (P<0.0001). A similar pattern was observed after 6 weeks of the injection in a different cohort, where control animals showed 35.4±6.1 RNA/cells, and the shRNA group only had 24.1±5.2, representing 32% drop in the transcripts for that group (P<0.0001). This initial protocol allowed us to verify that the gene expression has been knocked down at critical time points, encouraging further studies using shRNA to evaluate whether attenuation of Nav1.6 expression in rhythmogenic preBötC neurons impairs breathing, which could resolve the longstanding question regarding the putatively rhythmogenic role of INaP.
Source Tracking based on Microbial Communities: Sample Collection and ddPCR/qPCR
Authors: Orellana Marilyn , Veenstra Brendan , Veine Cameryn , Vollmert Grace , Pikaart Michael
Published: May 2022

Fecal contamination serves as a major public health threat, especially in drinking or recreational waters. This is particularly concerning because it promotes the exposure of communities to harmful pathogenic bacteria. Identifying the source of contamination serves as an important context when mitigating the effects of the pollution or preventing it from initially entering the water. Microbial source tracking (MST) is employed to achieve the quantification of dominant sources of fecal contamination. Specifically in this study, a library‐independent method was utilized in the sample level detection of host‐associated 16s rRNA markers. Among the most prevalent bacteria in human and animal gut microbiomes is the phylum Bacteroidetes,which prove to be extremely useful in MST assays. TaqMan primers and probes targeting host‐independent (universal) Bacteroidetes(BacUni), human‐host specific (HF183), and pig host specific (Pig2Bac) were utilized. Dye‐based SYBR assays targeting avian bacteria Helicobacter (GFD) were also investigated. Solid fecal samples were acquired from a local petting zoo and surrounding beach sites. Water samples from various lakes, streams, and wastewater treatment plants were also obtained. Samples were filtered using membrane filtration technology and DNA was isolated using DNEasy PowerSoilkit. Upon DNA isolation, the concentration of DNA was measured using NanoDrop and the samples were subjected to qPCR or ddPCR assays. Results indicate effective primer/probe selectivity. Cross‐reactivity between samples and primers demonstrated that the targets were not exclusive to the presumed host and could overlap. Future research includes testing primers on samples from a wider range of animals and water sources. Additionally, a comparison of qPCR results to those from ddPCR and quantifying any differences within or between the two.
Dramatization via Zoom to Teach Complex Concepts in Physiology
Authors: Carvalho Helena , Halpin Patricia A. , Scholz‐Morris Elke G. , Carvalho Rosa
Published: May 2022

Our aim is to present DramaZoom as an innovative student‐centered teaching method and to discuss how theater/drama improve comprehension of physiology concepts. In‐person dramatization,that requires physical contact, was adapted into a series of physiology videos using dramatization to teach complex topics in endocrinology. Three instructors simultaneously collaborated with a drama teacher on Zoom at three different institutions to create the DramaZoom videos. In six videos, instructors mimic organs and body systems to teach hypothalamus‐hypophysis interactions and the corresponding hormones. First year medical students at VTCSOM received a packet with six questionnaires corresponding to the six videos. Although students had no prior preparation or access to other material, they demonstrated significant (P<0.05) improvement from pre‐ to post‐tests. Data from all pre‐ and post‐tests combined (pre 65±2.2 and post 91±2.0) showed significant (p<0.5) improvement in knowledge. Detailed results show for Hypothalamus‐Hypophysis Relationship video pre 56±3.9, post 92±9.2; Neurohypophysis and Antidiuretic Hormone video pre 70±4, post 96±2.4; Growth Hormone and Adenohypophysis video pre 58±2.7, post 93±2.1; Adrenal Gland Cortex and Adenohypophysis video pre 72±5.7, post 95±2.0; Adenohypophysis and Thyroid video pre 58±7.0, post 88 ± 4.7; Steroid and Non‐steroid Hormones video pre 83±5.9, post 93±4.8 (data given in percent of correct response ± SE). Students' comments were very positive, stating that the videos were fun to watch, and they recommended them for visual learners. It was remarkable to observe immediate student engagement with peer discussion after they watched each video. In conclusion, DramaZoom videos provide a learning experience that creates an opportunity for a fun class that improves knowledge and leads to spontaneously student interactions. Future research will be done to investigate long‐term retention of content.
RAS Imbalances Account for Baroreflex Dysfunction and Neuroinflammation Induced by Postpartum Endotoxemia in Weaning Preeclamptic Rats
Authors: Abuiessa Salwa A. , Helmy Mai M. , El‐Gowelli Hanan M. , El‐Gowilly Sahar M. , El‐Mas Mahmoud M.
Published: May 2022

We recently reported that postpartum endotoxemia induced by lipopolysaccharides (LPS) provokes greater attenuation of arterial baroreflexes in weaning preeclamptic (PE) rats. Considering the role of renin‐angiotensin system (RAS) in PE programming, we investigated whether pharmacologic RAS modulation alters PE programming of baroreceptor dysfunction induced by endotoxemia. PE was induced by depriving pregnant rats of nitric oxide via gestational L‐NAME administration. RAS modulation involved gestational treatment with Ang 1‐7, losartan, pioglitazone, or combined losartan/pioglitazone. Arterial baroreflexes were assessed with the vasoactive method, which measures negative and positive chronotropism following blood pressure manipulations evoked by phenylephrine (PHE) or sodium nitroprusside (SNP). Slopes of the baroreflex curves were recruited as indicators of baroreflex sensitivity (BRSPHE and BRSSNP). All therapeutic regimens, but Ang 1‐7, abolished the rises in blood pressure seen in weaning PE dams. In contrast to no effect for losartan or pioglitazone, the shifts in baroreflex curves and reductions in BRSSNP in weaning PE/LPS mothers were reversed in dams treated gestationally with Ang 1‐7 or losartan/pioglitazone therapy. Alternatively, the concomitantly impaired BRSPHE was solely improved by the combined losartan/pioglitazone therapy. Immunohistochemical studies revealed upregulated TLR‐4/ACE expression and downregulated ACE2 expression in brainstem neuronal pools of PE/LPS rats. These effects were distinctly affected by gestational therapies as Ang 1‐7 reversed the altered brainstem expression of TLR‐4/ACE2, while gestational losartan/pioglitazone reversed the augmented ACE expression. Together, gestational targeting of offensive (ACE/Ang II (suppression) and defensive (ACE2/Ang 1‐7, facilitation) arms of RAS offers a reprogramming strategy against brainstem neuroinflammation and consequent baroreceptor depression caused by endotoxemia in weaning PE dams.
Gabapentinoids Attenuate Naloxone Reversal of Heroin‐Induced Ventilatory Depression
Authors: Flynn Shawn M. , France Charles P.
Published: May 2022

A growing body of epidemiological evidence suggests increasing misuse of gabapentinoids (gabapentin and pregabalin, calcium channel inhibitors approved to treat some neuropathic pain conditions) in people with opioid use disorder. The number of prescriptions for gabapentinoids has risen rapidly over the past decade, with the majority of these prescriptions being for off‐label indications. Over the same period, gabapentinoids have become increasingly prevalent in opioid overdose deaths. Despite these trends little research has evaluated potentially harmful interactions between gabapentinoids and opioids. This study evaluated the effects of gabapentin and pregabalin on the ventilatory depressant effects of heroin, and the reversal of heroin‐induced ventilatory depression by naloxone. Five male Sprague Dawley rats were given pregabalin (1‐32 mg/kg), gabapentin (10‐100 mg/kg), or saline i.v. 30 minutes prior to receiving increasing doses of heroin while ventilation was monitored using whole‐body plethysmography. Sessions began with a 30‐minute baseline period followed by a test period during which rats received infusions of heroin or saline at minutes 0, 3, and 6 with heroin infusions resulting in cumulative doses of 0.1, 0.32, and 1 mg/kg (i.v.), respectively. Naloxone (0.0056‐0.01 mg/kg) or saline was administered i.v. 5 minutes following the final infusion of heroin. Minute volume, the product of tidal volume (mL/inspiration) and respiratory rate (breaths/minute), was the primary outcome of this study. Heroin dose‐dependently reduced minute volume with 1.0 mg/kg reducing minute ventilation on average to 42% of baseline. Neither pregabalin nor gabapentin altered the ventilatory depressant effects of heroin. When given 5 minutes following the 1.0 mg/kg cumulative dose of heroin, naloxone dose‐dependently reversed heroin‐induced decreases in minute volume with 0.0056 and 0.01 mg/kg naloxone restoring ventilation to 95 and 160% of baseline levels, respectively. Interestingly, pretreatment with gabapentin or pregabalin dose‐dependently attenuated the ability of naloxone to reverse heroin‐suppressed minute volume, with the largest dose of gabapentin and pregabalin almost completely blocking the effects of 0.0056 mg/kg naloxone. Both gabapentin and pregabalin attenuated the ability of naloxone to restore ventilation following administration of heroin in rats but did not alter the effects of heroin alone. These findings suggest that gabapentinoids might diminish the effectiveness of naloxone to reverse opioid overdose in humans, a possible explanation for the prevalence of these drugs in opioid overdose deaths. It will be important to determine whether similar interactions occur between gabapentinoids and naloxone for reversing the effects of other mu opioid receptor agonists such as fentanyl.
Probing the Central Role of Phosphatidylinositol Synthesis in Lipid Metabolism of Eukaryotic Cells
Authors: Mandal Amrita , Kim Yeun Ju , Pemberton Joshua , Balla Tamas
Published: May 2022

Phosphatidylinositol (PI) is the precursor of all phosphoinositides (PPIns), a class of regulatory phospholipids that play key roles in cellular physiology. PI is synthesized in the ER by a single enzyme, phosphatidylinositol synthase (PIS). PI may have important roles of its own in membranes where PPIns are not produced such as in the ER and mitochondria. Disruption of PIS in yeast and zebrafish is lethal and PIS has been postulated to be an essential gene in studies of haploid cells. Therefore, it is difficult to evaluate the processes where this enzyme, or its lipid product, PI plays a critical role. To overcome this problem, we generated stable HEK293 cell lines in which a doxycycline (Dox)‐inducible PIS‐GFP transgene was stably integrated in a safe‐harbor site of the genome and the endogenous PIS locus was inactivated via CRISPR/Cas9 genome editing. We verified the knock‐out of the endogenous PIS and the inducible expression of PIS‐GFP by western blot analysis and sequencing of genomic DNA. Using these cells, we found that PIS enzymatic activity assayed in ER membranes prepared from cells after Dox deprivation was indistinguishable from the assay blank. Dox treatment of cells elevated PIS activity in the isolated ER membrane although it was still lower than that found in the membranes prepared from parental HEK293. Surprisingly, in spite of the undetectable low PIS activity, Dox‐deprived cells still contained about 50% of PI as assessed by equilibrium labeling with 3H‐inositol and retained their PPIns distribution as shown by confocal microscopy using GFP‐tagged PPIns binding modules. This disparity between PIS activity and PI availability was observed even under culture conditions that deprived cells of any external PI source. Such cells were subjected to lipidomic analysis to assess the overall lipid changes associated with the reduction of PI synthesis and to identify processes that require PI. This analysis confirmed the ~50% PI loss and also revealed reprogramming of specific lipid metabolic pathways. Current work focuses on the possibility that alternate source(s) of PI exist and on the question of what lipid synthetic pathways show adaptation to the greatly reduced or missing PIS activity. These experiments have the promise of a better understanding of how PI lipids determine the overall lipid landscape and homeostasis of eukaryotic cells and may unravel new molecular targets that can be exploited for therapeutic interventions in human metabolic and lipid disorders.
Characterization of Post‐translational Modifications of Mitochondrial RNA Polymerase and Mitochondrial Ribosomal Protein L12
Authors: Paluch Katelynn V. , Platz Karlie R. , Gross Matthew , Dodge Helen , Dittenhafer‐Reed Kristin E.
Published: May 2022

Mitochondria play an important role in energy production and cellular metabolism. Mitochondria contain their own DNA (mtDNA), which encodes 13 subunits necessary for oxidative phosphorylation. Over 1500 other mitochondrial proteins, including the 77 remaining subunits required for oxidative phosphorylation and the machinery required for transcription and translation of mtDNA, are encoded by the nuclear genome. Thus, the nuclear and mitochondrial genomes must communicate to respond to the energetic needs of the cell. The mechanism of this communication is unclear. The mitochondrial proteome, including the transcriptional machinery, is subject to post‐translational modifications (PTMs) such as phosphorylation of serine, threonine, and tyrosine and acylation of lysine. We hypothesize that PTMs of the mitochondrial transcriptional machinery regulate mitochondrial gene expression, akin to mechanisms controlling nuclear gene expression. Transcription of mtDNA requires three nuclear‐encoded proteins: mitochondrial transcription factor A (TFAM), transcription factor B2 (TFB2M), and mitochondrial RNA polymerase (POLRMT). An accessory factor, mitochondrial ribosomal protein L12 (MRPL12), is thought to stabilize POLRMT and may promote transcription. Prior experiments in our lab show phosphorylation mimics of TFB2M have significantly reduced binding affinity for mtDNA and exhibit transcription initiation defects in vitro. Using mass spectrometry POLRMT was previously shown to be acetylated at one lysine and phosphorylated at nine amino acids, while MRPL12 contains five acetylated lysines and one phosphorylated threonine. The biochemical function of these modifications is unknown. PTMs were studied by using site‐directed mutagenesis to replace the amino acid of interest to mimic acetylation (lysine to glutamine) or phosphorylation (threonine to glutamate) of POLRMT and MRPL12. Mutated proteins were purified and their mtDNA promoter binding affinity was determined by fluorescence anisotropy experiments. Fluorescence anisotropy revealed that POLRMT PTM mimics had little effect on mtDNA binding. However, when WT MRPL12 was co‐incubated with WT POLRMT mtDNA binding affinity was enhanced by 30%. Increased binding affinity was lost with MRPL12 PTM mimics. WT and PTM mimics of MRPL12 were also overexpressed in mammalian cell lines. mtDNA transcript levels and mtDNA content were measured using quantitative PCR. mtDNA content was largely unchanged while some transcript‐dependent effects were observed in the presence of MRPL12 protein mutants.
Probabilistic Reinforcement Schedules in Neuropsychiatric Medications Development
Authors: Kangas Brian D.
Published: May 2022

Anhedonia, the loss of pleasure from previously rewarding activities, is a core symptom of several diverse neuropsychiatric conditions, including major depressive disorder, bipolar disorder, post‐traumatic stress disorder, schizophrenia, and substance use disorders. Unfortunately, despite its transdiagnostic prevalence, no effective therapeutics exist to treat anhedonia. This is due, in part, to inconsistent assays across clinical populations and laboratory animals, which hamper treatment development. To bridge this gap, recent work has capitalized on exposing humans, rodents, and nonhuman primates to identical asymmetric probabilistic schedules of reinforcement to objectively quantify their responsivity to reward. In these computerized tasks, subjects make visual discriminations and probabilistic contingencies are arranged such that correct responses to one alternative are rewarded more often (rich) than correct responses to the other (lean). Under these conditions, both healthy humans and laboratory animals consistently develop a response bias in favor of the rich alternative. However, patients with neuropsychiatric disorders often exhibit blunted biases, which correlate with current, and predict future, anhedonia. Likewise, laboratory animals exposed to early‐life adversity or chronic stress have been shown to be less sensitive to asymmetric probabilistic contingencies. In addition, pharmacological challenges with candidate therapeutics known to enhance hedonic tone have produced similar desirable outcomes across species. Taken together, this quantitative framework offers a highly translational approach to assaying reward responsiveness to accelerate treatment development for neuropsychiatric disorders involving anhedonia.
Mice With Monoallelic GNAO1 Loss Exhibit Reduced Inhibitory Synaptic Input to Cerebellar Purkinje Cells
Authors: Feng Huijie , Yuan Yukun , Williams Michael R. , Roy Alex J. , Leipprandt Jeffery , Neubig Richard R.
Published: May 2022

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. Gαo, the most abundant membrane protein in the brain, mediates downstream signaling pathways by itself, or via regulating free Gβγ subunits. To determine the role of Gαo protein in regulating synaptic transmission, we use electrophysiology to study cerebellar molecular layer interneuron ‐ Purkinje cell synaptic transmission in a haploinsufficient loss‐of‐function (LOF) Gnao1 mouse model, manifest with movement abnormalities and enhanced seizure susceptibility. We hypothesize that a LOF Gαo protein will increase GABA release through presynaptic Gαo ‐ coupled receptors.

Inhibitory post synaptic currents (IPSCs) and excitatory post synaptic currents (EPSCs) were recorded using standard patch clamp techniques. Spontaneous IPSCs (sIPSCs) were isolated with glutamatergic receptor inhibitors CNQX and AP‐5. Spontaneous EPSCs (sEPSCs) were isolated with the GABAergic receptor inhibitor bicuculline. Miniature PSCs were isolated with the additional perfusion of tetrodotoxin. Cerebellar molecular layer inhibitory neurons were labeled with the Kit‐eGFP method and observed under an inverted epifluorescence microscope. Gαo and Gβ protein expression levels from flash‐frozen mouse brain tissues were determined using Western Blot.

Instead of increase, we observe a significant reduction in both sIPSC (40%) and mIPSC (75%) frequency but not amplitude in the Gnao1 mutant mouse model. Both frequency and amplitude of sEPSCs and mEPSCs remain unchanged. On Western blots, levels of Gαo were reduced by about half, as expected, while Gβ levels were unchanged from control. To determine whether this reduction is due to the loss of cerebellar inhibitory interneuron populations, we compare cerebellar interneuron number in the cerebellar molecular layer between Gnao1 mutant mice and their wildtype littermate control. There is a modest reduction (13%) in interneuron number of cerebellar molecular layer in the Gnao1 mutant mouse.

The observed reduction in IPSC frequency is not related to Gαo ‐ coupled GABAergic receptors. Also, the modest change in cerebellar interneuron counts does not seem to account for the marked reduction in IPSC frequency. These observations, and the reduced Gαo expression in brain with the normal levels of Gβ, strongly support that a model with a haploinsufficient LOF Gnao1 mutant mainly functions through receptor‐independent regulation of free Gβγ subunits to suppress inhibitory neurotransmitter release in Gnao1 mutant cerebellum.
Mapping Allosteric Sites on Protein Kinases Critical in MAPK Signaling
Authors: Fera Daniela , Lin Sabrina , Naagaard Daniel , Chen Han , Finkelstein Maxwell T. , Glatz Gwendolyn , Hayashi Scout , Hotan Quynn , Lau Benjamin , LeBlanc Julia , Li Kevin , Parker Miller Emma , Ye Ming , Zaidi Hussain
Published: May 2022

The mitogen‐activated protein kinase (MAPK) pathway plays a critical role in controlling cell cycle progression and cell proliferation. This Ras‐Raf‐MEK‐ERK pathway is constitutively activated in over 80% of melanomas, as well as in some pancreatic, colon, lung, ovarian, and kidney cancers. Anticancer drugs targeting proteins earlier in the pathway, such as B‐Raf and MEK, often lose efficacy due to the development of resistance. Thus, drugs that target allosteric sites hold promise if used in combination with ATP‐competitive inhibitors that have already been developed. We have been working on identifying allosteric sites on key kinases of this pathway that could be used for future drug discovery efforts.

We previously found that the alpha‐G helix of B‐Raf is critical for its interaction with MEK and that mutations of several residues along this helix could completely abrogate binding and downstream phosphorylation activity in vitro. With this information, we further analyzed the crystal structure of the B‐Raf‐MEK complex to generate mutations in MEK to determine if the same region of MEK that contacts the B‐Raf alpha‐G helix is important for binding ERK. Additionally, since no complex structure of the MEK‐ERK complex was available, we generated mutations in different regions of the C‐lobe of ERK as a starting point to identify a region of contact. We tested the effects of our mutations on binding using pull downs and biolayer interferometry. We also assessed phosphorylation levels of ERK in vitro.

Our MEK mutants displayed no difference in binding to B‐Raf, but did have altered binding to ERK. This suggests that the modes of MEK binding to B‐Raf versus ERK differ. Moreover, our mapping of the ERK interface has led to the identification of a residue on the helix spanning residues 232‐245 that, when mutated, significantly reduces both binding to MEK and phosphorylation of ERK. Additional work is needed to determine if our mutation affects the conformation of a nearby alpha helix of ERK and as a result affects MEK binding or if it is due to a direct effect. Nevertheless, these results demonstrate that MEK‐ERK binding and downstream activity can be altered by targeting sites outside their catalytic region.
The Effects of ß‐caryophyllene on Pain‐Evoked and Pain‐Depressed Behaviors in Rats with Chronic Inflammatory Pain
Authors: Chin Christopher Y. , Ron Katiana , Fernandez Alyssa , Kandasamy Ram
Published: May 2022

The antinociceptive effects of major cannabinoids such as ∆9‐tetrahydrocannabinol (THC) and cannabidiol (CBD) have been extensively studied in rats. These studies have led to formulations of THC and CBD for human use; however, humans use different strains of Cannabis that contain several hundred different compounds. The contribution of these compounds to pain relief produced by Cannabis is unclear. ß‐caryophyllene (BCP) is one compound found in the essential oils of Cannabis. Despite some early studies, the extent to which these compounds produce pain relief in assays of pain‐evoked behaviors (i.e., von Frey and Hargreaves tests) and pain‐depressed behaviors (i.e., home cage wheel running) is unclear. We hypothesized that BCP would inhibit mechanical allodynia and thermal hyperalgesia as well as restore depressed wheel running activity in male Sprague‐Dawley rats with inflammatory pain. Three different doses of BCP (10, 30, and 100 mg/kg) or vehicle were administered to rats via an intraperitoneal injection after hindpaw inflammation induced by an intraplantar injection of Complete Freund’s Adjuvant (CFA). Neither the low dose (10 mg/kg) nor the medium dose (30 mg/kg) of BCP reversed mechanical allodynia of the inflamed hindpaw after intraperitoneal injection. However, a high dose of BCP (100 mg/kg) reversed mechanical allodynia on the von Frey test; however, this dose did not reverse thermal hyperalgesia. A hindpaw injection of 0.1 mL CFA decreased wheel running activity as is consistent with a painful stimulus. However, neither 30 mg/kg BCP nor 100 mg/kg BCP restored pain‐depressed wheel running in injured rats. These same doses of BCP did not affect wheel running in uninjured control rats. Therefore, a high dose BCP produces pain relief, although it only does so against mechanical allodynia. BCP does not restore normal activity. This suggests that although pain may be eliminated following BCP administration, a return to normal levels of activity may not be possible which raises questions about the utility of BCP to treat pain. Future studies of the pain‐relieving effects of Cannabis constituents must include tests of many pain‐related behaviors to understand dose‐response relationships and their therapeutic potential.
Knockdown and Inhibition of Spleen Tyrosine Kinase Decreases mTORC1 Activity and Enhances Lysosomal Biogenesis and Autolysosomal Maturation in Pancreatic Cancer Cell Lines
Authors: Pan Michelle , Hua Kevin , Villait Akash , Zuger Angelina , Rafati Minoo , Chen Ryan , Singh Anurag
Published: May 2022

In cancer, oncogene dependency is a phenomenon where a dominant driver oncogene promotes tumor cell proliferation and survival, and loss of this oncogene results in tumor cell death and, eventually, tumor regression. KRAS, a GTPase that regulates cell growth and proliferation, is an oncogene constitutively activated in over 90% of pancreatic cancer. However, clinically effective inhibitors of KRAS have been unsuccessful so efforts have been focused on identifying other potential targets associated with the KRAS signaling network. Spleen tyrosine kinase (SYK), which is expressed at high levels in KRAS‐dependent pancreatic cancer cell lines, may be one of these targets. Our data indicate that SYK activates the mechanistic target of rapamycin kinase complex 1 (mTORC1), which promotes protein translation and cell growth. SYK activation also leads to decreased autolysosome count. In connecting SYK activation with increased mTORC1 activity and decreased autolysosome count, we hypothesis that the MiT/TFE transcription factors are involved. We propose that SYK inhibition in pancreatic cancer cells leads to reduced mTORC1 activity, which reduces phosphorylation of MiT/TFE transcription factors. The unphosphorylated MiT/TFE transcription factors may then enter the nucleus to activate genes for lysosomal biogenesis and autophagy. Autophagy is a process that recycles cellular macromolecules during nutrient deprivation by fusing autophagosomes and lysosomes, produced from lysosomal biogenesis, to generate autolysosomes. From our experiments, we were able to show that SYK inhibition blocks mTORC1‐dependent phosphorylation of MITF and TFEB transcription factors, members of the MiT/TFE family. MITF and TFEB activation leads to increased autophagy due to autolysosomal biogenesis and accumulation. In summary, our studies of the SYK‐mTORC1‐autophagy pathway provide support to investigate SYK as a candidate therapeutic target for pancreatic cancer treatment.
emx2is Required for Nephron Segment Development in the Zebrafish Pronephros
Authors: Nguyen Khoa , Wingert Rebecca
Published: May 2022

The empty spiracles homeobox gene 2 (emx2)encodes a transcription factor that is expressed during neuronal, auditory, olfactory, and renal development, but its role in the latter has not yet been fully elucidated. Here, our objective is to utilize the zebrafish animal model to investigate the functions of emx2 during the development of nephrons, which are the structural and functional units of the kidney. Using whole mount in situ hybridization (WISH), we detected emx2transcript beginning at the 6 somite‐stage in renal progenitors and this expression continued throughout the process of forming discrete proximal convoluted tubule (PCT) and proximal straight tubule (PST) segment regions at 24 hours post fertilization (hpf). Based on this spatiotemporal pattern of emx2 expression in renal progenitors, we hypothesized that emx2is essential for segmentation of the nephron into discrete proximal and distal regions. To investigate this, we generated emx2‐deficient embryos and compared them to wild‐type (WT) controls at various time points from 24‐96 hpf. Knockdown of emx2resulted in head necrosis and diminished yolk sac extension at 24 hpf, as well as development of smaller head, eye and body, pericardial edema, abnormal fin growth, yolk ball inflammation and vascular hemorrhage in the central nervous system from 2‐5 days post fertilization. We used WISH to stain for each nephron segment in fixed animals and then quantified segment length and cell number. WISH analysis revealed a significantly reduced podocyte area, diminished PCT and reduced multiciliated cell numbers in emx2‐deficient embryos compared to WT embryos. WISH results also revealed secondary phenotypes, such as reduced fin bud area and somite in emx2‐deficient embryos. Additionally, Alcian Blue staining at 96 hpf revealed jaw abnormalities and pharyngeal arch malformation in emx2‐deficient embryos. O‐Dianisidine staining showcased vascular hemorrhage in the eyes and head of emx2deficient embryos. We concluded that emx2is essential for kidney segmentation in zebrafish, especially in podocyte and PCT formation. Additionally, emx2deficiency causes secondary jaw, fin, cartilage blood accumulation and muscle phenotypes. Future studies will delineate the cellular defects in nephron development in emx2 deficient embryos and determine the relationship between Emx2 and pathways which are essential for proximal segment ontogeny, such as retinoic acid signaling.
Advanced Maternal Age Impairs Uterine Artery Remodelling in the Pregnant Rat
Authors: Wooldridge Amy L. , Pasha Mazhar , Chitrakar Palehswan , Kirschenman Raven , Spaans Floor , Cooke Christy‐Lynn M. , Davidge Sandra T.
Published: May 2022

Advanced maternal age (AMA, ≥35 years) pregnancies are increasingly common, and are at increased risk for pregnancy complications. We previously showed that ex vivo uterine artery constriction due to increasing intraluminal pressures (myogenic tone) was greater in arteries of AMA vs. young pregnant rats. Aging impairs vascular reactivity and increases vascular stiffness via structural changes (e.g. increased collagen), however, the impact of AMA on adaptations to pregnancy in the uterine artery is unknown. We hypothesized that functional and structural uterine artery adaptations to pregnancy are impaired in aged compared with young rats.

Pregnant young (4 months) and aged (9 months; ~35 years in humans) rats were studied on gestational day 20 (term=22 days) with age‐matched non‐pregnant rats. Uterine arteries were isolated and mounted in a pressure myograph. Active tone (calcium present in buffer, n=6‐10) and mechanical properties (circumferential stress and strain; calcium absent, n=10‐24) were assessed. Myogenic tone, circumferential stress and strain were calculated from active and passive curves. Collagen and elastin in the tunica media and intima (combined) were visualized with Masson’s trichrome (collagen) or Verhoeff’s (elastin) stains (n=4‐6). Data are mean area under the curve (AUC, myography) or % positive area (histology) ± SEM; analyzed by two‐way ANOVA with Sidak post‐hoc tests (significance: p<0.05).

Between 4‐100 mmHg, myogenic tone did not change with age or pregnancy. However, arteries from young pregnant rats displayed forced vasodilation at higher pressures (100‐160 mmHg) that did not occur in arteries from other groups (p<0.0001). Circumferential stress and strain (Figure 1) increased with pregnancy (p<0.0001) but was significantly lower in arteries from pregnant AMA compared to young pregnant dams (p<0.05). Collagen and elastin (% positive areas) did not change with age or pregnancy overall, however, arteries from young non‐pregnant rats had greater collagen:elastin ratios than those from aged non‐pregnant rats (p=0.021). Arteries from aged non‐pregnant, young pregnant and AMA rats had similar ratios.

This study illustrates that aging impacts both structural and functional adaptations of uterine arteries during pregnancy. The lower compliancy of arteries from AMA rats may be due to increased crosslinking of the existing collagen within the artery, as it was not explained by collagen:elastin ratio. These structural differences may contribute to the functional differences, potentially reducing uteroplacental blood supply. Thus, impaired uterine artery adaptations may increase the risk of pregnancy complications in women of advanced age.
DANCR acetylated‐KLF5 induces cisplatin resistance in triple‐negative breast cancer by inhibiting the transcription of p27Kip1
Authors: Tang Jianming
Published: May 2022

Cisplatin is used as a common chemotherapy drug in triple‐negative breast cancer. However, although growing evidence demonstrates that its chemoresistance becomes a important obstacle for curing of triple‐negative breast cancer, its molecular mechanism remains unclear. Here we detected that Krüppel‐like factor 5 (KLF5) depletion reduce cisplatin chemoresistance both in triple‐negative breast cancer cells in vitro and in vivo. Further experiments showed that long non‐coding RNA DANCR binds with KLF5 and then induces the acetylation of KLF5 at lysine 369 (K369), which upregulates KLF5 protein level, contributing to cisplatin chemoresistant. Additionally, DANCR/KLF5 signalling pathway renders chemoresistant hypersensitive to cisplatin that inhibiting p27 transcription. Our study uncover a novel DANCR/KLF5/p27 signalling pathway to mediate cisplatin chemoresistant in triple‐negative breast cancer and provide a new molecular target for treatment of triple‐negative breast cancer.
Nicotine and THC Effects on Rodents
Authors: Parekh Grace , Trejo Joseph
Published: May 2022

Nicotine is a highly addictive stimulant that causes an increase in blood pressure, heart rate, and inflammation in the lungs. The use of vaping devices has a grand effect on the youth today. Vapes and e‐cigarettes are used for recreational purposes, yet this may affect adolescents by weakening their respiratory and immune systems. Nicotine and THC both have a strong impact on vapes. Experimental models in rodents suggest differences in behavior and physiological conditions when exposed to nicotine with or without THC. These changes can include differences in weight, bone density, bone strength, and respiratory rates. The aim of this study is to determine the effects of nicotine in the presence and absence of THC on the respiratory and immune systems in mice. Over the course of 12 weeks, both groups were treated with glycerin and propylene glycol. The 19 mice in the experimental group received nicotine on top of glycerin and propylene glycol. Animals were exposed to respective environments for 5 min/day, 2 days/week for a total of 12 weeks. Respiratory, minute volume, tidal volume, bone scans, fat percentage, and weights of the mice were collected weekly during the 12‐week period. The vital organs were harvested for further examination: these tissues included serum, lung, brain, and femur. Data analysis showed significance in weight and fat percentage. Conclusions upon THC in addition to nicotine are in the process of being completed.
Pharmacokinetics and distribution of cathine and cathinone in rats
Authors: Attafi Ibraheem M. , Alamir Ahmad M. , Jeraiby Mohammed A. , Oraiby Magbool E. , Shaheen Emad S. , Juraybi Ismail A. , Attafi Mohammad A. , Albeishy Mohammed Y. , Hakami Ahmed M. , Khardali Ibrahim A. , Korashy Hesham M.
Published: May 2022

Khat is a stimulant plant that contains stimulant chemicals such as cathine and cathinone, which abuses to induce euphoria, alertness, and motor activity. This study was carried out to determine the pharmacokinetic and distributions of cathine and cathinone following a single dosage of khat extract in rats. Practically, a total of 24 adult male Wistar albino rats aged between 8 and 12 weeks (250‐300 g weight) were randomly selected and divided into six groups 4 rats each. After a single dosage (2000 mg/kg body weight) administered by oral gavage, each group was sacrifice at specific time that schedules to be 0.5, 1, 2.5, 5, 12, and 24 hours for group number 1, 2, 3, 4, 5, and 6, respectively. Specimens were obtained as tissues and separated serum for consequences analysis. The mass spectra profiles of the interested analytes were detected by using two different types of liquid chromatography mass spectrometry; (i) Ultra‐performance liquid chromatography in coupled with with quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF/MS) system, (ii) High performance liquid chromatography ion trap mass spectrometry (HPLC‐IT/MS) system. Cathine and cathinone were qualified and quantified in serum, brain, lung, heart, liver and kidney following a single khat administration dosage. The highest tissue levels were reach within 0.5 hour in the lung, liver, and heart for cathine and within 0.5 hour in the heart for cathinone. Interestingly, more than 60% of the serum content of cathine and cathinone reached the peak in the brain within 2.5 hours with mean residence time (MRT last) 2.31 – 3.31 hours. Moreover, at the level of the neurotransmitters such as epinephrine, dopamine, norepinephrine and serotonin, their presence were detected in a delayed, prolonged and organ specific manner. This findings provided further basis for experimental, clinical, and forensic studies.
The Role of Lymphocyte Cytosolic Protein 1 in a Mycobacterium Marinum Infection
Authors: Conrad Will , Remish Emma , Sahin Ceylin K. , Grimes Beau
Published: May 2022

Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent due to the lack of effective treatments. The goal of this project is to identify if there are host genes that affect the disease outcome. We decided to study the influence of the Lymphocyte Cytosolic Protein 1 (LCP1) gene in zebrafish larvae infected with mycobacterium marinum. To understand the immune response in the fish we observed the bacterial burden in fish that had LCP1 (heterozygotes) and in fish that did not (knockout mutants). M. marinum was injected into the caudal vein of the zebrafish larvae and the fish were infected. Once the fish were fixed and euthanized, images of the bacterial infection were taken using a fluorescent microscope. The bacteria was tagged with tdTomato, a red fluorescent protein which allows for the bacteria to glow red under the microscope. The images were then analyzed, and the bacterial burden was quantified using ImageJ to count the number of glowing pixels. Following the imaging of the infected larvae, the fish were genotyped. The DNA from the larvae was extracted through a hotSHOT PCR protocol, then LCP1 Exon 2 was amplified through PCR amplification. The PCR product was then cut with the Bsl‐I enzyme to show where the gene was expressed, gel electrophoresis was then run to analyze where the Bsl‐I cut and identify whether the fish was a heterozygote or knockout mutant. We then matched the suitable images of the fish to the genotype and found that fish with LCP1 may have a higher rate of infection. Understanding why fish with LCP1 have a higher bacterial burden could provide an insight to the role LCP1 plays in the human body’s immune response. The future directions of this project are to observe more fish in order to replicate the findings and to test other phenotypes including granuloma size, macrophage motility, and extracellular growth in order to have a better understanding of LCP1's role in the disease outcome. Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent.