bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physical and Mechanistic Characterization of Tardigrade Cryptobiotic States in Response to Environmental Stressors
Authors: Joseph Kara M. , O'Dell Hayden M. , Clark Trace A. , Crislip Jessica R. , Smythers Amanda L. , Hicks Leslie M. , Kolling Derrick 
Published: May 2022

Tardigrades, known more commonly as ‘water bears,’ are semi‐microscopic invertebrates that possess a remarkable ability to withstand extreme stress. This stress tolerance is inferred via cryptobiosis, a collection of quiescent states enabling long‐term survival to extreme stressors through temporary metabolic suspension and upregulation of protective biomolecules. While cryptobiotic abilities span taxa across all kingdoms, the tardigrades ability to survive desiccation, freezing, oxygen starvation, fluctuating osmotic pressure, and ionizing radiation (using anhydrobiosis, cryobiosis, anoxybiosis, osmobiosis, and irradiation‐induced dormancy, respectively) is matched by none.Tardigrade cryptobioses, particularly those that form tuns, seem to occur via two distinct but connected processes, one of which facilitates and/or initiates muscular contraction and one of which expels internal water stores. However, the mechanisms through which these cryptobioses are initiated, maintained, and regulated are still unknown. Additionally, while research has shown distinctions between cryobiosis and anhydrobiosis, it is unclear to what extent tun‐forming cryptobioses (i.e. anhydrobiosis and osmobiosis) are distinctly regulated. Furthermore, while anhydrobiosis and cryobiosis have been well characterized, other forms of cryptobiosis have not been empirically explored.Our work has focused on charactering the physiological and metabolic distinctions between cryptobioses in Hypsibius exemplaris,a model tardigrade species. We have mapped all known cryptobiotic states through the implementation of confocal microscopy and biochemical characterization. Together we have shown structural and metabolic distinctions between tun‐forming and non‐tun cryptobiotic states. These quantitative measurements provide insight into mechanisms corresponding to protective physiological changes and represent the first known empirical analysis of the five cryptobiotic states.
Characterization of An Inducible p65 Knockout Mouse Model for Studying Glioblastoma
Authors: Trang Amy , Alptekin Ahmet , Rajpurohit Surendra , Arbab Ali , Bradford Jennifer 
Published: May 2022

Glioblastoma (GBM) is a malignant brain cancer that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor promoting M2 phenotype which encourages tumor growth. However, there are limited studies about the specific effect(s) that microglia have on GBM thus requiring further research into how these cells affect GBM tumors.Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor‐associated cells that are involved in sustaining GBM tumors could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor‐kappa B (NF‐κB) pathway, which has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on characterization of the p65fl/fl/CX3CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF‐κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF‐κB pathway, should be deleted in microglia due to the C‐X3‐C chemokine receptor 1 (CX3CR1) promoter. Tamoxifen binds to the estrogen receptor on the cre recombinase (creER) so that the cre recombinase can recombine the floxed p65 (p65fl/fl) gene in microglia. Characterization of this particular mouse model is necessary to determine if the floxed p65 gene is effectively deleted in microglia from these mice. Liquid chromatography‐mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen‐treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the floxed p65 gene in isolated microglia compared to p65 knockout mice given the vehicle control. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl/CX3CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggested that the floxed p65 gene is partially deleted in microglia of the tamoxifen‐treated p65 knockout mice compared to the vehicle control mice.
The Training‐Induced Increase in Critical Power Is Strongly Related to Changes in Vascular Function in Young Adults
Authors: Gifford Jayson R. , Wallace Taysom , Marchant Erik , Kaluhiokalani Jamie , Hyldahl Robert , Hancock Chad 
Published: May 2022

Homeostatic or steady‐state conditions are necessary to sustain exercise. When steady state cannot be achieved, muscle fatigue quickly grows, and task failure promptly occurs. Critical Power (PCRIT) is the highest power output at which steady‐state conditions can be achieved during a given exercise and is strongly related to aerobic capacity. Endurance training increases PCRIT, but the extent to which changes in vascular function, exercise blood flow and mitochondrial function are related to training‐induced changes in PCRIT is unknown.PurposeDetermine the relationship between training‐induced changes in blood flow, mitochondrial function and PCRIT.Methods20 untrained adults participated in either 6 weeks of high‐intensity knee extension exercise (3x per week, n=10) or 6 weeks of sham therapy (3x per week of sham heat therapy, n=10). PCRIT and work‐prime (W′) were determined during single‐leg knee extension (KE) before and after training. Maximum exercise blood flow (Doppler Ultrasound during maximum KE), resistance artery function (passive‐leg‐movement (PLM) technique) and maximum mitochondrial respiration (i.e., State 3 respiration of permeabilized muscle fibers biopsied from the vastus lateralis) were also assessed before and after training.ResultsPCRIT increased by 28% in the exercise group (Figure A, P=0.01), but did not change in the sham group (Figure A). W′ did not significantly change with the intervention (P=0.23). Maximum exercise blood flow (P=0.03), peak flow during PLM (P=0.02), and maximum coupled mitochondrial respiration (i.e., State 3 Respiration, P=0.03) all tended to increase in the exercise group, but not the sham group (P=0.82, 0.18, 0.76, respectively). As illustrated in Figures B, C and D, Pearson correlation indicated that the change in PCRIT was related to the changes in maximum exercise blood flow (R2=0.29, P=0.01), PLM Peak Flow (R2=0.54, P<0.01), and State 3 respiration (R2=0.29, P=0.03).ConclusionsWhile changes maximum exercise blood flow and maximum coupled mitochondrial respiration were related to training‐induced changes in PCRIT, the change in resistance artery function best predicted the training‐induced increase in PCRIT. While not tested in this study, resistance artery function may potentially relate to PCRIT by improving the precision of oxygen delivery and reducing oxygen deficit at the start of exercise. Interventions seeking to improve PCRIT and exercise tolerance, should consider focusing on strategies to enhance resistance artery function in the exercising muscles.
PP1cβ dephosphorylates cardiac myosin by MYPT‐dependent and independent mechanisms
Authors: Lee Eunyoung , Liu Zhenan , Nguyen Nhu , Nairn Angus , Chang Audrey N. 
Published: May 2022

In beating hearts, cardiac muscle myosin is phosphorylated at its regulatory light chain (RLC) at 0.4 mol phosphate/mol RLC. This level of constitutive phosphorylation is necessary for normal cardiac function, and is maintained by balanced activities of the dedicated cardiac muscle‐specific myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). The MLCP is best characterized in the smooth muscle, and is comprised of the regulatory myosin phosphatase target subunit MYPT1, a catalytic subunit PP1cβ, and an accessory subunit of unknown function M20. In smooth muscles in vivo, MYPT1 and PP1cβ co‐stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, the ubiquitously‐expressed MYPT1 and the striated muscle‐specific MYPT2 are both expressed in the cardiomyocyte, but the contributions to physiological regulation of cardiac myosin dephosphorylation by PP1cβ are unclear.Using a conditional knockout of PP1cβ, we tested the hypothesis that the main catalytic subunit for cardiac MLCP is PP1cβ, and that its activity toward phosphorylated cardiac RLC in vivo is dependent on regulation by striated muscle‐specific MYPT2. We measured the effects of the knockout on the expression levels of other regulators of cardiac RLC phosphorylation, and a cardiac myofibrillar phosphatase assay was developed to measure MYPT‐regulated and MYPT‐independent phosphatase activities toward phosphorylated cardiac myosin. We report that 1) PP1cβ is the main isoform expressed in the cardiac myocyte, 2) cardiac muscle pathogenesis in PP1cβ knockout animals involve upregulation of PP1cα and MYPT1 in activated myofibroblasts, 3) the protein stability of endogenous mouse cardiac MYPT1 and MYPT2 are not dependent on the expression of PP1cβ, and 4) phosphorylated cardiac myosin in intact myofibrillar preparations are dephosphorylated by both myosin‐targeted and soluble MYPT‐independent PP1cβ activities. These results contribute to defining properties of the cardiac MLCP in vivo.
Cellular localization, immunological function and biophysical characterization of Mycobacterium tuberculosis Methyltransferases
Authors: Nazim Tasmin 
Published: May 2022

BackgroundTuberculosis is the 13th leading cause of death and the second leading infectious killer after COVID‐19 (above HIV/AIDS) around the world. In 2020, an estimated 10 million people fell ill with tuberculosis (TB) worldwide. Extensively drug‐resistant TB (XDR TB) is a rare type of MDR TB that is resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second‐line drugs (CDC). To avoid another pandemic from happening after covid‐19 we must work on drug discovery and vaccines to prevent TB from spreading.ObjectiveThis work is done on mycobacterium tuberculosis, the causative agent of tuberculosis, using laboratory experiments and computational biological tools to characterize methyltransferase proteins of Mtb. This study is targeted to find out any potential drug or vaccine target in Mtb.MethodWith the help of bioinformatic tools a hypothetical methyltransferse protein was selected from previously reported variety of 121 methyltransferses according to the domain configuration. The protein was verified by the thermal stability, Protein conformational changes, localization, signal peptide chain, structure prediction and function, mutational analysis and molecular docking.ResultsRv3366 gene was selected based on its secretory characteristic and localisation in mitochondria among 121 methyltransferases. SignalP4.1 and Mitoprot ll were used for the cleavage site prediction. Protein modelled by online server has 95.6% allowed region and validated by Ramachandran Plot Statistics from Richardson's lab.The biophysical characteristics of the gene were also identified using methyltransferase assay, thermal denaturation assay, spectrophotometry, fluorescence assay. Alarm assay was done for quantitation of viable cell number in proliferation and cytotoxicity in the presence of the target protein. MitoTracker Red CMXRos kit was used to verify the localisation of the protein in mitochondria.ConclusionThis study covers essential identification of hypothetical methyltransferase that will help in targeting dataset for future studies leading to drug target and TB control of MTB or probably even MDRTB.
Fatty Gastrocnemius Muscle: A Case Report
Authors: Liu Siyu , Kostek Matthew , Omstead Kailey 
Published: May 2022

BackgroundThe gastrocnemius muscle is a superficial two‐headed muscle located in the posterior compartment of the leg, consisting of two heads: medial head and lateral head. The gastrocnemius muscle plays a significant role in our daily activities because its function is plantar flexing the foot at the ankle joint and flexing the leg at the knee joint. We report a very rare case of fatty gastrocnemius.MethodsThe abnormal gastrocnemius muscles were encountered during the dissection of the lower limbs of a 63‐year‐old female cadaver. For histological examination, muscular samples were sectioned from the top, middle, and bottom of two heads of both legs. A total of six muscular slides, nerve slides, artery slides were all stained with Hematoxylin and Eosin (H&E) stains. Slides were analyzed using light microscopy.ResultFor anatomy analysis, the medial heads of both gastrocnemius muscles of the lower limbs are completely infiltrated by adipose tissue. But the lateral heads of gastrocnemius muscles on both sides are almost healthy. Only the bottom of the lateral head of gastrocnemius muscles shows adipose tissue, but the middle and topdo not. Additionally, the medial heads connect to the tibial nerve and arteries normally. No abnormal adipose tissue was observed in other muscles throughout the body. For histological analysis, the entire medial heads and the bottom lateral heads are replaced by adipose cells, but the middle and top of the lateral heads are healthy muscle cells. No abnormalities or pathologies were observed in the nerves or arteries.ConclusionBoth medial heads of the gastrocnemius muscle appear to have become adipose tissue. While no underlying cause was identified it appears the pathology was spreading to the medial head.
Inflammatory phenotype and T‐cell mitochondrial reactive oxygen species in young adults with major depressive disorder
Authors: Grotle Ann‐Katrin , Darling Ashley M. , Saunders Erika F. , Fadel Paul J. , Greaney Jody L. , Trott Daniel W. 
Published: May 2022

BackgroundMajor depressive disorder (MDD) is prevalent in young adults and projected to rise further with the COVID pandemic. Notably, MDD is associated with an increased risk of cardiovascular disease (CVD) and mortality. Although the mechanisms are unclear, evidence supports chronic systemic inflammation as a major contributor to CVD risk in older adults with MDD. However, whether an inflammatory phenotype is present in young adults with MDD prior to the onset of CVD and other comorbidities is equivocal. Recent work has implicated the sympathetic nervous system and specifically norepinephrine as a potent driver of inflammation via stimulating an increase in T‐cell mitochondrial reactive oxygen species (T‐cell mitoROS). Given previous findings of elevated plasma norepinephrine and inflammation in MDD, the purpose of this study was to examine the potential for a T‐cell mitoROS pro‐inflammatory pathway in young otherwise healthy adults with MDD. We hypothesized that young adults with MDD but otherwise free of clinical disease would have augmented circulating pro‐inflammatory cytokines and T‐cell mitoROS compared to young healthy adults without MDD (HA).MethodsAll participants underwent a structured diagnostic neuropsychiatric interview to determine the presence or absence of MDD based on the DSM‐5 diagnostic criteria for MDD. Systemic pro and anti‐inflammatory cytokines and T‐cell mitoROS were measured in 14 healthy young adults with MDD of mild‐to‐moderate severity (non‐medicated; age: 23±2 years) and 11 HA (age: 22±1 years). Serum cytokines were detected using LEGENDplex Human Essential Immune Response panel and assessed by flow cytometry. Seven serum cytokines related to the norepinephrine‐T‐cell mitoROS‐inflammation pathway were measured. Immune cells were isolated from whole blood by red blood cell lysis. T‐cell mitoROS was measured by labeling with antibodies against CD3+ (total T cells) and mitoROS targeted fluorogenic dye (MitoSoxRED) and assessed by flow cytometry. All data are expressed as mean ± SE.ResultsFor pro‐inflammatory cytokines, there were no differences between MDD and HA for IL‐6 (41±18 pg/mL; 92± 66 pg/mL, P=0.66), IL‐17A (7±3 pg/mL; 17±8 pg/mL, P=0.13), TNF‐α (19±9 pg/mL; 36±18 pg/mL, P=0.20), IFNγ (120±70 pg/mL; 201±137 pg/mL, P=0.34) or IL‐2 (5±2 pg/mL; 13±6 pg/mL, P=0.25), respectively. Likewise, no differences were found in anti‐inflammatory cytokines between MDD and HA for IL‐10 (18±9 pg/mL; 78±57 pg/mL, P=0.19) or IL‐4 (33±15 pg/mL; 95±46 pg/mL, P=0.13), respectively. In contrast, T‐cell mitoROS was significantly elevated in MDD (34402±13353 a.u) compared to HA (6258±2942 a.u, P<0.05).ConclusionThese preliminary findings suggest T‐cell mitoROS but not circulating cytokine profiles are different between young adults with and without MDD. Thus, an elevated T‐cell mitoROS may represent an early marker of immune system alterations and inflammation in MDD.
Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis
Authors: Katzen Jeremy , Rodriguez Luis , Tomer Yaniv , Babu Apoorva , Zhao Ming , Murthy Aditi , Morley Michael , Mulugeta Surafel , Pelura Timothy , Rosen Glenn , Beers Michaell F. 
Published: May 2022

Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial “transitional state” enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown.We previously reported the in vivo modeling of PF utilizing inducible, knock‐in expression of a clinical Surfactant Protein C (SP‐C) mutation in alveolar type 2 cells (AEC2). Expression of the SP‐C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury.Using two clinical SP‐C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis.We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro‐fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.
CRISPR‐ethics in the Undergraduate Classroom using CCR5 Gene
Authors: Santisteban Maria S. , Applewhite Leanne , Natvig Lydia , Challa Anil K. 
Published: May 2022

Biology education has undergone revolutionary changes in the last two decades. Research on how students learn, personal experiences, and the ultimate recommendation of the Vision and Change report that “Students should have opportunities to participate in authentic research experiences”has moved instructors to adopt active learning strategies that emphasize concepts and skills over details, such as CUREs, where students learn science by doing science.Active learning should engage students to allow them to develop core competencies. However, even a well‐designed CURE may not fully engage students if they don’t see the relevance. CRISPR‐Cas9 makes an excellent tool to bring authentic research experience in the molecular biology laboratory because is contemporary, the concept is attractive to students, and provides a common set of technologies to teach molecular biology. Multiple iterations are possible by changing the research questions being addressed.In 2018, after the inaugural CRISPR‐Cas workshop offered in advance at the ABLE meeting, this framework was adopted to teach a summer Molecular Biology course at UNC Pembroke and the research question has been modified every year. This past summer students at UNC Pembroke and the University of Alabama at Birmingham worked on a project to produce various knockouts the human CCR5 gene in vitro, in an attempt to mirror the infamous experiment of the “Chinese twins” whose genomes were edited to be HIV resistant. The CRISPR‐Cas9 context was effective at engaging students and provided opportunities for discussions around ethics. Students in the two institutions connected twice towards the end of the course, once to reflect on their gains and shared experiences, and another time to discuss ethical implications. As part of the assessment, regarding the ethics, students created two products: 1‐ a reflection on the potential broader social and ethical implications of CRISPR‐editing, and their personal views on the subject before and after taking the course; 2‐ a letter to their congressperson where they explain the CRISPR‐Cas9 technology and the bioethical dilemma associate with its use for genome editing and describe and support an action plan that they want the congressperson to take.In addition to becoming familiar with the CRISPR‐Cas9 technology and potential applications, the students learned about the central dogma and mastered many techniques of molecular biology (primer design, PCR, in vitro transcription, nuclease digestions, and polyacrylamide and agarose electrophoresis). They were introduced to several web platforms, databases, and computer programs, such as Ensembl, Benchling, CRISPRscan, SnapGene and used electronic notebooks.Beyond the practical and conceptual learning, the approach proved valuable in engaging students through the ethics discussions and by feeling part of a larger effort, which should nurture their science identity.