bluuebunny/crossref_metadata_2025 huggingface dataset:
PDB‐101: Molecular Explorations through Biology and Medicine
Authors: Zardecki Christine , Goodsell David S. , Dutta Shuchismita , Voigt Maria , Burley Stephen K.
Published: May 2022

PDB‐101 is an online portal for teachers, students, and the general public to promote exploration of the structural biology of proteins and nucleic acids (pdb101.rcsb.org). Learning about the diverse shapes and functions of these biological macromolecules helps to understand all aspects of biomedicine and agriculture, from protein synthesis to health and disease to biological energy.

Why PDB‐101? Researchers around the world are studying these molecules at the atomic level. These 3D structures are freely available at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. This website builds introductory materials to help beginners get started in the basics of biomolecular structure and function (“101”, as in an entry level course) as well as resources for extended learning.

Since 2011, PDB‐101 has been developed by the RCSB PDB, a global resource for the advancement of research and education in biology and medicine. Along with our Worldwide PDB collaborators, RCSB PDB curates, annotates, and makes publicly available the PDB data deposited by scientists around the globe. The RCSB PDB then provides a window to these data through a rich online resource with powerful searching, reporting, and visualization tools for researchers. This information is then streamlined for students and teachers at PDB‐101.

Features include the ongoing Molecule of the Month series, educational materials such as paper models, posters, molecular animations, educational curricula and more. The section “Guide to Understanding PDB Data” is a primer for detailed PDB‐specific information: PDB Data, Visualizing Structures, Reading Coordinate Files, scientific methods for structure determination, and more.

PDB‐101 also runs annual Video Challenges for high school students. Participants create short videos that tell molecular stories that connect structural biology and medicine. Previous topics have included HIV/AIDS, diabetes, and antimicrobial resistance. The 2022 challenge will focus on Molecular Mechanisms of Cancer.

PDB‐101 activities are evaluated using user surveys, feedback from in‐person activities, and website analytics. In 2020, PDB‐101 hosted >850,000 users and >2.6 million page views.
Inhibiting Actin Polymerization Increases Tension Cost in Porcine Airway Smooth Muscle
Authors: Jensen Cole W. , Han Young Soo , Sieck Gary C.
Published: May 2022

The polymerization of globular to filamentous actin in smooth muscle occurs upon activation and is associated with cytoskeletal remodeling. Remodeling of the cortical cytoskeleton in this manner allows for tethering of contractile proteins and force transmission to the extracellular matrix. Muscle contraction involves ATP hydrolysis and cross‐bridge cycling, which are both sensitive to internal load. During isometric activation of smooth muscle, ATP hydrolysis rate peaks initially but decreases with time, reaching a steady‐state, while maintaining maximum force. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle contraction. We hypothesized that the time‐dependent decrease in tension cost and ATP hydrolysis are associated with actin polymerization and a subsequent increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, ATP hydrolysis rate and isometric force were measured simultaneously after activation with maximum Ca2+ (pCa 4.0). Two strips were tested from each animal. One strip was treated with Cytochalasin‐D (Cyto‐D; 1 μM for 30 min), an inhibitor of actin polymerization, and one was an untreated, time‐matched control. We found that peak and steady‐state isometric force decreased by ~40%, and peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively, following Cyto‐D treatment. Thus, tension cost increased by ~100% and ~60% at peak and steady‐state, respectively. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate relates to an increase in internal load on cross‐bridges via actin polymerization. Inhibition of actin polymerization induced by Cyto‐D in smooth muscle impairs contractile protein tethering and therefore reduces internal loading, resulting in increases in tension cost, ATP hydrolysis rate and faster cross‐bridge cycling.
Meckel’s Cartilage and Mandibles: Effects of the Fgfr2 C342Y mutation on development of the lower jaw in a Crouzon syndrome mouse model
Authors: Romano Natalie A. , Durham Emily , Kawasaki Kazuhiko , Wu Meng , Kawasaki Mizuho , Pitirri M. K. , Motch Perrine Susan M. , Richtsmier Joan A.
Published: May 2022

The Fgfr2cC342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in fibroblast growth factor receptor 2 (FGFR2), equivalent to the FGFR2 mutation commonly associated with Crouzon syndrome. Crouzon syndrome is an autosomal dominant condition characterized by craniofacial anomalies. The FGFR2 C342Y mutation results in constitutive activation of the receptor and is associated with up‐regulation of osteogenic differentiation. Although premature closure of the coronal suture is the focus of most studies of Crouzon syndrome, cartilage elements of the skull are also known to be affected by this mutation. Fgfr2cC342Y/+ Crouzon syndrome mice commonly exhibit malocclusion of the jaws, most likely due to abnormal craniofacial form. The current paradigm of mandibular development assumes Meckel’s cartilage (MC) provides a model for mandibular development. Malformation of the mandible can precipitate a plethora of complications including disrupting development of the upper jaw and the palate, impediment of the airway, as well as altering the functional occlusion necessary for proper mastication. Here we investigate the relationship of Meckel’s cartilage to mandible development. Using Fgfr2cC342Y/+ Crouzon syndrome mice and their unaffected littermates (Fgfr2c+/+ mice) at embryonic days 15.5 and 17.5 we explore the form of Meckel’s cartilage and the developing mandible as Meckel’s cartilage begins to degrade. A histological assessment of the chondrocytes within Meckel’s cartilage reveals few differences in cell size between genotypes at either timepoint, however there are more chondrocytes present in the most anterior aspects of the cartilage in Fgfr2cC342Y/+ mice at E15.5. No differences between genotypes were found in osteoblast (ALP) or osteoclast (TRAP) activity of the mandibles at E15.5 but osteoclast activity was greater in unaffected Fgfr2c+/+ mice at E17.5. These data reveal that further investigation of Crouzon syndrome mice can uncover differential effects of the C342Y mutation in FGFR2 in cells other than those surrounding the coronal suture.
Natural Compounds as a Promising Therapeutic Agent for SPOP Downregulated Breast Cancer
Authors: Araujo Rincon Mariana , Burleson Marieke
Published: May 2022

Breast cancer is the second most deadly type of cancer for women in America. Due to this, there is a pressing need to investigate and develop a better treatment plan for those afflicted by it. There are numerous studies that show a downregulation of the E3 ubiquitin ligase Type POZ Protein (SPOP) for up to 70% of breast cancer patients. This demonstrates that, in breast cancer, SPOP is a vital tumor suppressor gene. In previous research projects, our lab has shown that SPOP targets GLI3 for ubiquitination and degradation which subsequently places a restraint on SHH signaling. Furtermore, we have shown that SPOP mutations lead to a upregulation of GLI3 and subsequent hyperactivated SHH signaling in prostate cancer. Since SPOP is downregulated in a large number of breast cancer patients, we hypothesize that SPOP downregulated breast cancer tumors would also display hyperactivated SHH signaling. Additionally, we hypothesize that SPOP downregulated breast cancer patients will benefit from therapeutics that specifically target the SHH signaling pathway. To characterize the effect of SPOP downregulation in breast cancer, we used a lentivirus with shRNA complementary to SPOP to generate a stable MCF‐7 SPOP knockdown cell line. Proliferation assays were utilized to first confirm that SPOP knockdown promotes enhanced cell proliferation. Next, quantitative PCR was used to demonstrate that SPOP knockdown leads to an upregulation of GLI3 target genes. To find a novel treatment strategy for SPOP downregulated breast cancer, a natural compound library was used. Through the screen and subsequent quantitative PCR analysis, we identified novel therapeutic natural compounds that specifically target SPOP downregulated MCF‐7 cells in a manner that involves disruption of GLI3‐dependent SHH signaling. Our findings thus give necessary insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.
Radiography Suggests a Fibrous Tubular Sleeve Distal to a Cartilaginous Anlage in a Limb with ‘Absent Fibula’ Represents a Traction
Authors: Krisch Maria , Levinsohn Mark , Hootnick David , Ogden Kent , Katzman Philip
Published: May 2022

Background

Dissections of congenitally shortened limbs displaying congenital fibular deficiency have revealed pathologic cartilaginous bodies and fragments along the course of the “absent” fibula consistent with remnants of a fibular anlage (Wagstaffe, 1875; Freund, 1936; Harmon & Fahey, 1937; Thompson et al., 1957). Bands distal to the anlagen have traditionally been reported as fibular calcaneal collateral ligaments.

Objective

This project reinterprets the composition of the posterolateral ankle band frequently found in patients with congenital shortened limb (CSL) with fibular deficiency based on advanced radiographic criteria.

Patient and Method

Bilateral thin slice computed tomography (TSCT) imaging was performed on the distal lower extremities of a 43‐year‐old female patient with unilateral congenital shortened limb with fibular “absence” for operative planning. The images were imported into virtual modeling system 3D Slicer and various tissues in the region were measured and compared for attenuation using the Hounsfield unit (HU) scale.

Results

The radiological study of the posterolateral ankle band in the presence of a limb with a diminutive fibular anlage exhibiting a normal medullary density revealed a tubular structure sharing attenuation readings with nearby adipose tissue at its core and fibrous tissue at its periphery.

Conclusion

The tubular structure of the posterolateral ankle band with an adipose core, surrounded by fibrous tissue in the presence of a retained primitive fibular anlage, supports the assertion that fibular absence in this congenitally shorted limb is a radiographic misnomer, since a dystrophic anlage persists in its place. Additionally, previously identified fibulocalcaneal structures (or bands) may actually represent a stretched periosteal sleeve caused by traction exerted on the anlage by the paired lower tibia.

These findings support the hypothesis that this “ligament” structure is the distalmost tubular remnant of an otherwise normally formed cartilaginous scaffold of the fibular anlage. The anlage has failed to ossify to form a normal fibular long bone structure, due to embryonic arterial dysgenesis and failed vascular invasion during the sixth‐seventh weeks of embryonic development.
Brain‐derived Neurotrophic Factor in Duchenne Muscular Dystrophy Myopathy
Authors: Kristensen Jeffrey , Gansereit Lauren , Nguyen Van Thuan , Brula Imamulhaq , Hill Braxton , Galindo Cristi L.
Published: May 2022

Brain‐derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB) are crucial for neuronal growth and survival, and loss of this signaling nexus invariably leads to neurodegeneration. In a small cohort of Duchenne muscular dystrophy (DMD) patients we found that BDNF blood levels positively correlated with better heart function, and carriers of the common BDNF single nucleotide polymorphism rs6265 (Val66‐Met) exhibited significantly worse skeletal muscle function and earlier loss of ambulation, compared to non‐carriers. We thus hypothesized that BDNF/TrkB signaling delays progression of myopathy in DMD. To test this hypothesis, we administered the TrkB agonist 7,8‐dihydroxyflavone (DHF) to mdx mice in their drinking water for 26 weeks, beginning at 8 weeks of age. Based on functional assessment using the hang test, DHF treatment preserved skeletal muscle function compared to vehicle‐treated controls. Trichrome staining of gastrocnemius and cardiac tissues also showed reduced levels of fibrosis, which correlated with BDNF expression levels. Using humanized BDNF polymorphic mice, which have the Val66‐Met mutation, and mice with a point mutation in the equivalent position of murine BDNF (Val68‐Met), we found that skeletal muscle strength was significantly reduced in mice with the Met allele, compared to Val/Val littermate controls. Although Val/Met and Met/Met mice weighed considerably more than control mice, they exhibited significantly increased activity, as measured by voluntary wheel running. Based on transmission electron microscopy, individual gastrocnemius muscle fibers of Met/Met mice were smaller and less organized compared to those of Val/Val. Finally, we crossed Val66‐Met mice with mdx mice to assess the effects of the BDNF polymorphism on the dystrophic background. As was found for DMD patients who harbor the BDNF rs6265 polymorphism, dystrophic mice with the Met allele exhibited worse skeletal muscle function compared to noncarriers. Considered together, our results indicate that BDNF plays a protective role in dystrophic muscle and might represent a novel therapeutic candidate for DMD myopathy.
High Sodium Intake Differentially Impacts Brachial Artery Flow Mediated Dilation When Evaluated with Reactive Versus Active Hyperemia
Authors: Decker Kevin , Weggen Jennifer , Hogwood Austin , Darling Ashley , Chiu Alex , Richardson Jacob , Garten Ryan
Published: May 2022

OBJECTIVE

This study sought to determine if a high sodium (HS) intake attenuates upper limb flow mediated dilation in response to reactive (RH‐FMD) and sustained (SS‐FMD) hyperemia, and the role of oxidative stress in this response. Our hypothesis is HS intake will similarly impair upper limb vascular function when assessed as both RH‐FMD and SS‐FMD, and that this dysfunction will be reversed following acute antioxidant consumption.

METHODS

Ten young, salt resistant participants (9 Males, 1 Female) consumed a 7‐day HS (6,900 mg/day) or a 7‐day low sodium (LS: 2,300 mg/day) diet in a randomized order. On the last day of the diet, brachial artery RH‐FMD (evaluated after 5 minutes of cuff occlusion) and SS‐FMD (handgrip exercise) were performed following consumption of placebo (PL) and repeated two hours after the consumption of antioxidants [AO; (1000 mg of vitamin C, 800 IU of vitamin E, and 400 mg of alpha lipoic acid]. Handgrip exercise (HG) was performed in 3‐minute workloads, increasing by 8 kg until failure, at a rate of one contraction per second. RH‐FMD responses were evaluated as percentage change from baseline and SS‐FMD was evaluated as brachial artery shear‐to‐dilation slopes assessed across all HG workloads achieved.

RESULTS

Between diets, sodium consumption and excretion were significantly greater in the HS condition (p < 0.05 for both), but mean arterial blood pressure was unchanged (LS 85 ± 5; HS 84 ± 7 mmHg; p = 0.5). RH‐FMD was significantly lower during HS compared to LS (p = 0.01), in the placebo condition but not during the AO condition (p = 0.7), with a significant interaction present (HS+PL: 5.9 ± 3.4; HS+AO: 8.2 ± 2.7; LS+PL: 8.8 ± 4.7; LS+AO: 7.0 ± 2.1 %; p = 0.01) due to a significant increase in the HS+AO condition. A comparison of shear‐dilation slopes during SS‐FMD revealed no differences between sodium interventions (p = 0.3), AO condition (p = 0.1), or any interaction effect (p = 0.7) [HS+PL: 0.0031 ± 0.0013; HS+AO: 0.0026 ± 0.0012; LS+PL: 0.0028 ± 0.0012; LS+AO: 0.0022 ± 0.0010; mm.seconds‐1.kg‐1].

CONCLUSION

Independent of changes in MAP, a HS intake impaired peripheral vascular function when assessed via RH‐FMD, which was restored with AO consumption, potentially implicating elevated oxidative stress as a contributor to this dysfunction. However, SS‐FMD, evaluated via HG exercise, was not altered by a HS intake or AO consumption. In conclusion, 7‐days of a HS intake appears to have disparate effects between the two peripheral vascular function techniques, implicating an inability of HS intake to impact exercise‐induced BA dilation.
Identification of gut bacterial factors linked to altered host susceptibility to acetaminophen‐induced liver injury
Authors: Yang Xiaotong , Cho Sungjoon , Lee Hyunwoo , Jeong Hyunyoung
Published: May 2022

Acetaminophen (APAP) overdose causes acute liver injury. We previously demonstrated that differential gut microbiota modulates the host susceptibility to APAP‐induced liver injury in mice and subsequently identified 16 metabolites associated with altered susceptibility to APAP hepatotoxicity. This study aims to examine whether these metabolites modulate APAP hepatotoxicity. In primary mouse hepatocytes cultured in high‐galactose medium, phenylpropionic acid (PPA) and 3‐(4‐hydroxyphenyl)‐propionic acid (HPPA) significantly decreased APAP‐induced cell necrosis whereas 5‐aminovaleric acid (5‐AV) worsens it. In mice, PPA (in drinking water at 4 % w/v) for four weeks and HPPA (intraperitoneal injection, 50 mg/kg 2 hours prior to APAP) significantly alleviated the APAP‐induced liver injury. PPA, HPPA and 5‐AV were rarely detected, if any, in the tissue (portal vein serum or liver) of germ‐free mice, suggesting that that gut microbial production is a major determinant of host exposure to these metabolites. The biosynthetic pathways for these metabolites have been reported in bacteria. Indeed, mouse cecal bacteria readily converted L‐phenylalanine, L‐tyrosine and L‐proline to PPA, HPPA and 5‐AV, respectively. Bacterial conversion of L‐Phe to PPA is known to be mediated by the phenylalanine reductive metabolism pathway (called “Fld pathway”) or phenylalanine ammonia lyase. Examination of the deuterium‐labeled metabolites (d6‐PPA and d7‐PPA; produced via Fld pathway and phenylalanine ammonia lyase, respectively) after incubating mouse cecal bacteria with deuterium‐labeled L‐Phe (d8‐Phe) revealed that Fld pathway is the main route of PPA production from L‐Phe in mouse cecum. Interestingly, previously reported bacterial genes in Fld pathway were not detected in the mouse cecum. Also, the in vitro rate of d6‐PPA production by the cecal bacteria did not correlate with the differential levels of basal PPA levels in mouse cecum contents. The bacterial genes encoding the Fld enzymes as well as factors modulating its activities are remained to be identified. Taken together, this study presents multiple gut bacterial factors that are involved in gut microbiota’s modulation on APAP‐induced hepatotoxicity. Our next step is to study the underlying mechanisms for altered susceptibility to APAP hepatotoxicity by these metabolites.
Loss of Transcription Factor 19 (Tcf19) Impacts Metabolic Response to Stress
Authors: Blumer Joseph T. , Han Jee Young , Ricciardi Keelin , Saghafi Samuel T. , Beric Anya , Yang Grace H. , Lodh Sukanya , Fontaine Danielle A. , Davis Dawn B.
Published: May 2022

Type 1 and Type 2 diabetes (TID and TIID) differ in etiology, but both have decreased functional pancreatic β‐cell mass. Genome‐wide association studies identified transcription factor 19 (Tcf19) as a potential causal gene for both TID and TIID. Tcf19 is expressed in both humans and rodents, most highly in the pancreatic islet and upregulated in mouse models of non‐diabetic obesity. We showed that TCF19 is necessary for β‐cell proliferation and survival in INS1 cells. Thus, we hypothesized that TCF19 regulates β‐cell mass developmentally and in adaptive response to stress. A germline whole‐body knockout (wbTcf19KO) of Tcf19 mouse model was generated. wbTcf19KO and control (C57BL6/N) mice were fed a chow diet. Lean wbTcf19KO are metabolically similar to controls other than significantly higher body weights. Markers of proliferation (Ki67) and β‐cell identity (Pdx1, Nkx6.1, Nkx2.2) were significantly decreased while markers of pro‐apoptosis (Chop) and DNA damage response (Bak, Gadd45a, Dtx3l) were significantly increased in islets from wbTcf19KO mice. DNA damage is significantly elevated in wbTcf19KO islets as measured by y‐H2AX Western blot. Islet size distribution is significantly altered in wbTcf19KO islets, skewed by many very small islets. wbTcf19KO and control male mice were challenged with high fat diet (HFD; 1‐week & 10‐week) feeding. These mice failed to appropriately upregulate proliferation markers (Ki67, CyclinD2) in islets after 1‐week of HFD. Glucose intolerance and elevated fasting glucose develop in these mice after 10‐weeks of HFD. Female mice, less susceptible to diet induced hyperglycemia, were stressed with long‐term high fat high sucrose diet (HFHS) feeding to induce insulin resistance. After 8‐weeks of HFHS feeding wbTcf19KO females became significantly more glucose intolerant than control HFHS fed female mice. Ongoing studies will examine the impact of Tcf19 knockout on β‐cell mass during pregnancy in these HFHS‐fed females. Overall, loss of Tcf19 reveals changes in proliferation and DNA damage, which may be critical in stress‐induced β‐cell mass regulation.
Isoform‐selective PI3‐kinase Inhibition Confers Partial Resilience to Cocaine Cessation‐induced Anxiety‐like Behavior
Authors: Barbee Britton R. , Gourley Shannon L.
Published: May 2022

Phosphoinositide 3‐kinase (PI3K) is a multi‐subunit signaling complex that phosphorylates phosphoinositides, membrane‐embedded second messengers that are critical for synaptic and structural plasticity of neurons. Cocaine potentiates PI3K‐Akt‐mTOR cascade activity, and this activation persists beyond the period of drug exposure. The PI3K p110β isoform is neuronally enriched and able to control PI3K signal propagation, allowing for manipulation of PI3K activity in a more targeted manner than broad‐spectrum PI3K inhibition. Cessation of cocaine use triggers anxiety‐like behavior in humans and rodent models, and anxiety can be a causal factor in relapse. Here, we used viral‐mediated gene silencing to reduce expression of p110β in the dorsomedial prefrontal cortex (dmPFC). Isoform‐selective PI3K inhibition mitigated anxiety‐like behavior triggered by acute cocaine. Interestingly, however, a history of repeated cocaine exposure occluded this resilience, presenting an opportunity to compare immediate‐early gene expression between cocaine‐vulnerable and cocaine‐resilient mice. We examined 22 brain regions and found that resilient mice – those displaying less anxiety‐like behavior – displayed lower immediate‐early gene expression in the claustrum and lateral hypothalamus. We next found that chemogenetic stimulation of the claustrum induced anxiety‐like behavior. Future studies will determine whether suppressing p110β in the dmPFC combats anxiety‐like behavior via connections with the claustrum. Our findings suggest that isoform‐selective PI3K inhibition mitigates cocaine cessation‐elicited anxiety‐like behavior, likely via coordinated brain regions and circuits.
Loss of Club Cell Creb Mitigates IL‐1B–mediated Muco‐obstructive Phenotypes in Male Murine Airways
Authors: Sponchiado Mariana , Bonilla Angelina L. , Mata Luz , Liao Yan‐Shin , Fagan Amy L. , Moncada Victor M. , Reznikov Leah R.
Published: May 2022

Goblet cell metaplasia and/or mucus hypersecretion are hallmark features of multiple lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and pulmonary fibrosis. Interleukin 1β (IL‐1B) is a proinflammatory mediator that increases expression of the major gel forming mucins in the airways, Muc5b and Muc5ac, in part through activation of the cAMP response element binding protein (Creb) transcription factor. IL‐1B also induces Spdef, a key regulator of goblet cell differentiation from airway club cells. Here we tested the hypothesis that elimination of murine club cell Creb mitigates muco‐obstructive phenotypes induced by IL‐1B. Mice with floxed Creb1 were bred to Scgb1atm1(cre/ERT)Blh to generate mice with conditional loss of Creb1 from club cells. Wild‐type and transgenic male mice (7‐9/group) littermates received intranasal IL‐1B (0.5 ng/inhalation) or saline (control) for four consecutive days, with tamoxifen administration occurring on days 1 and 3 to induce Cre recombinase expression. Periodic acid–Schiff staining in lung samples showed that IL‐1B increased the number of goblet cells in central airways compared to saline controls. This effect was blunted by conditional loss of Creb1. Similarly, flexiVent measurements demonstrated that IL‐1B increased airway resistance (R), Newtonian resistance (Rn), tissue elastance (ERS), tissue damping (G) and tissue elasticity (H). Loss of club cell Creb1 mitigated all effects of IL‐1B on airway mechanics. Additionally, though the percentage of granulocytes in the bronchoalveolar lung lavage fluid was not increased by IL‐1B, loss of club cell Creb1 decreased the percentage of granulocytes in the bronchoalveolar lung lavage fluid independent of treatment (i.e., saline or IL‐1B). These findings suggest that the muco‐obstructive effects of IL‐1B are dependent upon club cell Creb1 and provide novel insight for therapeutic strategies.
Alcohol and L‐3‐hydroxyacyl‐CoA dehydrogenases activities of human Aβ‐binding alcohol dehydrogenase and type 10 17β‐hydroxysteroid dehydrogenase
Authors: He Xue‐Ying , Dobkin Carl , Brown W. Ted , Yang Song‐Yu
Published: May 2022

Type 10 17β‐hydroxysteroid dehydrogenase (17β‐HSD10) is a mitochondrial homo‐tetrameric protein necessary for brain cognitive function. It was first isolated as a human brain short‐chain 3‐hydroxyacyl‐CoA dehydrogenase (SCHAD), of which the primary structure is almost identical with that of endoplasmic reticulum associated Aβ binding protein (ERAB). The finding of generalized alcohol dehydrogenase activities in ERAB prompted its renaming as the Aβ‐peptide binding alcohol dehydrogenase (ABAD). Both reflect some dubious features of 17β‐HSD10 and recognized as alternative symbols of 17β‐HSD10, even if they were just a subunit of 17β‐HSD10 only. 17β‐HSD10 was encoded by HSD17B10gene whose mutations resulted in developmental disabilities such as infantile neurodegeneration. A 6xHis‐tag adding at the N‐terminus of 17β‐HSD10 would not substantially affect its catalytic function. Desired 6xHis‐HSD10 mutants can be readily made to study how a mutation not only interfering the neurosteroid metabolism but also the metabolism of acyl thioesters. Results of this study indicated that 17β‐HSD10 was not involved in the ketone body metabolism. Furthermore, it is impossible for anyone to reproduce the published generalizedalcohol dehydrogenase data. The clarification of the confused ABAD story may invigorate this research field, and open new approaches to investigate HSD17B10 gene‐related disorders including developmental disabilities, Parkinson disease, Alzheimer disease and some cancers.
The Dissection Experience of Orthopedic Resident: A Comparison between Soft‐Preservation and Formalin‐Fixation Embalming Techniques
Authors: Arce Jazmin E. , Contreras Erik , Vaghani Parth , Goyal Kanu , Balta Joy Y.
Published: May 2022

Objective

In a recent study, it was found that residents will feel more confidence, independence and fluency in the operational procedures presented to them throughout their career if introduced to the learning first on a human donor (James et al., 2020). This study is a comparison of alcohol preserved to standard formalin preserved through regional‐based donor dissection completed by orthopedic residents. The results from this study will help inform orthopedic residencies to consider different preservation techniques of human donors in teaching their future students through dissection for better learning outcomes.

Methods

Orthopedic Residents filled out a dissection survey after they had dissected for each week over a course of six weeks. Residents were expected to perform at least one dissection on a formalin‐based donor and one on an alcohol persevered donor for accurate comparison in data collection. The survey used a likert scale format ranging from 1 to 5. The survey questions range from bone, tendon, muscle, ligament, nerve, vessel quality; ease of dissection; similarity to in vivo tissue, ease of finding specific structures, etc. Descriptive statistics were used to describe the differences in resident opinion/experience between the two types of donors.

Results

A total of 24 residents participated in the dissector survey over the six weeks. Residents compared ease of dissection, ease of identification, odor of chemical, skills acquired, joint range of motion, tissue elasticity, donor comparability to live patients, time spent and overall learning preferences. 88.3% of residents in the dissections stated that overall they favored dissecting from alcohol preserved donors. When residents were asked to evaluate the differences in the donor preservations, residents favored alcohol preservation as a better way to stimulate their learning to in vivo tissue.

Conclusions/Implications

Orthopedic Residents ultimately agree that the alcohol preserved donors are more effective when it comes to identifying the human body framework through dissection. The results suggest that orthopedic residency programs should take into consideration the preservation techniques when teaching their students anatomy from human donors.
Dopamine‐driven Increase in IL‐1β in Myeloid Cells is Mediated by Differential Dopamine Receptor Expression and Exacerbated by HIV
Authors: Matt Stephanie , Nolan Rachel , Manikandan Samyuktha , Agarwal Yash , Channer Breana , Canagarajah Joanna , Runner Kaitlyn , Nickoloff‐Bybel Emily , Gaskill Peter J.
Published: May 2022

The neurological complications of HIV infection, known as neuroHIV, remain prevalent even in individuals on antiretroviral therapy (ART). Although the mechanism(s) underlying neuroHIV remain undefined, current data suggest that neuroinflammation is central to the development of HIV neuropathogenesis. Neuroinflammation can be exacerbated by substance use disorders, which are highly comorbid with HIV infection and substantively worsen clinical outcomes. Despite distinct mechanisms of action, all substances of abuse increase central nervous system (CNS) dopamine, suggesting that dopamine is a common mechanism by which addictive drugs potentiate neuroHIV. Our published data show that dopamine increases the production of inflammatory cytokines such as IL‐1β in human microglia and macrophages, but the mechanisms by which dopamine drives inflammation are not clear. Studies indicate that D1‐like dopamine receptors (DRD1, DRD5) mediate inflammatory activity while D2‐like dopamine receptors (DRD2, DRD3, and DRD4) mediate anti‐inflammatory activity, and we have shown that human macrophages and microglia have higher expression of D1‐like receptors. Therefore, we hypothesize that dopamine‐mediated production of IL‐1β in CNS myeloid cells is regulated by the ratio of different dopamine receptor expression levels and can be exacerbated by HIV infection. Our data in primary macrophages indicate that DRD1 expression is necessary for dopamine‐mediated increases in IL‐1β, but that changes in DRD2 expression can alter the magnitude of the dopamine‐mediated increase in IL‐1β. We confirm this in human microglia cell lines, showing that dopamine only increases IL‐1β gene and protein expression in microglia with a high ratio of D1‐like receptors to D2‐like receptors. Further, antagonizing dopamine receptor expression with the pan‐dopamine receptor antagonist flupentixol or decreasing the D1‐like/D2‐like ratio by overexpressing DRD2 diminishes the dopamine‐mediated increase of IL‐1β. We also show that the effects of dopamine on IL‐1β are potentiated in the presence of HIV infection in both microglial cell lines and iPSC‐derived microglia. Ongoing studies are examining the role of epigenetic regulation in the effects of dopamine and HIV on inflammation in these myeloid systems.

Due to the prevalence of both substance use disorders and the increasing use of dopamine‐modulating therapeutics, a detailed understanding of dopamine‐mediated changes in inflammation will be critical to effectively tailor ART regimens to HIV‐infected individuals using these drugs.
The New Faculty Myth‐busters: Pharmacology Educators and Curators of Information
Authors: Hernandez Mark , MacGregor Gordon G. , Parker Robert W. , Helms Kristen
Published: May 2022

Introduction

As we move towards completion of a second year of remote medical pharmacology instruction due to the Covid‐19 global pandemic, osteopathic medical students are also dealing with changes in the USMLE Step‐1 and/or COMLEX Level‐1 with the new Pass/Fail System in 2022. Unlike previous generations, Gen Z are inundated with an abundance of digital information that is readily and freely available, including professional advice from external sources which are not part of the formal curriculum (unspecified social networks, handed down Anki cards, etc.). Misinformation and/or misconstrued or unqualified advice for rapidly mastering pharmacology material over time can lead to development of myths which can be found in comments provided within the student evaluation of faculty. Identifying trends and/or myths in the feedback provided by the students can serve as a meaningful tool for faculty self‐reflection and will also help faculty steer students away from common misinformation as well.

Methods

The aggregate data reported in Medical School Year Two Questionnaire All Schools Summary Reportpublished by the AAMC at the end of the second year of medical education (Years 2014‐2021) was used to develop a construct of trends in medical student usage of YouTube and/or Wikipedia as a source for medical information. Faculty involved in the instruction of pharmacology at the Alabama College of Osteopathic Medicine convened to review student feedback of pharmacology faculty collected between 2020 and 2021. Student feedback was examined to identify facts and myths, and these were further categorized to develop approaches to address these concerns in the future.

Results

The use of Wikipedia has continually declined and the reliance on YouTube has increased as a resource of medical knowledge. Although this was already happening before the pandemic, the shift to other unspecified resources for medical knowledge during remote learning is of concern. In our institution, the review of pharmacology specific feedback demonstrated common myths within the following categories: 1) A lack of consistency in instructor and resources used (formal and informal), 2) perceived misconceptions on what is relevant (basic science versus clinical), 3) expectations for learning only what is perceived to be high yield. Approaches to preventing misperceptions for the future were identified for each category.

Conclusions

Pharmacology educators are curators of information. Evidence‐based clinical pharmacology information is changing faster than it can be updated in texts and the sanctioned board preparation resources that our students utilize. In the same manner, misinformation can rapidly spread via online forums and/or from handed down outdated resources from past students. Therefore, when pharmacology educators re‐engage medical students in‐person in the classroom, they will also have to more diligently serve as myth busters to combat misinformation and misperceptions.
Bacteriophage Isolation, Characterization and Bioinformatic Genetic Analyses against Actinomycetes Gordonia rubripertincta and Nocardia asteroides
Authors: Stockert Jessica E. , Mitchell Jon C.
Published: May 2022

Two of the biggest issues in the healthcare system today are antibiotic resistant bacteria and biofilms. Antibiotic‐resistant bacterial strains are increasing, and current treatment options have become limited forcing medical facilities to search for alternatives. Recent studies utilizing phage therapy have provided promising results supporting the use of bacteriophages to treat bacterial infections. (Hatfull, 2021). A bacteriophage is a virus that only infects prokaryotic bacterial cells and therefore, potentially used to treat bacterial infections without risk of infecting the patient’s eukaryotic cells. This research focuses on the isolation and microbiological characterization of bacteriophages isolated from environmental samples against the Actinomycete host, Gordonia rubripertincta. Gordonia species are typically found in soil and aquatic habitats and have recently been identified as primary agents of infections in both humans and animals (Pope, et al., 2020). Due to these organisms’ ability to form biofilms, infections have been found following various surgical procedures and applications of catheters (Arenskötter, et al., 2004). Following a Gordonia‐enrichment protocol, standard viral lab techniques such as serial dilutions, pick‐a‐plaque, and spot‐plating were performed resulting in pure viral colonies. Genomic DNA was isolated and sequenced by the Pittsburgh Bacteriophage Institute. Genomes were annotated using Phamerator™, PECAAN™, and NCBI BLAST bioinformatics programs. Host‐range specificity studies and gene knock‐out experiments were conducted using phylogenetically‐related Actinomycetes to assist understanding of the molecular interactions between phage and host throughout the infection cycle. Several bacteriophages were identified which exhibit both lytic and temperate life cycles against known clinical, biofilm‐producing Actinomycete bacteria Nocardia asteroides and N. corallina.This research exemplifies the potential of using phage therapy to create an alternative treatment for antibiotic‐resistant bacteria and biofilms within the healthcare system.
Individualized Estrogen Dosing in Women Undergoing Premenopausal Bilateral Oophorectomy
Authors: Harvey Ronee E. , Baker Sarah E. , Kapoor Ekta
Published: May 2022

Early menopause (prior to age 46 years) due to bilateral oophorectomy results in an abrupt loss of estrogen and other ovarian hormones and an increased risk of cardiovascular disease (CVD). Administration of estrogen partially mitigates this increased CVD risk; however, optimal dosing strategies for lowering CVD risk have not been studied. Current standard care is to use a dose of estrogen higher than that used for vasomotor symptom treatment in women after natural menopause in order to approximate the physiologic estradiol levels in premenopausal women (standard care); however, estradiol levels are not generally measured. An individualized approach that titrates estradiol levels to premenopausal hormone levels (individualized dosing) may be better for CVD risk mitigation in this younger population.

The goal of this study was to compare the effect of estradiol dosing strategies (standard care vs. individualized dosing) on subclinical predictors of CVD in women with early menopause due to bilateral oophorectomy.

We studied a total of 18 women at baseline and 6 months following surgical and medical intervention: 6 women underwent bilateral oophorectomy and were treated with standard care (age 41±2 years), 7 women underwent bilateral oophorectomy followed by estrogen therapy dosage based on individualized dosing (39±2 years), and 5 women served as time controls (did not undergo bilateral oophorectomy and were studied during the early‐follicular phase of the menstrual cycle at both visits; 34±3 years). At each time point, we assessed parameters of subclinical CVD including resting aortic blood pressure, carotid‐femoral pulse wave velocity, and blood pressure changes during a handgrip test and a cold pressor test.

At baseline, serum estradiol levels were similar amongst groups (p>0.05). After 6 months, women who received individualized dosing of estrogen therapy had higher serum estradiol levels (179±23 pg/ml) than women who received standard care (53±25 pg/ml, p=0.01) and women in the control group (78±27 pg/ml, p=0.03). Systolic and diastolic aortic blood pressures did not change between baseline and 6 months, nor did they differ amongst groups at any time point (p>0.05 for all). There were no group or time differences in the change in mean blood pressure during the handgrip exercise or cold pressor test across 6 months (p>0.05 for all).

Over a 6‐month follow‐up, despite differences in serum estradiol levels following two different estradiol dosing strategies after bilateral oophorectomy, there were no group differences or detrimental changes in subclinical CVD parameters across time. It is yet to be seen if differences in these parameters will be apparent following a longer duration after bilateral oophorectomy.
Dual Pathways for B. anthracis Asparaginyl‐tRNA Formation
Authors: McElhoe Nicole , Sheppard Kelly
Published: May 2022

In Bacillus anthracis, there are two distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct route, asparaginyl‐tRNA synthetase (AsnRS) directly ligates Asn to tRNAAsn. The indirect pathway is a two‐step process where a non‐discriminating‐AspRS (ND‐AspRS) attaches aspartate (Asp) to tRNAAsn which is them amidated by GatCAB, an amidotransferase. In this pathway, Asn is ultimately synthesized on the tRNA using a complex called the transamidosome which is made up of the ND‐AspRS, tRNAAsn, and GatCAB. B. anthracis encodes two distinct AspRSs with one of bacterial origins and the other acquired through horizontal gene transfer from archaea. The archaeal AspRS serves as the non‐discriminating enzyme for tRNA‐dependent asparagine biosynthesis. We have successfully overproduced in E. coli and purified the enzymes for both pathways to further characterize the two routes under various physiologically relevant conditions. The research provides a better understanding as to why this pathogen acquired an archaeal aspartyl‐tRNA synthetase and encodes two distinct pathways for asparaginyl‐tRNA synthesis.
The Effect of Lipolysis Inhibitor Acipimox on Brown Adipose Tissue Bioenergetics and Uncoupling Protein Abundance in Severely Burned Rats
Authors: Rontoyanni Victoria G. , El Ayadi Amina , Prasai Anesh , Wang Ye , Bhattarai Nisha , Vardarli Emre , Porter Craig , Murton Andrew J.
Published: May 2022

Introduction

Uncoupling protein 1 (UCP1) activation in brown adipose tissue (BAT) contributes to the hypermetabolic stress response to burn trauma. Strategies that target BAT UCP1 could be beneficial in controlling burn‐induced hypermetabolism. Acipimox, an inhibitor of adipose tissue lipolysis, has been shown to be effective at blunting burn‐induced lipolysis and expression of UCP1 protein in rodent white adipose tissue. The impact of acipimox on BAT bioenergetics and uncoupling protein abundance is currently unknown. We hypothesized that acipimox would blunt burn‐induced UCP‐1 dependent respiration and UCP‐1 protein levels in BAT.

Methods

Sixty‐four male Sprague‐Dawley rats received a 60% TBSA scald burn (n=32) or sham procedure (n=32) and were subsequently randomized to receive either acipimox (n=16/group; 50 mg/kg/day i.p.) or vehicle (n=16/group) daily for 7d. BAT was harvested from the interscapular fat pad on day 7 after burn and mitochondrial respiratory function assessed by high‐resolution respirometry. GDP‐sensitive respiration was assayed as a direct measure of UCP1 function. UCP1 and adenine nucleotide translocator (ANT2) protein levels were determined by western blot.

Results

UCP1‐dependent and UCP1‐independent uncoupled respiration in BAT was reduced at 7d after severe burn, independent of treatment (injury effect, P<0.01). Post‐burn, UCP1‐dependent respiration accounted for 65% of total uncoupled respiration compared to 59% in sham rats (injury effect, P<0.05), independent of treatment. Acipimox appeared to exert a lowering effect on UCP1‐dependent respiration (treatment effect, P=0.07) but not on UCP1‐independent respiration. Neither burn nor treatment with acipimox had a significant effect on UCP1 protein levels. Protein levels of ANT2, a carrier protein involved in fatty acid‐induced uncoupling, were greater in response to acipimox (treatment effect, P<0.05).

Conclusion

BAT UCP1 function than UCP1 protein content is greater in severely burned rats. Acipimox appears to reduce UCP1‐dependent respiration, independent of UCP1 protein levels. The effect of acipimox on BAT ANT2 protein content may partly explain the absence of an effect on UCP1‐independent respiration, but this warrants further investigation.

The protocol was approved by the University of Texas Medical Branch IACUC.
VEGFR2 signals with VE‐cadherin and PECAM to facilitate leukocyte transendothelial migration
Authors: Sullivan David P. , Fu Tao A. , Weber Evan W. , Gonzalez Annette P. , Dalal Prarthana J. , Rutledge Nakisha S. , Muller William A.
Published: May 2022

The migration of leukocytes out of the blood and into tissue is a critical component of the inflammatory process. To gain access to the target tissue and exert their beneficial or deleterious effects, leukocytes undergo a complex series of adhesive interactions with endothelial cells, which line the vessel wall. In the putative committed step in this process, the leukocyte must find the junction between adjacent endothelial cells and crawl through. This process, commonly referred to as transendothelial migration (TEM), has long been known to require homophilic interaction between PECAM on leukocyte and the endothelial cells. Recently our group showed that the process also involves calcium signaling originating through the cation channel TRPC6.

We hypothesized that PECAM may be functioning in TEM using some of the interactions recently shown to be involved in its role in the mechanotransduction of fluid sheer stress. Perhaps engagement of leukocyte PECAM with endothelial cell PECAM at the cell border provides the sheer stress in this case. Here we report the unexpected role of a macromolecular PECAM complex that facilitates signaling to TRPC6 in TEM. Specifically, we show that endothelial cell PECAM binds to VEGFR2 and VE‐cadherin via the transmembrane domain of VE‐cadherin. Swapping the VE‐cadherin transmembrane domain with that of N‐cadherin abrogated TEM. Likewise, deletion of the cytoplasmic c‐terminal tail of PECAM also abolished this signaling cascade. Stimulation of this complex with antibodies against PECAM induces the phosphorylation of VEGFR2 on tyrosine Y1175. Mutation of that critical residue to phenylalanine (Y1175F) prevented VEGFR2 phosphorylation and blocked TEM. Furthermore we show that this complex signals specifically to PLC1γ (and not PLCβ3) leading to its phosphorylation. Blocking its function with the inhibitor U73122 also blocked TEM. Diacylglycerol liberated by PLCγ1 activates TRPC6.

We also confirmed the role of VEGFR2 in vivo using to separate models of inflammation. First, we examined the recruitment of neutrophils to inflamed skin using the croton oil dermatitis model. In control animals, neutrophils are robustly recruited to and migrate across post‐capillary venules. In VEGFR2 knockout (KO) mice, most neutrophils are found inside the vessel with a corresponding decrease in those found out in tissue. Similarly, live animal imaging (intravital microscopy) of the inflamed cremaster muscle showed no defect in neutrophil rolling or adhesion in the VEGFR2 KO animals, but a profound disruption in their ability to undergo TEM compared to control wild type mice.

Taken together, these data confirm the role of a VEGFR2/PECAM/VE‐cadherin mechanotransduction complex that signals through PLC1γ to facilitate leukocyte TEM upstream of the action of TRPC6. Uncovering this signaling pathway opens up several new targets for potential therapeutic intervention.