bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics and ROS emission in the Heart and Kidney Cortex and Outer Medulla
Authors: Tomar Namrata , Sadri Shima , Zhang Xiao , Kandel Sunil , Yang Chun , Audi Said , Cowley Allen W. , Dash Ranjan K.
Published: May 2022

Rationale

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) is determined by the availability and utilization of particular respiratory substrates. Although the heart and kidney represent the two major energy consuming organs in the body, the substrate dependency of their mitochondrial OxPhos has not been characterized for these two organs within the same species and same individual animal.

Method

The effects of different substrate combinations on the kinetics and efficiency of OxPhos were characterized in mitochondria isolated from the heart, renal cortex, and renal outer medulla (OM) of adult Sprague‐Dawley rats. The substrate combinations included pyruvate+malate (PM), glutamate+malate (GM), palmitoyl‐carnitine+malate (PCM), alpha‐ketoglutarate+malate (AM), and succinate±rotenone (Suc±Rot). Mitochondrial bioenergetics (O2 consumption and membrane potential) and H2O2 emission were studied using two protocols: addition of a fixed [ADP] and sequential additions of incremental [ADP] in the presence of different substrates to determine how these substrates influence mitochondrial functional markers in each of these tissues.

Results

Results show that the kinetics and efficiency of mitochondrial OxPhos are highly dependent on the substrates used and this dependency is tissue‐specific. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates used in comparison to kidney mitochondria. Renal cortex mitochondrial respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. It was also found that heart mitochondria do not synthesize ATP efficiently from ADP with Suc in the absence of Rot (blocker of complex I). This may be due to (i) accumulation of oxaloacetate (OAA) and/or (ii) occurrence of reverse electron transfer (RET). In contrast, cortex and OM mitochondria achieved the same respiratory rates with Suc±Rot suggesting that efficient conversion of ADP to ATP by kidney mitochondria can occur in the presence of Suc. Similar differences were observed in mitochondrial membrane potential. However, differences in H2O2 emission in the presence of Suc±Rot were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. The bioenergetics and H2O2 emission data observed with Suc±Rot indicate that OAA accumulation and RET play a more prominent regulatory role in heart mitochondria than in kidney mitochondria.

Conclusion

This study provided novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial bioenergetics and H2O2 emission in a tissue‐specific manner.
F1A‐CreERT2 Mice: Targeting and Tracing Cardiomyocytes with Fgf1 Expression in Adult Mouse
Authors: Chiu Ing‐Ming , Chung Yu‐Fen , Chen Mei‐Shu , Wang Chi‐Kuang , Jiang Si‐Tse , Hsu Yi‐Chao
Published: May 2022

Fibroblast growth factor 1 (FGF1) regulates many biological and homeostatic processes, including obesity and metabolic syndrome. In mice, Fgf1 gene contains at least three upstream promoters and their respective adjacent untranslated exons that spliced to the first protein coding exon mutually exclusively, giving rise to different Fgf1 mRNA variants. Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line Fgf1A promoter (F1A)‐driven inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A‐CreERT2 mice, similar to that of Fgf1A mRNA. The F1A‐CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ‐positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2‐mediated recombination in the tissues is monitored through LacZ‐positive signals, indicating the in situ localization of F1A‐positive cells. Consistently, these F1A‐positive cells with RFP‐positive signals or LacZ‐positive blue signals were co‐localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte‐specific activation of Fgf1A promoter. Our data suggested that the F1A‐CreERT2 mouse line could be used for time‐dependent and lineage tracing of Fgf1A‐expressing cells in vivo. F1A‐CreERT2 line is a valuable tool for in vivo lineage tracing of Fgf1A‐expressing cells during heart development and will facilitate the elucidation of the physiological and pathological role of Fgf1A in heart failure and metabolic syndrome.
Characterizing Novel Kinases as an Authentic Research Experience in the Undergraduate Biochemistry Lab
Authors: Hall Bonnie L. , Jordan Sarah
Published: May 2022

The protein databank (PDB) contains 183,000 structures, with approximately 4200 of those currently classified as having “unknown function”. Based on structures with known function(s) in the PDB, about 2500 of those proteins are expected to be enzymes. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) consortium has developed a series of modules that guide undergraduate students through in silicoprediction of the function of proteins of “unknown” function, along with modules for in vitro confirmation of these predictions. We used these modules as part of a kinase‐focused course‐based undergraduate research experience (CURE). Kinases were selected because of their abundance in humans (over 500 members of the kinase superfamily) and their involvement in almost every aspect of cellular function. Multiple putative kinases were characterized by undergraduates, using the in silicoBASIL modules (which utilize BLASTp, Pfam, SPRITE and Dali). Specific potential substrates were then identified using SwissDock and/or AutoDock Vina (via PyRx). Students were able to successfully overexpress the putative kinases and subsequently purify them using affinity chromatography. Coupled in vitro kinase assays were performed first using the well‐characterized Abl kinase and an appropriate peptide substrate. Students then adapted the coupled kinase assay to evaluate the potential substrates they had identified. Students reported that having an integrated laboratory experience, with the ability to make their own decisions based on their in silicodata, gave them a strong sense of ownership of their projects. Additionally, the techniques they learned were all used to support their hypotheses, which helped them understand the details of why and how each method was performed. Overall, they described it as a positive experience and one of their more interesting laboratory courses.
Hyperglycemia and Obesity Play a Different Role in the Pathogenesis of Colitis in Diabetic Mice
Authors: Bu Heng‐Fu , Subramanian Saravanan , Geng Hua , Wang Xiao , Du Chao , Tan Xiao‐Di
Published: May 2022

Obesity, diabetes, and related manifestations have a large impact on global health. Evidence shows that these pathological disorders are highly associated with gastrointestinal (GI) diseases such as colitis. However, the impact of diabetes‐related hyperglycemia and obesity in the development of colitis is uncertain. In this study, we aimed to study the role of hyperglycemia with or without obesity condition in the pathogenesis of colitis in diabetic mice. For this purpose, we designed two mouse models of diabetes in male mice (9 weeks old). In the first model, B6 db/db mice with diabetic and obese phenotype or wild‐type B6 mice were subjected to dextran sulfate sodium (DSS, 40 kDa) treatment in drinking water for one week to induce colitis. In another model, we treated wild‐type B6 mice with streptozotocin (STZ, 175 mg/kg) or saline (control) to induce a hyperglycemia/diabetic phenotype without obesity. Eight weeks after induction of diabetes by STZ, mice were given DSS in their drinking water to examine whether prolonged hyperglycemia condition affects colitis development. The blood glucose, disease activity index (DAI, incorporating weight loss, stool consistency, and GI bleeding) of colitis and intestinal histological changes were monitored throughout the study. Interestingly, we found that DSS treated db/db mice with both diabetic and obese phenotype showed very mild symptoms (DAI 4.5 vs DAI 8.5, P<0.05), and significantly less body weight loss (1.87 % vs 19.04 %, P<0.01) compared to the DSS treated wild‐type B6 mice. In meanwhile, DSS treated STZ‐induced hyperglycemia mice without obese phenotype exhibited severe symptoms of colitis compared to DSS treated wild‐type B6 mice (DAI 2.4 vs DAI 0.64, P<0.01) beginning at day 3 of DSS treatment. They were found to have a delayed recovery from the intestinal injury and an increase in mortality rate after the withdrawal of DSS compared to control groups, suggesting that the long‐term hyperglycemia predisposes diabetic mice to gut inflammation. Collectively, prolonged hyperglycemia/diabetes enhances the susceptibility to intestinal injury. In contrast, the obese condition seems to have a different role or delaying the development of colitis in diabetic individuals. The underlying mechanisms by which diabetes and obesity affect the pathogenesis of colitis in diabetic mice are to be studied in the future.
Skeletal muscle alpha actin (ACTA1) acetylation negatively regulates muscle function in response to obesity
Authors: Lopez Adrian , Romanick Samantha , Stewart Travis , Madan Aditi , Baker Josh , Cammarato Anthony , Ferguson Bradley
Published: May 2022

Post‐translational modifications (PTMs) of proteins are an increasingly important area of study that contributes to our understanding for protein function in the control of health and disease. Acetylation, for example, has long been associated with histone modifications to control gene expression, yet emerging omics data demonstrates that acetylation occurs across the proteome. Our most recent work further shows global shifts in non‐histone protein acetylation within the left ventricle (LV) in response to diet‐induced obesity. Despite these advances in omics technologies, our understanding for how non‐histone protein acetylation regulates cardiac biology remains a ‘black box’. In this study, we examined the functional importance for skeletal muscle alpha actin (ACTA1) acetylation in the control of muscle function. For this, we used actin‐motility assays to examine the biophysical changes between acetylated ACTA1 and the other myofibrillar proteins involved in cardiac contraction and relaxation (e.g. myosin, tropomyosin). Here, we show that ACTA1 acetylation increased actin‐myosin binding, decreased actin sliding velocity and increased Ca2+ sensitivity. These data suggest that ACTA1 acetylation significantly alters muscle function. Of note, we previously published that lysine (K) residue 50 (K50) of ACTA1 was acetylated, similar to other proteomics reports. Interestingly, the positively charged K50 on ACTA1 sits juxtaposed to the negatively charged aspartic acid (D) residue 391 of the cardiac myosin binding protein (cMy‐BP). cMy‐BP is essential for actin‐myosin interactions, and this suggests an important role for ACTA1 K50 acetylation in the control of muscle contraction. Thus, we knocked out endogenous actin (Act88F) from the indirect flight muscles (IFMs) of D. melanogaster and inserted an Act88F K50Q mutant, which mimics acetylation specifically at this site. Previous reports have shown that IFMs behave similarly to cardiac muscle. Phenotypically, the K50Q flies behave similar to their wild‐type counterparts. However, current studies are underway to examine changes in flight as well as purify the K50Q actin for actin‐motility assays to examine biophysical changes in myofibrillar proteins. Combined, our data suggests that ACTA1 acetylation negatively impacts muscle function in response to obesity, current work will determine the implications for K50 actin acetylation in this phenotype.
FABP1: A Potential Biomarker for Nephropathy in Type II Diabetes
Authors: Prabhakar Sharma , Deshmukh Hemalata , Cooper Darbie
Published: May 2022

Aims of the study

Nephropathy is an important and frequent complication of diabetes mellitus. Diabetic nephropathy (DN) is also the leading cause of end stage renal disease in most parts of the world. Albuminuria is the hallmark of DN although recent observations suggest that non‐albuminuric nephropathy accounts for as much as 40% of all cases of DN. There is a need for additional and more accurate biomarkers for diagnosis and prognosis of DN. Fatty acid binding protein 1 (FABP1) is normally expressed in the liver but also in the kidney in human and animal DN models. However, it has not been detected in normal rat kidneys. We hypothesized that FABP1 is consistently elevated in the kidneys of experimental nephropathy of type II diabetes and could be a diagnostic biomarker and may have a potential pathogenic role.

Methods

Male and female ZSF1 rats were used as an experimental model for nephropathy in type II diabetes and CD rats (non‐diabetic rats) were used as controls. Animals were studied from 8th week and sacrificed at 40 weeks. Kidneys were harvested and tissue homogenates were used for total RNA extraction for Next Gen sequencing and protein extraction for western blots. Serum and urine samples were also collected at the start and end of the study to quantitate the renal function and excretion of markers.

Results

The ZSF1 rats spontaneously developed obesity, diabetes and hyperlipidemia as expected. Male rats developed more severe hyperglycemia, renal failure and proteinuria (data previously communicated) than females. Next Gen Sequencing of kidney tissue revealed amongst other findings a 1396‐fold increased expression of FABP1. Examination of kidney tissue protein homogenates for the expression of FABP1 showed increased expression especially in the male ZSF1 rats. Examination of urine samples for the presence of FABP1 by ELISA showed remarkably increased levels (12 to15‐fold in males and 6 to 7‐fold in females) increase compared to CD rats. (See figure 1)

Conclusions

Both male and female ZSF1 rats developed metabolic syndrome and nephropathy although the disease was much more severe in male rats. FABP1 was expressed more in male rat kidneys compared to female and control rat kidneys. Additionally, the urine excretion of FABP1 was increased in both male and female ZSF1 rats compared to control rats. These data suggest FABP1 may be a potential biomarker in DN. Additional studies are warranted to examine the mechanisms driving the robust renal FABP1 production/excretion and the possible pathogenic role and potential of FABP1 as a therapeutic target in DN.
Folding of the SARS‐CoV‐2 RNA Polymerase by the Cytosolic Chaperonin CCT
Authors: Smith Theresa , Wang Shuxin , Kwon Yujin , Reid Ann Aubrey , Robison Richard , Shen Peter , Willardson Barry
Published: May 2022

The ongoing Covid‐19 pandemic, caused by the highly transmissible SARS‐CoV‐2 virus, constitutes the worst global public health crisis of the past century. Although effective vaccines have been developed, administration has been slow, new variants continue to emerge, and there remains a lack of effective antivirals to treat severe cases. Thus, there remains a significant need to understand the mechanisms of SARS‐CoV‐2 infection and replication to identify new potential therapeutic targets. To this end, we investigated whether SARS‐CoV‐2 might depend on the host cell chaperone system, particularly the essential cytosolic chaperonin, CCT, to fold or assemble any of its proteins. CCT has previously been shown to be required for the replication of several other viruses including reoviruses and zika virus. We screened likely candidates among the SARS‐CoV‐2 proteins for co‐immunoprecipitation with CCT and identified an interaction with the RNA‐dependent RNA polymerase, Nsp12. To confirm Nsp12 was a substrate, we depleted cells of CCT, then transfected with Nsp12 and observed a 40% decrease in Nsp12 expression in CCT‐depleted cells compared to the control. This decrease is consistent with what we have observed for other known CCT substrates. Additionally, CCT‐depleted cells infected with live SARS‐CoV‐2 produced a 50% decrease in viral titer compared to controls, indicating that CCT is important for viral replication. A preliminary cryo‐EM structure of the Nsp12‐CCT complex shows a very large mass identifiable as Nsp12 inside of CCT that extends from the equatorial domains up and out through one of the two folding cavities. The structure stands to both reveal an important step in SARS‐CoV‐2 replication and to answer a long‐standing question regarding how CCT can accommodate very large substrates.
Characterization of Putative Transcriptional Regulator Orf90
Authors: Enquist Kathryn , Madrigal Vanessa , McLaughlin Krystle
Published: May 2022

The rapid emergence of antibiotic resistant bacteria is a pressing concern, threatening public health and food security worldwide. Antibiotic resistance commonly originates through conjugative plasmid transfer (CPT), where plasmids are transferred and replicated between bacteria. Although the overall mechanism of CPT is known, many plasmid‐encoded proteins essential for CPT remain unstudied. One such unstudied plasmid encoded protein is orf90, a putative transcriptional regulator located on the multiresistant plasmid pSK41 from Staphylococcus aureus. Methicillin resistant S. aureus(MRSA) is considered a serious threat by the Centers for Disease Control and Prevention (CDC), and how it acquires resistance plasmids is still an open area of study. In this investigation, we sought to gain valuable insight into the structure and character of orf90. We successfully expressed and purified two orf90 constructs, an untagged version (His‐orf90) and a maltose binding protein tagged version (His‐MBP‐orf90). We hypothesize that orf90 will bind the pSK41 origin of transfer and that one of our constructs will yield usable crystals for structure solution. Crystallography trials for His‐Orf90 did not yield macromolecular crystals, possibly due to the sample purity. Binding of His‐Orf90 to a 205 bp region of pSK41 containing the conserved origin of transfer (oriT) was confirmed via EMSA (Electrophoretic Mobility Shift Assay). Additionally, crystallization screens with His‐MBP‐orf90 identified three favorable conditions for crystallization. We have shown that orf90 binds to the pSK41 oriT and that His‐MBP‐orf90 is a promising crystallization construct. Characterizing pSK41 Orf90’s structure and function will further enhance our understanding of CPT, potentially yielding useful data for research aimed at combating antibiotic resistance.
Cystine‐stapled Helical Peptide from Withania somnifera Is Highly Stable and Cell‐permeable
Authors: Tang Fan , Loo Shining , Kam Antony , Tam James P.
Published: May 2022

The alpha‐helix is a common structure motif found in proteins but rare in peptides, particularly as a single helix. Peptide stapling is a synthetic strategy to constrain the helical conformation of a short peptide, generally through covalent linkage of their side chains. They are also known to be cell‐penetrating and capable of inhibiting intracellular protein‐protein interactions. However, naturally‐occurring stapled peptides are rare. Previously, we discovered lybatides which contain a single helix stapled by cystine. Moreover, lybatides form a new family of cysteine‐rich peptides with novel disulfide connectivity. Here, we report the identification and characterization of a new member of cystine‐stapled helical peptide, wisotide (wS1) from Withania somniferawhich is commonly known as Indian ginseng. Sequencing by mass spectrometry showed that wS1, with eight cysteine, contains 31 amino acids. Structural determination by NMR showed that wS1 displays the lybatide‐like disulfide connectivities to support an α‐helix. Similar to lybatides, the highly disulfide‐braced wS1 is resistant to degradation by high temperature, low pH, and proteolysis. Importantly, the anionic cystine‐stapled wS1 is cell‐penetrating. Database mining further revealed that >50 lybatide‐like sequences are found in plants. Taken together, wisotide wS1 and the cystine‐stapled peptides could represent useful leads of peptide biologics to probe intracellular protein‐protein interactions.
Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome
Authors: de Bovi Pontes Carolina , Jefferys Cody , Bedwan Muhamad , Ciesielska Elena J. , Ciesielski Grzegorz L.
Published: May 2022

Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post‐replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.
Intra‐ and interspecific structural variation in the archosaur lung: insights from 3D imaging and segmentation
Authors: Schachner Emma R. , Lawson Adam B. , Martinez Aracely , Echols M S. , Diaz Raul E. , Grand Pre Clinton A. , Hedrick Brandon P.
Published: May 2022

Innovations in three‐dimensional (3D) imaging and segmentation have facilitated unprecedented levels of anatomical investigation into the detailed structures of the respiratory system that are often difficult to study in situ. Recent hypotheses of homology between crocodilians and birds have facilitated quantitative comparative analyses of bronchial trees and in situ models reveal new complexities in the relationship between the respiratory and skeletal systems. Here we quantitatively compare the bronchial trees of two crocodilians, the American alligator (Alligator mississippiensis) and Cuvier’s dwarf caiman (Paleosuchus palpebrosus) with select birds, including the ostrich (Struthio camelus), the African gray parrot (Psittacus erithacus), and the red‐tailed hawk (Buteo jamaicensis). Notably, the relative distances from the carina to the secondary bronchi measured are conserved, indicating a possible ancestral or constrained trait. With respect to interspecific avian comparisons, we found grossly observable variation within a single taxon in air sac morphology (e.g., P. erithacus), as well as substantial differences between the individual taxa via segmented surface models – particularly in the expansions of the interclavicular sacs, the extent of the diverticula, and the size of the abdominal sacs. Furthermore, we found that specific sac contribution to postcranial pneumatization varies substantially across our dataset. Individual specimens imaged for this study also revealed multiple pathologies, including scoliosis, foreign objects inside the animals, and broken bones, which have been incorporated into the anatomical models for clinical surgical atlases that are under development. While these data are preliminary, they provide a framework for larger scale comparisons and hypotheses of the ancestral archosaurian pulmonary system.
Inhibition of Cardiac Glucose Transport Corrects Diabetic Cardiomyopathy Even Without Alleviation of Hyperglycemia
Authors: Mia Sobuj , Kyriazis Ioannis D. , Sidiropoulou Rafailia , Hill Daniel , Mantalaris Athanasios , Drosatos Konstantinos
Published: May 2022

Healthy hearts rely more on fatty acid (FA) rather than glucose utilization. It remains unclear whether diabetic cardiomyopathy (DbCM) is accounted for by glucotoxicity or lipotoxicity. Previously, we discovered that either insulin deficiency or insulin resistance causes FOXO1‐KLF5 activation in human and murine hearts, which drives cardiac lipotoxicity and oxidative stress. Now, we investigate how glucose activates cardiac KLF5 and causes DbCM. We hypothesized that higher cardiac glucose content in diabetes potentiates FOXO1‐KLF5 activation and causes glucolipotoxicity.

We mimicked Type‐1 diabetes (T1D) in C57BL/6 mice via 5 daily intraperitoneal injections of streptozotocin (STZ). In contrast to late T1D (12 Wks post‐STZ), cardiac KLF5 expression levels are not increased in the early T1D (4 Wks post‐STZ). However, mice developed cardiac dysfunction in early T1D. Seahorse analysis in adult cardiomyocytes isolated from mice with early T1D showed suppression of FA dependence in the expense of higher glucose dependence compared to non‐diabetic mice. To confirm whether hyperglycemia or higher cardiac glucose content accounts for cardiac dysfunction in early T1D, we applied anti‐hyperglycemia treatment (Dapagliflozin, DAPA, SGLT2 inhibitor) or GLUT1 inhibition (STF‐31). Either of the two treatments restored cardiac FA dependence and prevented both glucose preference and cardiac dysfunction in early T1D. GC‐MS analysis in hearts of mice with late T1D, which have shifted back to increased FA dependence, showed increased cardiac glucose content ‐presumably unused glucose‐ and subsequent increase of cardiac KLF5, as previously shown, which exacerbates cardiac dysfunction. New data revealed that GLUT1 levels were increased in late T1D compared to early T1D, as well as that this is driven by KLF5 activation. The lack of activation of KLF5 expression in early T1D, which reverses in late T1D, is mirrored by FOXO1 transcriptional activity as shown by expression of FOXO1 targets and lower FOXO1 acetylation in late T1D, which is controlled by Sirtuin‐1. Analysis of hearts from mice with late T1D and a human cardiomyocyte cell line (AC16) that was treated with high glucose indicated higher Sirtuin‐1 expression and Sirtuin‐1 binding on FOXO1. Accordingly, prevention of glucose transport to hearts of diabetic mice via treatment with either DAPA or STF‐31 for 12 Wks lowered cardiac expression of KLF5 and its targets and improved cardiac function.

Conclusively, DbCM begins in early T1D with lower mitochondrial FA utilization that is compensated by higher glucose utilization and is exacerbated in late T1D with activation of SIRT1‐FOXO1‐KLF5 axis that causes combined lipotoxicity and glucotoxicity. Inhibition of GLUT1 alleviates DbCM via prevention of both early glucose dependence and late KLF5 activation.
Liver Fatty Acid‐Binding Protein (LFABP) Ablation Drives Hyperplastic Expansion of Subcutaneous Adipose Tissue in Male Mice Fed High‐Fat Diet
Authors: Diolintzi Anastasia , Zhou YinXiu , Fried Susan K. , Polunas Marianne , Sidossis Labros S. , Storch Judith
Published: May 2022

Liver fatty acid‐binding protein (LFABP) functions both in intracellular lipid trafficking in liver and small intestine, and in systemic energy homeostasis regulation. We found that despite the marked obesity of high fat‐fed LFABP null (LFABP‐/‐) mice compared to their wild‐type (WT) counterparts, they display a ‘metabolically healthy obese’ (MHO) phenotype characterized by normoglycemia and normoinsulinemia, increased spontaneous physical activity, and a resistance to high‐fat diet (HFD)‐induced decline in exercise capacity. They also display increased muscle fatty acid oxidation suggesting that adipose tissue lipolysis is enhanced. To gain insight into the effects of Lfabp ablation on the quality of adipose tissue, we determined the mass and cellularity of the inguinal (iWAT) and epididymal (eWAT) white adipose tissues.

LFABP‐/‐ mice fed HFD for 12 weeks weighed 23.7% more (BW, g: 41.7 LFABP‐/‐ vs 33.7 WT, p=0.006, n=5) and their fat pads tended to be heavier (iWAT, g: 1.1 LFABP‐/‐ vs 0.8 WT, p=0.060, n=5; eWAT, g: 1.4 LFABP‐/‐ vs 1.8 WT, p=0.065, n=5). Intriguingly, as shown in Figure 1, adipocyte size of iWAT adipocytes is smaller than that of the WT mice (44.6pL LFABP‐/‐ vs 188.1pL WT per cell, p=0.009, n=5). Adipocyte number was substantially increased in the iWAT of LFABP‐/‐ (23.6 LFABP‐/‐ vs 4.6 WT x 106, p=0.001, n=5). In contrast, the eWAT adipocyte size in the LFABP‐/‐ is comparable to WT (210pL LFABP‐/‐ vs 268 pL WT per cell, p=0.293, n=5), and eWAT fat cell number was highly variable and trended higher in the LFABP‐/‐ mice (9.8 LFABP‐/‐ vs 4.7 WT x 106, p=0.059, n=5).

Because LFABP is not expressed in adipose tissue, our data suggest that its ablation promotes interorgan signaling that may limit hypertrophy and drive hyperplasia in expansion of potentially metabolically beneficial subcutaneous iWAT. More studies are warranted to elucidate the marked differences in cell size and the apparent depot‐dependent effects of LFABP‐/‐ deficiency, as well as the mechanisms by which it modulates adipocyte growth and function.
Do crocodilians have an air‐conditioning unit in their skulls? Experimental test of purported thermoregulatory function of the dorsal temporal fenestrae in the American alligator
Authors: Ramirez Breanna Alexis S. , Sandoval Sara , Sparks Jon , Elsey Ruth , Owerkowicz Tomasz
Published: May 2022

The classic view of dorsal temporal fenestrae (DTFs) in diapsids posits that they provide attachment sites for jaw adductor muscles. A recent hypothesis suggested that DTFs in archosaurs may play a role in selective brain thermoregulation, but did not provide supporting experimental data. We tested this hypothesis in two years‐old juveniles of the American alligator. We used chronically implanted thermocouples to monitor brain (Tb) and core (Tc) temperatures in constrained but unanaesthetised animals during acute heat exposure. Animals were first equilibrated to either 15°C (n=9, body mass range 200‐563 g) or 30°C (n=6, body mass range 173‐362 g), and then exposed to a radiant heat source (a 75W incandescent light bulb suspended 20cm above the animal). As experimental treatment, we used insulating covers (custom‐made of adhesive foam tape and aluminium foil) to obscure either the DTFs or a corresponding skull surface in subsequent trials. We expected that obscuring the DTF (as opposed to covering the skull roof) would result in Tb rising more slowly in animals at 15°C (i.e., interfering with selective brain warming), and would result in Tb rising faster in animals at 30°C (i.e., interfering with selective brain cooling). We found no statistically significant differences in the rate of brain warming between DTF‐covered vs. skull‐covered treatments in animals at either 15°C (0.541°C/min vs. 0.683°C/min) or 30°C (0.356°C/min vs. 0.358°C/min). Our results suggest that DTFs, at least in small (<1 kg) alligators, do not function as thermal windows for heat exchange with the environment to afford them selective brain temperature control. However, given that the DTF surface area scales with positive allometry on brain mass (Mb^1.25) in the American alligator, further experiments are needed to determine whether DTFs acquire a thermoregulatory function in larger crocodilians.
Modeling Surgical Management of Uterine Atony Induced Postpartum Hemorrhage using Cadaver Dissection
Authors: Richter Emily , Fogarthy Tannice , Ravikumaran Nair Rakesh
Published: May 2022

Introduction

There is a well‐documented disconnect that medical students experience when applying preclinical anatomical knowledge to clinical applications.1 According to the United States National Inpatient Sample, postpartum hemorrhage (PPH) carries an overall incidence of up to 3% of all U.S. deliveries; this study designates uterine atony as the most common etiology of PPH, causing at least 80% of PPH cases.2Thus, fourth year medical students preparing to enter into the specialty of obstetrics and gynecology should have in vivo exposure to the surgical management of uterine atony induced PPH prior to beginning residency. Modeling arterial ligation of the anterior division of the internal iliac artery using a cadaver model is a practical approach that most resembles an in vivo illustration of PPH surgical management while also maintaining a safe, controlled environment.

Objective

In efforts to provide fourth year medical students with exposure to PPH management, create a guide for ligation of the internal iliac artery’s anterior division using a cadaver model.

Material and Methods

Dissection was completed by two fourth year HWCOM medical students anticipating entry into the specialty of obstetrics and gynecology. After completion of anterior abdominal wall dissection and peritoneal entry, GI organ were retracted for full visualization of genitourinary structures. The following arteries were identified and skeletonized in the following fashion: common iliac artery, internal iliac artery, anterior division of the internal iliac artery, umbilical artery, obturator artery, and uterine artery. The anterior division of the internal iliac artery was identified and ligated 2 cm below the bifurcation of the common iliac artery to avoid unintentional ligation of the superior gluteal artery. A picture of the arterial ligation site was captured.

Results/Images

Proper arterial ligation site of the internal iliac artery’s anterior division can be visualized in the appendix.

Conclusion and Significance/Implication

Surgical management of PPH requires both anatomical knowledge of arterial and pelvic anatomy but also spatial familiarity of structures in vivo. Performing the surgical management of PPH on a cadaver model could provide fourth year medical students an opportunity to learn the appropriate sites and steps and surgical arterial ligation in a less pressured environment before entering residency. Future implications of this study could combine virtual learning tools, such as anatomy mobile applications, to further aid in medical education’s exposure to PPH management and associated anatomy.
GARLAQs vs. Multiple Choice Questions: Do First‐Year Medical Students Perform Similarly on their Anatomy Examinations?
Authors: Thom Mitchell L. , O'Neill Steven , Crofton Andrew , Wish‐Baratz Susanne
Published: May 2022

PURPOSE

Medical students at the Case Western Reserve University School of Medicine are assessed on their knowledge of anatomy using an essay exam that tests clinically relevant anatomy/radiology concepts (Gross Anatomy, Radiology, and Living Anatomy Questions, or GARLAQ). GARLAQs are uniquely designed to facilitate student collaboration, as such these essay questions are given to students months prior to examination. This format has raised concerns about student memorization and a lack of in‐depth understanding of GARLAQ answers. The purpose of the present study is threefold: 1) to examine whether students perform similarly on GARLAQs and Multiple Choice Question (MCQ) examinations, 2) to assess whether students perform similarly to the GARLAQs on MCQs throughout the hierarchy of Bloom’s Taxonomy, 3) to collect qualitative data on student opinions of both assessment formats.

METHODS

First‐year medical students will be assessed during the gastrointestinal unit of their clinical anatomy curriculum. Volunteers will be recruited from a class of 184 students. A crossover study design will be employed, wherein participants will be randomly placed into one of two cohorts: 1) first complete the required GARLAQ exam, second the MCQ exam, or 2) first complete the MCQ exam, second the required GARLAQ exam. A paired t‐test will determine if there is a difference in overall examination performance for each respective student. Each MCQ will be categorized using Bloom’s Taxonomy. A paired t‐test will compare each student’s mean scores by Bloom’s category to their GARLAQ performance. Participants will also complete a post‐test survey in which they will be asked to rate their opinions about contrasting exam modalities on a 5‐point Likert scale.

RESULTS

Data collection is currently ongoing and will be completed in February, 2022

CONCLUSION

Information gained through review of student assessment scores and responses can guide future curricular changes at our institution in a cycle of continuous improvement.
Enhanced antimycotic activity of nanoconjugates from fungal chitosan and Saussurea costus extract against resistant pathogenic Candida strains
Authors: Alshubaily Fawzia A.
Published: May 2022

Targeting the control of pathogenic Candida spp., especially the fungicides resistant strains from C albicans and C glabrata, nanoconjugates from the biopolymer (chitosan) and costus root extract (Saussurea costus) was synthesized and characterized. Chitosan was extracted from the grown mycelia of Aspergillus niger and characterized with high deacetylation degree of 91.2% and moderate molecular weight of 106.8 kDa. Synthesis of nanoconjugates from fungal chitosan/costus extract (NCt/CE) was conducted using ionic gelation technique; the resulted NCt/CE particles were characterized with mean diameter of 48 nm, positive zeta potentiality (+3.28 mV) and high stability. The infra‐red spectra of synthesized nanoconjugates indicated their strong biochemical cross‐linkage. The antimycotic activities, of the synthesized NCt, CE and their nanocomposite, were evaluated against standard and antibiotic‐resistant strains from C albicans and C. glabrata and revealed that the entire agents had notable antimycotic potentiality against all examined strains; the NCt/CE nanoconjugates had significantly stronger antimicrobial action. The scanning microscope imaging, of exposed resistant strains to NCt/CE, indicated their vigorous structural and morphological alterations and confirmed the antimycotic activity of the nanocomposite. NCt/CE nanoconjugates' synthesis could be exceedingly recommended as a natural, biodegradable and effectual antimycotic agent to control resistant pathogenic yeast strains.
Glucosidase Inhibitor Alleviates Inflammation and Fibrosis in Type‐1 Diabetic Kidney
Authors: Juin Subir Kumar , Pushpakumar Sathnur , Sen Utpal
Published: May 2022

Diabetic nephropathy (DN) is characterized by renal fibrosis leading to chronic kidney disease and end‐stage renal disease. A plethora of evidence suggests that chronic inflammation plays a crucial role in the development of renal fibrosis, i.e., the accumulation of extracellular matrix (ECM) proteins in the glomerular and tubulointerstitial region. Moreover, epithelial‐mesenchymal transition (EMT), increased macrophage infiltration, elevated pro‐inflammatory cytokines and pro‐fibrotic factors contribute to renal fibrosis. Previous studies have shown that excess ECM accumulation leads to fibrosis in type‐1 diabetic kidney. Nimbidiol is a diterpenoid derived from the medicinal plant ‘neem’ (Azadirachta indica), and is reported to have anti‐diabetic properties by inhibiting glucosidases. Further, ‘neem’ extracts are shown to regulate oxidative stress, inflammation and ECM proteins in different disease models. The aim of our study was to understand the regulatory role of the glucosidase inhibitor, Nimbidiol on renal inflammation and fibrosis and its underlying signaling mechanisms in type‐1 DN. Wild type C57BL6/J (WT) and type‐1 diabetic C57BL6/‐Ins2Akita/J (Akita) mice (12‐14 weeks) were treated without or with Nimbidiol (0.4 mg/kg/day) for eight weeks. In diabetic kidney, the expression of CD40, the M1 macrophage marker, was elevated along with pro‐inflammatory cytokines viz., TNFα, IL‐6, IL‐1β and pro‐fibrotic factors including MCP‐1, TGF‐β1, α‐SMA and collagen. Additionally, Akita mice exhibited downregulation of CD206, the M2 macrophage marker and anti‐inflammatory cytokine, IL‐10 and also E‐cadherin in the kidney. The changes were associated with excess collagen deposition in the glomerular and tubulointerstitial region of the diabetic kidney compared to that of WT mice. Furthermore, the expression of NF‐κB, p‐Smad2/3, p‐P38, p‐JNK and p‐ERK were elevated in the diabetic kidney compared to the WT mice. Nimbidiol reversed the above changes to alleviate renal inflammation and fibrosis in Akita mice. Together, our results suggest that Nimbidiol protects the diabetic kidney from macrophage‐mediated inflammation and fibrosis by inhibiting NF‐κB, TGF‐β1/Smad and MAPK signaling pathways.
The Polysaccharide of Ligustrum Lucidum Ait. Protected from Renal Fibrosis by Interfering with Angiotensin II Signaling
Authors: Zhang Jia‐li , Wang Na‐ni , Zhang Yan
Published: May 2022

Background

A number of preclinical advances have indicated the cellular and molecular mechanisms underlying renal fibrosis, such as the signaling of angiotensin II (Ang II), which is a bioactive peptide in renin‐angiotensin system. As one of kidney‐tonifying herbs, Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait. (Oleaceae) recorded as a tonic and dietary supplement, is a commonly prescribed component in traditional kidney‐tonifying herbal formula. This study aimed to clarify the polysaccharide component in FLL and investigate its potential role in preservation against renal fibrosis.

Methods

The crude polysaccharide of FLL was prepared and deproteinized by Sevag reagent. After lyophilization, the DEAE‐cellulose DE52 column and the Sephacryl S‐200 HR column were used for further isolation and purification of homogenous polysaccharide. The mice with unilateral ureteral obstruction (UUO) underwent daily intraperitoneal injection with FLL polysaccharide starting 7 days before UUO surgery until day 7 after surgery. The murine mesangial SV40‐MES13 cells were treated with vehicle, Ang II (10‐7 M) either with the absence and presence of angiotensin type I receptor blocker (ARB) (10‐6M), or with low dose (50 μg/mL, Poly‐L) and high dose (200 μg/mL, Poly‐H) of FLL polysaccharide.

Results

The purified homogenous polysaccharide (Poly) was explored from FLL and the carbohydrate content was 97.45%. A hypothetical structure for Poly was proposed considering the comprehensive data of methylation, monosaccharides, and NMR analyses. Treatment of UUO mice with Poly decreased kidney index and ameliorated glomerular sclerosis and interstitial fibrosis, as well as down‐regulated the expression of profibrogenic factor VEGF and matrix fibrotic protein fibronectin (FN). Furthermore, as the same effect to ARB, the pre‐incubation with Poly alleviated the Ang II‐induced up‐regulation of FN in mesangial cells by immunofluoresence staining. The protein expressions of FN and proinflammatory factors like MCP‐1 and Rantes were significantly lower in Poly‐treated cell group than those in Ang II treatment group.

Conclusion

The polysaccharide of Ligustrum lucidum Ait. was accurately explored with structure clarification. This polysaccharide component repressed angiotensin II‐induced fibrotic phenotype of renal mesangial cells and exerted beneficial effects on kidney of UUO mice.
Abnormal Relationship with the Styloglossus and Medial Pterygoid Muscles: A Description of Two Cases
Authors: Kim Pil‐Woo
Published: May 2022

Introduction

Lateral pharyngeal space is of great interest to dentists and surgeons. The pre‐styloid space is located in a portion of the lateral pharyngeal space. The pre‐styloid space has stylohyoid muscle, styloglossus muscle, stylopharyngeus muscle, and external carotid artery. Medial and lateral pterygoid muscles are located anterior to the pre‐styloid space. This study describes the relationship of the structures in the cases.

Hypothesis

The muscular attachment from the medial pterygoid muscle to the styloglossus muscle is unique. Morphological understanding with the external carotid artery of the cases will be helpful to prevent surgical mistakes.

Methods

The two cadavers were found to have a rare medial pterygoid and styloglossus during the dissection in 2020. It had been assessed in the adjacent structures.

Results

Both cases showed that the medial pterygoid muscle has a tendinous attachment from the inside to the styloglossus muscle. The first case’s tendon and muscle were 5mm wide, 3mm thick, and 10mm long and attached to 15mm proximal area of the styloglossus muscle (Fig. 1). The second case’s tendon was 5mm wide, 1mm thick, and 7mm long and attached to a 12mm proximal area of the styloglossus muscle (Fig. 2). Both styloglossus muscles were attached to the tip of the styloid process. Both styloid processes were more than 25mm long from the temporal bone (Fig. 2).

Both external carotid arteries passed through between stylohyoid and styloglossus muscles and ran behind the tendon and up to the head of the mandible (Fig. 1.C).

Conclusion

The medial pterygoid muscle attaches to the ramus of the mandible. The ramus has an accessory fascicle that attaches to the styloglossus muscle in the fetus. It may not entirely disappear after being born. The length of the styloid process may be a contributing factor in the development of eagle syndrome.

The styloglossus muscle plays an essential role in swallowing, mastication, and speech. The attachment involves the function of the motion of the mandible. The external carotid artery travels along with the accessory muscle‐tendon and needs to be considered during surgery.