bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensing Solvent in the Collapsed State of Glutamine‐Rich Domains
Authors: McGee Abigail Parker , Garelli Colton , Scott Adam , Ellis Jamie P.
Published: April 2020

Intrinsically disordered proteins (IDPs) challenge the widely accepted structure to function paradigm of protein behavior and are associated with important biological functions. These disordered domains are estimated to account for at least 30% of the proteome in eukaryotic genomes and are correlated with signal transduction and gene regulation, as well as certain disorders including some types of cancers, amyloidosis, heart disease, and neurodegenerative disease. While many IDPs perform their functions by folding with a partner protein, a subclass is prone to collapsed states, disordered interactions, and aggregation. We need to obtain a higher understanding of the biophysical attributes—level of structures versus collapse and behavior with water in comparison to sequences and biological traits—to characterize these IDPs and to extrapolate their functions. This project takes on two modes: We are (1) developing methods on a model protein known to form a collapsed state that traps water and (2) evaluating a small library of IDPs with distinct roles in transcription by the method developed. Studying these aggregation‐prone disordered domains is challenging. We utilize fluorescence methods to reach low concentrations, an environmentally‐sensitive fluorophore to evaluate the presence of water, and collisional quenching to follow the flow into the interior of the collapsed domain. Our studies of the control protein κ‐casein demonstrate that water is present in the collapsed state but is not able to freely exchange with the bulk solution. Our experimental library of IDPs were selected from auxin response factors (ARFs) from Arabidopsis thaliana. This family of transcription factors have three distinguishable sections: a DNA‐binding domain, a low complexity middle region, and a structured protein:protein interaction domain. My work is centered on the middle region as it is predicted to be disordered across the family but is distinct in abundance of glutamine depending on function. Transcription activators are consistently higher in glutamine than their repressive counterparts. Our final goal is to further investigate this thought by attaching fluorophores to the cysteines of the protein in order to compare the natural and denatured states across our selected proteins. After cloning select sequences from cDNA, we have optimized expression, purification, and fluorophore labeling of four proteins from both transcription activators and repressors. Our comparisons of these will expand our understanding of the sequence‐dependent biophysical traits in the context of their differing biological roles within the context of auxin response in plants.
Comparison of Giemsa and Wright’s Stain For Differential Analysis
Authors: Gistover Noah , Randolph Tim
Published: April 2020

Giemsa stain is available in most developing nations for malarial parasite detection. In the absence of Wright stain, Giemsa is also used to perform differential assessments. We are comparing Giemsa and Wright stains using buffers to determine the optimal staining protocol for differential analysis. We compared Giemsa with reverse osmosis drinking water (RO), deionized water (DI), buffer at pH 7.2, to Wright’s stain with RO and DI, using Modified‐Wright’s stain with buffer in an automated slide stainer as the control. Blood samples (n=3) were drawn from the research team and analyzed using these staining protocols. Wright’s stain with DI performed equivalent to control for white blood cell (WBC), platelets (PLT), and red blood cell (RBC). Undiluted Giemsa with DI showed good staining of WBCs and PLTs, and moderate staining of RBCs (grayish discoloration). Diluted Giemsa with buffer stained RBCs poorly (pale ghost cells) but stained WBC, and PLT well. Diluted Giemsa with DI demonstrated good staining of PLTs, poor staining of RBCs, and moderate staining of WBCs. Undiluted Giemsa with RO showed poor staining of RBCs with moderate staining of PLTs and WBCs. Diluted Giemsa with RO has poor staining of RBCs and WBCs, and moderate staining of PLTs. Wright with RO stained RBCs, WBCs, and PLTs poorly. We recommend using Wright stain with DI water for differential analysis but undiluted Giemsa with DI water is an adequate substitute.

Support or Funding Information

NG is supported by the DeNardo Education and Research Foundation grant.
A Mouse Model for Cerebro‐Costo‐Mandibular Syndrome (CCMS) with an Intronic Deletion in SnrpB
Authors: Alam Sabrina , Majewski Jacek , Majewska Loydie
Published: April 2020

SNRPB, an essential effector of the spliceosomal complex, is implicated in the splicing of mature mRNA and is mutated in a rare craniofacial disorder known as Cerebro‐costo‐mandibular syndrome (CCMS). Most CCMS patients carry point mutations in the highly conserved alternative exon 2 (AE2) of SNRPB, leading to increased inclusion of a pre‐termination codon (PTC) and reduced levels of the protein‐coding transcript. Given that SNRPB is implicated in all steps of splicing, it was thought that its requirement would be equivalent in all cells and tissues. However, CCMS patients predominantly present with micrognathia and rib defects with variable penetrance. We hypothesize that a subset of transcripts is highly sensitive to the levels of SNRPB during development and is tissue specific. Here, we are generating a mouse model for CCMS to uncover those transcripts that play an essential role in craniofacial development. Using the CRISPR/Cas9 gene‐editing system, we developed a mutant mouse line with a 61 base‐pair (bp) intronic deletion upstream of the AE2. Interestingly, a subset of the heterozygous and homozygous embryos and pups for this deletion show morphological abnormalities in the face, head, ribs, and limbs. Moreover, a significant percentage of the mutants with the 61‐bp deletion die in their first year of life. A subgroup of the heterozygous and homozygous mutant embryos show increased inclusion of the PTC containing AE2 and reduced SNRPB protein levels. We presume that this predominant inclusion of the AE2 induces non‐sense mediated decay resulting in decreased levels of SNRPB. As our data suggest that the 61 bp intronic sequence plays a critical role in regulating SNRPB levels in mice, we are currently investigating if similar intronic sequences in intron 2 of human SNRPB are involved in the preferential splicing of AE2. In addition, we generated a SnrpB conditional knockout mouse model, which will be used in parallel with the deletion model to investigate the cell‐type‐specific roles of SNRPB during craniofacial development. Our novel model for CCMS will further our understanding of the role of SNRPB in craniofacial development.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
The best of each capture – the combination of 3D laser scanning with photogrammetry for optimized digital anatomy specimens.
Authors: Dixit Ishan , Dunne Connor , Blumer Paige , Logan Curtis , Prakitpong Ratthamnoon , Krebs Claudia
Published: April 2020

Visualization of anatomical specimens has gone through an evolution from drawings to photographs and now 3D volumetric capture of dissected specimens. The technologies behind this 3D volumetric visualization are rapidly evolving and becoming more broadly accessible with improved hardware and software. There are two main approaches to 3D reconstruction: laser scanning and photogrammetry (PGM). Our previous work demonstrated that while laser scanning provides superior geometric accuracy, PGM delivers a more photorealistic texture. For accurate anatomical study, both a precise geometry and a photo‐realistic texture are necessary ‐ so we set out to combine both methods. We found that both methods individually and the combination capture method work best with rigid (plastinated or bony) specimens – but many dissected specimens are wet specimens, which tend to change in their geometry depending on how they are positioned on a surface for capture. We created a methodology to provide consistency and support for the geometry of wet specimens with an alginate cast and a rigging method for longer pieces such as vessels and nerves. The cast allows us to flip the specimen with minimal disturbance in order to capture all sides accurately with both 3D laser scanning and PGM. The two datasets are then combined in Artec Studio and Reality Capture software packages. The combination of these two methods results in a very accurate 3D geometric mesh of the specimen with a photorealistic texture, providing the most realistic 3D volumetric captures of complex anatomical specimens. The combination method is more time‐consuming than each of the methods individually and a greater level of care must be taken to prevent any misalignment between the two datasets. Many specimens can be captured in an accurate and satisfactory way with either PGM or 3D laser scanning alone, and some will require this combination capture. The advantages and limitations of each method need to be compared to the desired outcome. Our approach results in superior capture quality, especially for non‐rigid specimens. The digitization of both anatomical and pathological specimens can improve accessibility and distribution of high quality digital specimens for both education and research. Both ID and CD contributed equally to this work.
Changes in HAS3 and CaSR Expression Following Daily Neonatal CPAP in Mice
Authors: Mayer Catherine A. , Martin Richard J. , Pabelick Christina B. , Prakash Y.S. , MacFarlane Peter M.
Published: April 2020

Continuous positive airway pressure (CPAP) is a common mode of respiratory support for preterm infants. Recent studies from our lab, however, have shown that CPAP may contribute to the pathophysiology of airway hyperreactivity (AHR) associated with wheezing disorders in former preterm infants. The long‐term effects of CPAP on lung‐development, however, still remain largely unknown. Both hyaluronan synthase 3 (HAS3) and the calcium‐sensitive receptor (CaSR) have been associated with various respiratory morbidities. In the present study, we used a neonatal mouse model of daily CPAP treatment to investigate whether the long‐term effects on AHR are associated with changes in airway HAS3 and CaSR expression. Neonatal mice were exposed to CPAP (6cm H2O) for 3 hours per day for the first 7 postnatal days. Control animals experienced the same conditions as experimental animals only with no CPAP. On postnatal day 21, the lungs from these animals were harvested for either real time rt‐PCR or immunohistochemistry to assess changes in HAS3 and CaSR expression. We observed increased expression of multiple genes involved in hylauraonan synthesis, most notably has3, as well as increased CaSR expression, in the lungs of mice two weeks after CPAP ended when compared to control mice. The increased gene expression of has3 and CaSR was also reflected in increased protein expression as confirmed by immunohistochemistry. HAS3 was expressed predominantly in airway epithelium and the increased CaSR expression correlated with increased αSM actin. To further investigate the role of CaSR in CPAP‐induced AHR, CaSR KO mice (CaSR(−/−)) were exposed to CPAP and airway reactivity to methacholine challenge was assessed two weeks later (P21 days). Compared to control mice, AHR was not expressed in CaSR(−/−) mice suggesting resistance to CPAP treatment. These data suggest increased airway HAS3 and CaSR may be important modulators of CPAP‐induced AHR and remodeling.

Support or Funding Information

HLR01138402

HLR01056470
Significant role for intestinal urate transporters in host defense
Authors: Kazi Mirajul Hoque , Kuhns Victoria Halperin , Dixon Eryn , Woodward Owen M.
Published: April 2020

Humans, because of the loss of the uricase gene, have higher levels of serum urate (SUA) than most other mammals, prompting speculation of potential advantages of higher SUA levels. Others have proposed the role of urate as a chemical danger signal, and gout research has clearly shown urate crystal deposition stimulates a profound inflammatory response. Recently, we created a hyperuricemia mouse model through the insertion of a point mutation (Q140K) into the critical urate transporter ABCG2. Though these animals are hyperuricemic, they display no evidence of crystal deposition. In addition, we observed reduction in ABCG2 mediated intestinal secretion was key in the animal’s urate homeostatic mechanisms and the cause of the hyperuricemia. Thus, we hypothesized gastrointestinal infection may lead to dramatic changes in serum urate levels. In a mouse model of shigella infection we found 2‐fold increases in serum urate in infected mice. Because the intestines are a critical source of cytokines and other immune system signal, we next sought to better understand the role for urate. Is it a byproduct of infection or a signal that helps coordinate an immune response and host defense? To answer this question, we challenged the two mice genotypes, WT and the hyperuricemic Q140K, with intraperitoneally injected 100μg lipopolysaccharide (LPS, E. coli 055: B5). At baseline, pre‐LPS, a comparison between WT and the Q140K mice revealed no significant differences in our panel of cytokines. However, twenty‐four hours after LPS challenge showed significantly greater increases in the Th2 cytokines: IL‐5, ‐10, and IL‐13 (100%, 168%, and 100% respectively) in the hyperuricemic Q140K mice. We observed no differences in IL‐4 production, Th1 cytokines IL‐2, and TNF, or chemokines Ccl5, and Cxcl10 between the two genotypes. These results suggest the increased Th2 cytokine production in Q140K mice is due to hyperuricemia. In addition to the Th2 cytokines, vascular endothelial growth factor (VEGF) levels were increased by 2.3‐fold in Q140K mice as compared to WT in response to LPS challenge, potentially influencing the Th2 cytokine production and moderating immunopathology. We conclude hyperuricemia pre‐conditions the immune system, specifically Th2 cytokine, resulting in far greater responses to potential infectious pathogens and ultimately inhibiting disease progression.

Support or Funding Information

NIH, NIDDK
PTPRH mutations in NSCLC regulate EGFR phosphorylation
Authors: Andrechek Eran
Published: April 2020

The dysregulation of receptor tyrosine kinases (RTK) has garnered plenty of interest within the cancer field, and attention has begun to turn to phosphatases regulating RTK behavior. Under normal cellular conditions, protein tyrosine phosphatases remove phosphate groups from tyrosine residues, thus maintaining signaling homeostasis. In whole genome sequencing primary mouse mammary tumors from the polyoma virus middle T antigen (PyMT) mouse model, we found a mutation in the protein tyrosine phosphatase receptor type H (Ptprh) gene. Targeted resequencing of 45 mouse tumors showed a conserved heterozygous or homozygous mutation present in 80% of tumors. This C>T mutation results in a valine to methionine shift within one of the fibronectin domains of PTPRH. Previous literature has shown interactions between PTPRH and epidermal growth factor receptor (EGFR). To determine the relevancy of PTPRH mutations in human cancer, data from The Cancer Genome Atlas (TCGA) was analyzed and revealed PTPRH mutations in five percent of non‐small cell lung cancer (NSCLC) patients. Moreover, patients with a mutation in PTPRH were mutually exclusive from those with mutation or amplification of EGFR. We hypothesize a mutation in PTPRH results in a failure of PTPRH to dephosphorylate EGFR, resulting in inappropriate maintenance of downstream signaling pathways important for proliferation and evading apoptosis. Since NCSLC patients with EGFR mutations are successfully treated with tyrosine kinase inhibitors (TKI), we also hypothesize tumors with a mutation in PTPRH will be sensitive to TKIs. In support of this, we demonstrated mouse tumors with a mutation in Ptprh had increased phosphorylated EGFR (pEGFR). Furthermore, CRISPR mediated knockout of PTPRH in H23 NSCLC cells leads to increased pEGFR. Pathway signature analysis applied to microarray gene expression data from the Breast TCGA dataset (due to low sample size in the NSCLC dataset), and single sample gene set enrichment analysis applied to RNA sequencing data from the NSCLC TCGA dataset both predicted an increase in PI3K and AKT activity. This suggested the EGFR residue targeted by PTPRH was tyrosine 1197. Western blots on Ptprh mutant mouse tumors confirmed increased levels of pAKT. Additionally, immunohistochemistry for pEGFR 1197 revealed increased staining in mouse tumors with a mutation in Ptprh, with sub‐cellular location in the nucleus rather than the membrane. To determine whether TKIs may be an effective treatment for NSCLC patients who harbor a PTPRH mutation, H1155 and H2228 NSCLC cell lines with PTPRH mutations in the fibronectin and phosphatase domains respectively, were subjected to a dose response curve with the TKI osimertinib. These lines show significant growth differences as compared to the negative control cell line A427. While more work is needed to elucidate the role of mutant PTPRH in NSCLC, preliminary data suggests mutant PTPRH fails to dephosphorylate EGFR, and patients with a mutation in PTPRH may benefit from TKI therapy.
Direct Pathway For Bacillus anthracis tRNA Asparaginylation
Authors: Hernandez Jose Giron , Sheppard Kelly
Published: April 2020

Protein synthesis is essential for life and requires the correct pairing of amino acids to their cognate transfer tRNA (aminoacylation). To date, only two routes exist to attach asparagine (Asn), to it cognate tRNAAsn: the direct and the indirect pathways. The direct path uses asparaginyl‐tRNA synthetase to attach Asn to tRNAAsn when free Asn is present. The Asn is synthesized by asparagine synthetase A. The second pathway involves synthesizing Asn on the tRNA itself. The causative agent for anthrax, Bacillus anthracis, appears to encode both routes for Asn‐tRNAAsn formation. We would like to understand why B. anthraciscodes for both routes. This semester we have focused on the direct pathway. We report the successful purification of the asparagine synthetase A and asparaginyl‐tRNA synthetase along with activity measurements of asparagine synthetase A in order to characterize the direct pathway under different physiologically relevant conditions.

Support or Funding Information

National Science Foundation (MCB‐1615770)
Building Bridges Back to Back: 5 years of an outreach initiative in Anatomical Sciences and Medical Education between Brazil and US
Authors: Curcio Daniella , Laitman Jeffrey T.
Published: April 2020

The Building Bridges program has fostered collaborations and exchange experiences in Anatomical Sciences among students, researchers, and educators through lectures, colloquia, and student‐faculty exchange sessions. These have taken place in Sao Paulo (Santa Casa School of Medical Sciences), Ribeirao Preto (University of Sao Paulo School of Medicine, USP‐RP) and Rio de Janeiro (Federal University of Rio de Janeiro, UFRJ; Federal University of the State of Rio de Janeiro, UNIRIO; and the Brazilian National Academy of Medicine), since 2015. In August 2019, our group represented the American Association of Anatomists in London, at the 19th Congress of the International Federation of Associations of Anatomists (IFAA), contributing in a workshop addressing the topic International Connections of Anatomists in Global Health. Here, we comment on the outcomes of this workshop and report our upcoming endeavor in New York: the mini symposium Building Bridges Back to Back that will be hosted at the Icahn School of Medicine at Mount Sinai, December 2019. The meeting celebrates the 5th year anniversary of our international outreach initiative. It will bring together leaders from Medical Education from Brazil and connect other participants through videoconference channels. In the Medical Education session, the program includes the Dean of the Santa Casa School of Medical Sciences of Sao Paulo—Dr. Carlos Alberto Malheiros, as keynote lecturer. In the academic publishing session, the keynote lecturers will be the Director of the Graduate Program and Chief of the Surgery Department of the Medical Course at the Federal University of the State of Rio de Janeiro (UNIRIO), member of the Brazilian Academy of Medicine—Dr. Rossano Fiorelli; and the Editor in Chief of the Anatomical Record—Dr. Kurt Albertine. Within the two main sessions, Medical Education and Academic Publishing, the meeting will also cover new advances in Brazilian science, bringing leaders in education and research from different states in Brazil and the US. The event marks the launch of an international exchange program at the Icahn School of Medicine at Mount Sinai and Brazilian medical schools. Our goal is to offer opportunities for Brazilian students to experience medical learning in New York with the intent to help forge new leaderships and role models in medicine and health sciences in Brazil. We expect that this meeting will build the foundation to this exchange project and enhance the cooperation with the health care, medical and anatomical communities of the two countries.

Support or Funding Information

Meeting support funded in part by The Anatomical Record; Research reported funded in part by CNPq grant PDE #249582/2013–9
FGF1 receptor antagonist decreases biliary proliferation, fibrosis, and senescence in a mouse model of chronic cholestasis
Authors: O’Brien April , White Tori , Francis Heather , Meng Fanyin , Alpini Gianfranco , Glaser Shannon
Published: April 2020

Background

Acute or chronic cholestasis is a hallmark for a wide variety of liver diseases such as primary sclerosing cholangitis (PSC), alcoholic liver disease (ALD) and cholestasis of pregnancy. Available treatments for cholestasis are few and the subsequent scarring often necessitates the patients undergo a costly and risky liver transplantion. Fibroblastic growth factor 1 (FGF1) is involved in a wide range of cellular activities and has been previously shown to act in both a protective and detrimental manner as it participates in the fibrotic process of various tissue systems. Therefore, we aimed to evaluate the role of FGF1 in the progression of senescence, inflammation and fibrosis in an MDR−/− mouse model of PSC.

Methods

FVB and MDR−/− mice were treated with 100ng/kg/day for 7‐days with the FGF receptor inhibitor, AZD4547. Liver, serum, and pure cholangiocytes were obtained. Hepatic fibrosis was evaluated by H&E and Sirius Red staining, and qPCR for collagen‐1a. The inflammatory response was evaluated by qPCR in pure cells for IL1‐b, IL‐6, and IL‐33 and immunofluorescent staining of F4/80. Biliary proliferation was determined through IHC CK‐19 expression and qPCR for Ki67 and PCNA. Senescence was evaluated by qPCR for p53, p18 and p21. FGFR expression was determined by qPCR. FGF1 serum expression was measured using a commercially available ELISA kit and in OCT liver sections by immunofluorescence (IF). In vitro, human intrahepatic biliary epithelial cells (HIBepiC) were treated with 150ng/ml of either FGF1 or AZD4547 with or without the presence of 100ng/ml of LPS for 24 hr before evaluating inflammatory markers, biliary proliferation and markers of fibrosis.

Results

As expected, MDR−/− mice had a significant increase in biliary proliferation and fibrosis when compared to the FVB control. MDR−/− animals treated with AZD4547 experienced a significant reduction in fibrosis, with a subsequent decrease in collagen 1a1 gene expression. Treatment with AZD4547 also decreased senescence and inflammatory markers in MDR−/− mice evaluated by IHC and IF. Serum analysis showed a dramatic increase in FGF1 levels that were significantly reduced upon treatment. In vitro studies also had significant reductions in inflammatory and fibrotic markers.

Conclusion

FGF1 receptor antagonist decreased biliary proliferation, senescence, inflammation and fibrosis in cholangiocytes and surrounding liver in a mouse model of PSC. Targeting the FGFR pathway may provide a therapeutic option for patients suffering from various forms of chronic cholestasis such as PSC.

Support or Funding Information

NIH RO1 DK110035

R01 DK115184
Decline in Renal Hemodynamics During Pregnancy after Recovery from Ischemia Reperfusion Injury
Authors: Gillis Ellen E. , Sullivan Jennifer C.
Published: April 2020

Pregnancy is a unique physiological state that significantly alters renal hemodynamics. Renal demands are increased during normal pregnancy due to the large increase in plasma volume and cardiac output, resulting in increased glomerular filtration rate and decreased blood pressure (BP). Recent studies in our laboratory have reported that pregnancy after recovery from ischemia reperfusion (IR) injury results in poor maternal and fetal outcomes, including decreased fetal weight, increased fetal demise, and mild uremia in the dams. In the current study, we hypothesized that renal hemodynamics fail to adapt during pregnancy in these dams after recovery from IR. Female Sprague Dawley rats (10 weeks of age, n=3) were implanted with telemeters into the femoral artery for continuous BP measurements. Following 10 days of recovery, rats were randomized to receive either 45 minutes of warm, bilateral renal ischemia or sham surgery. Rats were then given 1 month to recover. Recovery from IR was confirmed by plasma creatinine and urinary protein excretion prior to mating. Vaginal smears were performed daily once mating began, with sperm on the slide indicative of gestational day 1. Glomerular filtration rate was calculated using creatinine clearance in the urine (24 hour urine collection from gestational days 19–20) and plasma creatinine on gestational day 20. BP decreased to a similar extent (7±1.2mmHg in control vs 8±1.2mmHg in IR dams) by gestational day 20, however the decrease in BP was delayed in the IR dams, resulting in an overall higher pressure load in the IR dams as determined by area under the curve analysis (2080±33 vs 2184±16, p<0.05, t‐test). Glomerular filtration rate was significantly higher in control dams compared to IR dams (3.1±0.7mL/min vs 1.6±0.1mL/min, p<0.05). These data suggest that after recovery from IR, the kidneys are unable to cope with the increased renal demands of pregnancy. Ongoing studies are measuring plasma volume expansion in this model to further explore the potential mechanisms by which IR predisposes these rats to poor pregnancy outcomes. We propose that plasma volume expansion, characteristic of normal pregnancy, is absent in this model, leading to decreased placental perfusion and poor fetal growth.
Exploring the scope of high throughput micro‐permethylation based glycomics.
Authors: Shajahan Asif , Supekar Nitin T. , Heiss Christian , Azadi Parastoo
Published: April 2020

The glycosylation plays important roles in various molecular and cellular functions such as cell signaling, adhesion, development, immune recognition, lubrication, cellular defense etc. The protein glycosylation also contributes towards the stability, serum half‐life, immunogenicity and more importantly, biological activity of therapeutic glycoproteins, including antibodies, vaccines, biomarkers etc. This justifies the increasing demand for the qualitative and quantitative characterization and validation of glycosylation profile on glycoproteins. However, profiling of glycosylation is challenging owing to the glycosylation microheterogeneity, non‐template driven synthesis and their lower abundances. We have recently developed a high throughput N‐ and O‐glycomics strategy through a microscale permethylation based glycan derivatization followed by mass spectrometry (MS). Through this strategy facile but detailed quantitative structural profiling of glycans in a high throughput fashion can be attained. Here, we are reporting the wide applicability of our high throughput glycomics on glycoconjugates from various sources, including human serum, mammalian cells, milk oligosaccharides and purified glycoproteins via an automated tandem ESI‐MSn acquisition platform. The released or free glycans are permethylated in microscale in a 96‐well plate or microcentrifuge tubes and analyzed by an automated ESI‐MSn system after a C18 tip‐based cleanup. An internal standard based method was also incorporated for the absolute quantitation of glycan species. We have employed an in‐depth automated structural characterization MS program for N/O‐glycans through an automated ESI‐MSn acquisition up to MS5 level. Through this automated process, detailed glycan structural topology can be identified including glycan termini, branching, isomers, sialic acid linkages, and bisecting GlcNAc on the glycans. This approach can facilitate profiling of glycosylation on large sample sets, including clinical glycan biomarkers, milk oligosaccharides, and other glycoprotein therapeutics.

Support or Funding Information

National Institutes of Health grant (No. 1S10OD018530) to Dr. Parastoo Azadi
Identifying Variants and Prevalence of the Superficial Brachial Artery – A Case Series
Authors: Bolden Alexys M. , Schmitt Emily A. , Alsharaf Fatemah , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

Superficial brachial artery (SBA) is the term used for when the brachial artery (BA) itself or an early branch from the BA travels superficial to the roots of the median nerve (MN) or the MN itself. The purpose of this study was to determine the prevalence of the aforementioned SBA variations, to identify early branching variants of the SBA, and to explore the appropriateness of the anatomical terminology of these SBA variants due to ambiguity in the nomenclature. Assessment of 87 cadavers (174 upper extremities) led to the discovery of nine (5.2%) SBA variations which were observed in a total of seven (8.0%) cadavers (five females, two males). Three different SBA variation types were observed: Variation 1 was identified when the entire BA ran superficial to the median nerve roots, present in 1 of 174 limbs (0.57%); Variation 2 was noted when the BA bifurcated proximal to median nerve roots, resulting in superficial brachial and brachial arteries, seen in 6 of 174 limbs (3.4%); and Variation 3, wherein the BA bifurcated distal to the median nerve roots at the location of an additional connection between the musculocutaneous and median nerves, also resulting in superficial brachial and brachial arteries in 2 of 174 limbs (1.1%). The branches of the BA were followed to discern their branching patterns distally. In the six examples described as Variation 2, four (67%) of the SBAs traveled distally to become the radial artery only (Variation 2A), and two (33%) traveled distally to become both the radial and ulnar arteries (Variation 2B). For the two examples identified as Variation 3, both of the SBA vessels turned into solely the radial arteries distally. Therefore, Variations 2A and 3 both travel distally to become solely the radial artery; however, there is confusion in the literature as to whether these SBA variants can also be called high origin radial arteries (HORAs). Therefore, clarification of the current criteria for naming these brachial artery variants will also be discussed. Due to the unusual locations of SBAs, these variant arteries may cause confusion during surgery and in the interpretation of imaging studies.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06]
Exercise‐enriched extracellular vesicles benefit brain health by decreasing protein aggregation
Authors: Geiger Paige C. , Deng Fengyan , Miller Joshua , Morris Jill , Vidoni Eric , Burns Jeffrey
Published: April 2020

Aerobic exercise results in improved brain health during both healthy aging and dementia. However, the precise biological mechanisms that drive exercise‐induced benefits for brain health remain unknown. One possible mechanism by which exercise may benefit the brain is by facilitating the delivery of molecular mediators within extracellular vesicles (EVs; exosomes and microvesicles). EVs are bilayer‐phospholipid enclosed vesicles that carry protein and microRNA cargo and are released into the blood from many organs and cells. EVs have been shown to regulate nerve regeneration, synaptic function and behavior. Importantly, when directly injected into rodent brains, EVs can effectively eliminate protein aggregates like amyloid‐β‐peptide (Aβ), which are closely linked to Alzheimer’s Disease (AD) pathology. We hypothesize that EVs isolated from human serum post‐exercise could effectively clear Aβ aggregates in neuronal cells. To test this hypothesis, we isolated EVs from cognitively healthy subjects and early onset AD subjects (8 non‐dementia subjects, ND and 4 early AD subjects) 15 minutes after maximal aerobic exercise (EX). EV size did not change with exercise in either ND or AD subjects, while EV concentration increased by 20% post‐EX in both groups. EV function was tested by incubating serum EVs with human neuronal SY5Y cells over expressing β‐amyloid precursor protein (APPswe). We found that post‐EX‐derived EVs were more effective at alleviating Aβ protein aggregation (40% decrease in aggregation compared to pre‐EX‐derived EVs). Heat Shock Proteins (HSPs), molecular chaperones that play a critical role in protein folding/misfolding, were increased in post‐EX EVs. These findings demonstrate a novel way in which exercise could benefit the brain, through the cell‐to‐cell signaling network of extracellular vesicle communication.

Support or Funding Information

Research support by the University of Kansas Alzheimer’s Disease Center.
Endogenous Promoters enable efficient CRISPR/Cas9 in a Tilapia Cell Line Model
Authors: Hamar Jens , Kültz Dietmar
Published: April 2020

Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control of the extracellular environment. The recent rise of CRISPR/Cas9 gene editing has provided an additional powerful tool to aid in these studies. For work focusing on fish species many fish cell lines now exist. However, conventional tools and methods for implementing CRISPR/Cas9 gene targeting in these cells doesn’t always provide sufficient results as seen in other animal cell models. A tilapia brain cell line (OmB) developed in our lab has proven useful for gene expression studies. Previous work with multiple conventional methods attempting to apply CRISPR/Cas9 failed to indicate genomic alteration at the targeted sites. Here we present a plasmid vector based system utilizing both Polymerase II and III tilapia endogenous promoters that enables proficient gene targeting. This system demonstrated efficient cleavage in most target sites attempted with efficiency as high as 80%. These tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically related fish species.

Support or Funding Information

This study was funded by a grant from NSF (IOS‐1656371) and BARD (IS‐48000‐ISR).
Methylmalonic Acid Compromises Energy Metabolism and Decreases the Expression of Neuronal Differentiation Genes in SH‐SY5Y Human Neuroblastoma Cells
Authors: da Costa Renata Torres , dos Santos Marcella Bacelar , de Almeida Raquel Pascott , Ribeiro César Augusto João
Published: April 2020

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L‐methylmalonyl‐CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and symptoms during metabolic decompensation commonly appear in newborns. Considering that the mechanisms underlying neurological alterations of this disorder are not fully understood, this study aimed to investigate the toxic effects of MMA in a neuroblastoma cell line presenting neuronal features. Undifferentiated and retinoic acid (RA)‐differentiated human neuroblastoma SH‐SY5Y cells were exposed to MMA (1–10mM) for 24 or 48 hours and cell viability, glucose consumption and mitochondrial metabolic function were analyzed. Additionally, the expression of some molecular markers of neuronal differentiation was also quantified after co‐exposition with 10 μM RA and 1 mM of MMA for 3 and 7 days.

MMA significantly reduced cell viability after 48‐hour period exposition in undifferentiated and RA differentiated SH‐SY5Y cells. It was also observed an increase in glucose consumption in cells exposed to MMA. Regarding oxygen consumption, there was an increase in respiratory parameters in RA differentiated SH‐SY5Y cells when compared to undifferentiated cells. MMA significantly reduced basal, ATP‐linked and maximal respiration, as well as the reserve respiratory capacity of cells after 48‐hour exposition in both undifferentiated and RA differentiated SH‐SY5Y cells. Regarding the effects of MMA in the neuronal differentiation process, it was observed that co‐exposition to RA and MMA significantly decreased the expression of enolase 2 (ENO2) and synaptophysin (SYP) genes after 7 days of exposition.

In summary, MMA cell viability and oxygen consumption in SH‐SY5Y neuronal cells, suggesting that this organic acid compromises cell respiration, at resting and reserve capacity states, which might influence the cell capacity to meet energetic demands. In addition, glucose uptake was increased after exposition to MMA, corroborating that aerobic metabolism is compromised. The expression of mature neuronal genes was also reduced, suggesting that MMA possibly compromises neuronal differentiation, which can be involved in symptoms presented by affected patients during brain development. Finally, our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.

Support or Funding Information

This study was supported by grants from FAPESP (2015/25541‐0), CAPES and UFABC.
Developing of a Muscle Atrophy Model
Authors: Garcia-Pelagio Karla , Martinez-Mendez Sheiren , Hinojosa-Rodriguez Cindy , Jimenez-Estrada Ismael , Segura-Alegria Bertha
Published: April 2020

Muscle atrophy is one of the most important and frequent problems observed in patients in Intensive Care Units following a surgery, immobilization, extended periods of bed rest as well as in sports medicine. All of these involves loss of muscle mass and decreased muscle strength which increases the risk for future health problems. Immobilization changes the function as well as structure of the peripheral neuromuscular system, and alters both protein degradation and synthesis. Therefore, effective therapeutic strategies to treat muscle wasting induced by immobilization are urgently required but first is necessary to validate and standardize the immobilization technique. In this work we develop a reproducible well‐characterized injury model using a modified Thomas‐ splint validated with mechanical force measurements, histology (H&E and NADH), morphology and ultrasound imaging. Forty young Wistar male rats were divided into two groups: 1) Control‐ no immobilized extremities (CON; n=10) and 2) Unilateral right hindlimb immobilization at 3, 7 or 14 days (Im 3, Im 7, Im 14; n=10 for each group). Results showed a significant increment in body weight for Im 7 and Im 14 compared to CON. Experiments performed in tibialis anterior (TA) of Im muscle showed morphological changes in muscle weight and muscle diameter compared to CON confirmed by Ultrasound imaging. These changes were accompanied with a decrement in peak isometric tetanic force (Po) of immobilized muscles of 20%, 41% and 28% at 3, 7 and 14 days post injury, respectively. Fatigue time for all Im muscles was higher than CON ones (Im 3= 23%, Im 7=33% and Im 14=42%). Metabolic changes using NADH technique denoted an early increase of oxidative fibers and a decrement of glycolytic fibers at 7 days post‐injury compared to CON. In here, we developed, standardized and characterized an immobilization technique that can be used as a first approach to use effective therapeutic strategies to avoid muscle atrophy.

Support or Funding Information

Supported partially by UNAM ‐PAPIIT RA207818 and CONACyT AI‐S‐17636 to KPGP.
OCT3 gated intracellular β1AR activation enhances excitation‐contraction coupling in heart
Authors: Wang Ying , Zhao Meimi , Shi Qian , Xu Bing , Xiang Yang Kevin
Published: April 2020

Rationale

β1AR‐adrenergic receptor (β1AR) plays a central role in sympathetic regulation of cardiac excitation‐contraction (EC) coupling. Stimulation of β1AR by catecholamines enhances cardiomyocyte contraction and Ca2+transient via the cAMP‐protein kinase A (PKA) pathway. Organic cation transporter 3 (OCT3) plays a role in cleaning up the extracellular catecholamines and transporting a portion of catecholamine into myocardium. Recent progress found that β1AR is also present at intracellular membranes. However, how the intracellular β1AR was activated, and the role of OCT3 in regulating intracellular β1AR signaling and EC‐coupling remains unknown.

Objective

To characterize the presence and activation of intracellular β1AR‐adrenergic receptors and their impact on cardiac EC‐coupling

Methods and Results

We used a genetic deletion of OCT3 to inhibit the entrance of catecholamine into the cells. We used isolated myocytes to measure β1AR‐induced contractility and Ca2+transients. We used PKA activity reporters to detect local activation via intracellular β1AR. We found that catecholamine‐induced inotropic effects were diminished in OCT3‐knock out hearts. OCT3 deficiency significantly attenuated norepinephrine (NE) induced PKA activities at the sarcoplasmic reticulum, myocyte contractility, and Ca2+transient. Additional data show β1AR associates with sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) at the sarcoplasmic reticulum (SR). Inhibition of OCT3 blocked the transportation of NE, and attenuated β1AR‐induced PKA activity at the SR and EC‐coupling.

Conclusion

Our finding demonstrates a previously unrecognized intracellular β1AR‐mediated local PKA activity at SR that is gated by OCT3. Upon catecholamine stimulation, this SR‐localized β1AR signal plays a critical role in promoting E‐C coupling and cardiac contractile function. Thus, internal β1AR signaling may presents a novel therapeutic target in cardiac diseases treatment.

Support or Funding Information

NIH HL113413VABX009200
The Multi‐functional Enzyme CHIP Regulates Microtubule Dynamics
Authors: Altinok Selin , McDonough Holly , Schisler Jonathan C.
Published: April 2020

The
Carboxy‐terminus of
Hsc70
Interacting
Protein (CHIP) controls several aspects of protein quality control machinery through its ability to function as a co‐chaperone, autonomous chaperone, and ubiquitin ligase. CHIP is ubiquitously expressed in all tissues, but interestingly, CHIP expression is higher in certain tissues such as heart, muscle and brain, suggesting an increased dependence on CHIP function in these organ systems. This concept is supported by studies that show how altered CHIP function associates with several ailments such as cardiac diseases, neurological disorders, and cancer. Microtubules (MTs) are a part of the cellular cytoskeleton that provides shape and structure to cells. In addition to these roles, MTs play a crucial role in cell division, migration, intracellular organization and trafficking. Dynamic assembly and disassembly of MTs upon various stimuli is important for the proper functioning of differentiated cells and is a process orchestrated by multiple proteins. Dysregulation in the MT reorganization machinery is implicated in a similar spectrum of diseases associated with the loss of CHIP function, however, there has yet to be a direct connection between CHIP and MT dynamics. We performed a proteomics screen on wild type and CHIP−/− mouse embryonic fibroblasts and identified differential regulation of proteins that regulate several aspects of MT function, such as STMN1 and PDLIM. Given the importance of CHIP in protein homeostasis, we hypothesized that CHIP is necessary for proper MT dynamics in response to cellular stimulations. We found that CHIP is required for the phosphorylation of stathmin, a regulator of MT assembly, and for the tubulin polymerization in C2C12 myoblast cells following insulin and glucose stimulation. This stimulation triggers an important MT‐dependent process to mediate glucose uptake. Furthermore, ex vivo studies using muscle fibers isolated from CHIP−/− mice failed to form MT lattices after glucose stimulation, consistent with a new role for CHIP in regulating MT in muscle. Altered MT dynamics, as well as CHIP loss of function, are also implicated in neurodegenerative diseases, particularly those associated with proteinopathies. To determine if CHIP contributes to MT regulation in neurons, we used an immunoprecipitation‐mass spectrometry approach and identified changes in interactions between CHIP and MT proteins during proteotoxic stress. Future experiments will focus on validating the interaction data and further dissecting the specific functions of CHIP that contribute to the regulation of MT dynamics using biochemical approaches and imaging. In conclusion, for the first‐time, our data couple CHIP to MT dynamics, and suggest that CHIP may contribute to cell protection by regulating the proteins that are vital to MT dynamics.

Support or Funding Information

Funding was provided by a grant to J.C.S. from the National Institute of Aging (R01‐GM061728).
Evaluation of Glutaredoxin 1 as a Novel Marker of Renal Damage after Ischemia‐Reperfusion Injury in Mice
Authors: Godoy Jose R. , O’Sullivan Meghan , Bethel-Lebel Herdest , Moncada Marcela H. , Illanes Oscar , Gellert Manuella , Lillig Christopher Horst
Published: April 2020

Renal ischemia‐reperfusion injury (IRI) is a significant cause of acute renal failure and delayed graft function in clinical patients. Tissue damage in renal IRI is caused in part by the activation of the immune response and the generation of reactive oxygen species (ROS), both taking place mainly during the reperfusion phase, once the blood supply to the tissue is restored. Glutaredoxin 1 (Grx1), a cytosolic protein that reduces protein disulfides as well as glutathionylated proteins, plays a protective role in ROS‐mediated damage to the cell. In a previous work, we detected increased levels of Grx1 in the urine of mice 24h after renal IRI
1. Recently, Grx1 was shown to be elevated in the serum of patients with chronic kidney disease
2. To determine the usefulness of Grx1 as a possible biomarker of ischemic renal damage we evaluated serum, urinary, as well as intra‐renal levels of Grx1 in a mouse model of renal IRI. Different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) were compared in order to follow Grx1 fluctuation levels in the kidney, serum, and urine. Serum Grx1 levels, analyzed by ELISA, were correlated with several renal function parameters such as serum creatinine, proteinuria, glomerular diameter, and tubular thickness. After IRI, Grx1 serum levels increased much earlier than creatinine (4h versus 24h). Western blot analyses showed a decrease of Grx1 in the kidney of mice with surgically induced IRI, compared to the sham group. Additionally, immunohistochemistry revealed an increased apical localization of Grx1 in cells of the distal convoluted tubules of the mice exhibiting IRI. Urinary levels of Grx1, even if increased in the first hours after renal IRI, were not significantly altered. These findings provide initial supporting evidence for the use of a redox protein such as Grx1 as a novel biomarker for assessing renal damage under experimental conditions. Further studies are necessary to validate these results.

Support or Funding Information

This work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine.