bluuebunny/crossref_metadata_2025 huggingface dataset:
Soil‐transmitted helminthic infection and nutritional status among urban slum children in Kenya
Authors: Suchdev Parminder Singh , Davis Stephanie , Ruth Laird , Worrell Caitlin , Wiegand Ryan , Montgomery Joel M , Foxe LeAnne
Published: June 2021

To evaluate the impact of soil‐transmitted helminth (STH) infection on nutritional status, we conducted a survey of 206 preschool (PSC) and 499 school‐aged children (SAC) randomly selected from CDC's population surveillance platform in the Kibera slum, Nairobi, Kenya. Hemoglobin, ferritin, inflammatory markers, retinol binding protein and malaria were measured from capillary blood; anthropometry and analysis of 3 stools for STH ova were performed. Approximately 40% of children had STH infection (39.9% PSC, 39.7% SAC), primarily Ascaris and Trichuris spp. Only 2.9% PSC and 1.0% SAC had heavy infection. Indicators of malnutrition among PSC and SAC were as follows: anemia 37.4% and 14.4%, iron deficiency (ID) 19.3% and 4.4%, vitamin A deficiency (VAD) 15.1% and 5.9%, and stunting 28.8% and 16.5%. Among PSC, STH was associated with VAD but not anemia, ID, stunting or inflammation. PSC with Trichuris infection were more likely to have ID than those without Trichuris (p=.04). The association between any STH and VAD remained significant in multivariate analysis, adjusting for age, sex, inflammation, maternal education and income (aOR=2.3, p=0.04). Our results suggest that STH infection is associated primarily with VAD, which may inform deworming and micronutrient supplementation programs.
Fat‐soluble vitamin status in response to non‐surgical weight loss in overweight post‐menopausal women
Authors: Prokopy Max , Booth Sarah L , Saltzman Edward , Yeum Kyung‐Jin , Nicklas Barbara , Shea M Kyla
Published: June 2021

Obesity is associated with an increased risk of fat soluble vitamin (FSV) deficiencies. The effect of dietary weight loss on FSV status is uncertain. We measured plasma concentrations of carotenoids, α‐tocopherol, retinol, phylloquinone, and 25‐hydroxyvitamin D (25(OH)D) in 112 overweight post‐menopausal women (mean(SD) BMI=33.1(3.7) kg/m2) who participated in a 20‐week hypocaloric weight‐loss intervention. Mean(SD) weight loss was −12.1(4.5) kg. All women received 200 IU/day vitamin D3. Except for 25(OH)D, the same FSVs were measured in adipose tissue. Using repeated measures ANOVA adjusted for the respective baseline FSV concentration and potential confounders, the following plasma FSVs increased significantly postintervention (all p<0.01): 25(OH)D (median increase 8.0 to 9.0 nmol/l), α‐carotene (0.09 to 0.13 μmol/l), and cryptoxanthin (0.11 to 0.13 μmol/l). The other FSV concentrations in plasma and adipose tissue generally increased but changes were not statistically significant (all p>;0.05). Modest weight loss achieved through caloric restriction was associated with modest increases in some FSV concentrations, although the clinical relevance is uncertain.

Grant Funding Source: NIH (grant R01‐AG/DK20583), Wake Forest University Claude D Pepper Older Americans Independence Center (P30‐AG21332), and Wake Forest University General Clinical Research Center (M01‐RR07122).
School‐based nutrition programs are associated with reduced child food insecurity in a longitudinal study among Mexican‐origin mother child dyads in Texas border colonias
Authors: Nalty Courtney , Sharkey Joseph R. , Dean Wesley R.
Published: June 2021

In 2011, nearly 3.9 million U.S. households with children experienced child food insecurity (CFI) as reported by parents. Yet, proxy reports may underestimate the true CFI prevalence. The study objectives were 1) understand mothers’ and children's (6–11 y) perspectives and experiences of CFI and its seasonal volatility, including the role of school‐based nutrition programs (SNP) and 2) assess CFI discordance among forty eight Mexican‐origin dyads in Texas border colonias. Promotora‐researchers administered standardized Spanish‐language food security instruments to dyads at two points in time during July 2010‐March 2011. Multilevel longitudinal logistic regression measured volatility in CFI while Cohen's kappa (κ) assessed discordance. SNP reduced odds of CFI by 74% (Odds ratio (OR) = 0.26, P < 0.01). Single head of household and Mexican ethnicity were associated with increased odds of CFI (OR = 4.63, P = 0.03) and (OR = 4.47, P < 0.01), respectively. Fair dyadic agreement was observed (κ = 0.21, P = 0.02). CFI lessened from summer to school year while SNP played a significant role. Mothers’ and children's perspectives and experiences of CFI ought to be measured at multiple occasions to obtain an accurate prevalence of the preventable condition.

Grant Funding Source: Robert Wood Johnson Foundation Healthy Eating Research
Spotlight on adipose tissue as a remarkable stem cell source for regenerative medicine and tissue engineering applications
Authors: Fradette Julie
Published: June 2021

Great scientific strides have been achieved upon discovering the great plasticity of adult mesenchymal stem cells. Such stem cells can be harvested from various sources, including subcutaneous adipose tissue which represents an almost ideal cellular reservoir. Cell therapies using adipose‐derived stem/stromal cells (ASCs) are being conducted in an increasing number of clinical trials such as osteoarthristis and congestive heart failure. The other field reaping the benefits of using ASCs is tissue reconstruction. The multipotency of these cells combined with different engineering strategies result in the in vitro production of a wide variety of human tissues. My research team is using ASCs as building blocks for the production of human tissue substitutes, including adipose tissue itself and skin. They feature a rich extracellular matrix produced by the mesenchymal cells themselves upon ascorbic acid stimulation, recreating a very physiological 3D environment. The reconstructed adipose tissues are functional and secrete important cytokines and growth factors such as leptin, Ang‐1, HGF, and VEGF. This secretome is modulated in a dose‐dependent manner following exposure to the inflammatory cytokine TNFα. Also, production of reconstructed skin using ASCs allowed us to investigate the fate of epithelial stem cells in these engineered tissues, before and after grafting. Supported by CIHR and NSERC.
Using the “Rate Your Plate” questionnaire to teach nutrition changed medical student eating habits
Authors: Valls Eric Vargas , Gadey Gautam , Zelis Robert
Published: June 2021

Over 56% of medical students across the US feel their nutrition related experiences are inadequate. Here we examined whether the addition of the “Rate Your Plate” questionnaire (RYP) (©2005 Brown University) during their second year fall integrated cardiovascular course, and knowledge of student personal 30 year risk of a cardiovascular event were useful in changing students’ eating behaviors. Thirty‐two students completed an unpublished 24‐item questionnaire (modified‐RYP ©2009 Brown University) about their eating habits in the spring of their first year. The same students then completed the questionnaire in the spring of their second year. Paired t‐test was used to analyze the difference in modified‐RYP scores. Pearson correlation coefficients were calculated for the Framingham 30‐year cardiovascular event risk and change in RYP score to examine whether risk knowledge may have changed eating habits. Mean scores at baseline and one year later were 57.19 and 58.97, respectively (Paired t‐test: P<0.01). Correlation coefficient between 30‐year relative risk and change in RYP score was ‐0.268 (ns). While medical students were eating healthy at baseline, introduction of the RYP questionnaire along with the nutrition curriculum during second year improved heart healthy eating habits. Surprisingly, knowledge of personal 30‐year cardiovascular event risk was not correlated with their change in RYP.
Developing integrated active learning activities for first year medical school curriculum: enhancing learning of the basic sciences by including clinically relevant material
Authors: Johnson William E , Estores David , Schocken Dawn
Published: June 2021

Over the past decade, there is an increasing emphasis on curricula incorporating more active learning activities and systems‐based teaching with a corresponding decrease in emphasis on traditional didactic lectures. In Course 4 (Gastrointestinal (GI), Renal and Endocrine Systems) anatomy labs that cover the GI system are integrated with other basic science disciplines and active participation. The class is divided in two groups: one group attends the gross anatomy laboratory, while students in the second group are divided into smaller subgroups that engage in active learning sessions with computerized patient simulation, ultrasound and radiology imaging with standardized patients, case problem solving involving relevant aspects of GI physiology, histology and embryology. The two groups switch during the next lab period. In order to promote learning and retention of important concepts, summaries of the anatomy laboratory exercises and cases of the week are provided during subsequent class meetings. Utilizing this integrated, multidisciplinary approach, the first year medical students reported a positive effect on their learning experience.
Structuring online course conversation increases discussion content, depth, and breadth
Authors: Shain Margaret E. , Matyas Marsha Lakes
Published: June 2021

Online programs are being increasingly used to provide teacher PD. Effective online programs engage teachers not only in acquiring knowledge but in active participation in a community of practice. Community interactions should increase understanding of effective pedagogies and provide help in planning applications. Creating online assignments that generate substantive interactions among a large number of teachers during asynchronous online discussions (AODs) is challenging. AODs have rich content (ideas, resources, and practices), wide breadth (engaging significant numbers of participants), and substantial depth (multiple layers of “back and forth” comments). Online discussions document and quantify the content, breadth, and depth of teacher conversations. In the APS Frontiers in Physiology PD program AODs, that were part of the teacher assignments, indicated that only those teachers who were among the first to post in a discussion had substantive responses attached to their posting. In this study, discussion assignments were restructured to promote AODs on initial postings by all teachers, even those who post later in the AODs. Results from 101 teachers showed significant increases in the content, breadth, and depth of AOD showing positive impacts on the overall program goals and objectives. This suggests that online learning environments may benefit from greater structure of AODs.
Pulmonary Hypertension Associated with Human Immunodeficiency Virus: Uncovering Viral Evolution via Bioinformatics
Authors: López‐Astacio Robert Alexis , Reno Samantha Marie , Schoen Rachel O. , Castro‐Flores Sonia , Almodóvar Sharilyn
Published: June 2021

Individuals with Human Immunodeficiency Virus (HIV) are prone to illnesses like pulmonary hypertension (PH), despite the success of antiretrovirals. PH affects more HIV+ individuals (0.05%) than uninfected (0.001%). The causes of HIV‐PH are unknown however, studies suggest that HIV proteins may play a role. Recent studies found specific HIV‐nef mutations associated with the PH phenotype in humans; others suggested that HIV Envelope (Env) induces PH‐like pulmonary vasculopathy in monkeys. We hypothesized that HIV‐PH‐specific mutations may also extend to env: HIV‐PH patients harbor Envelopes more fit to evade the immune system (more glycosylated), and more pathogenic (T‐tropic vs. M‐tropic), compared to controls. Preliminarily, we cloned HIV‐env and nef from blood of eight HIV+ consenting subjects, and analyzed the env and nef sequences in silico, using tools available in the Los Alamos National Lab. We found more glycosylated Env and T‐tropic viruses along with mutations in Nef in HIV‐PH samples, suggesting that mutations in HIV, in the context of Pulmonary Hypertension, may also implicate Env; further studies may confirm dual evolution of HIV Env‐Nef in a larger sample size. These data may help to gain insights into the mechanisms involved in HIV‐Pulmonary Hypertension.

This research was supported by UPR‐PRISE (NIH/NIGMS‐R25GM096955), NHLBI K01‐HL103196 (Almodóvar), and R01‐HL083491 (Flores).
Tubulobulbar complexes facilitate basal junction turnover in the seminiferous epithelium
Authors: Du Min , Young J'Nelle , De Asis Marc , Cipollone Jane , Roskelley Calvin , Takai Yoshimi , Nicholls Peter , Stanton Peter , Deng Wanyin , Finlay Brett , Vogl Wayne
Published: June 2021

Tubulobulbar complexes (TBCs) are cytoskeleton‐related structures that develop at sites of intercellular attachment in the seminiferous epithelium. It is increasingly evident that TBCs found apically in the epithelium play a role in junction internalization, but much less is known about TBCs that form at basal junction complexes. To test the hypothesis that the complexes facilitate basal junction turnover events, we demonstrate that they do form basally in rat Sertoli cells in primary cultures. Morphologically intact tight and gap junctions are observed in basal TBCs in rats. In addition, immunological probes for three types of junction proteins react with linear structures at the light level that we interpret as TBCs, and probes for EEA1 and Rab5 react in similar locations. Significantly, fluorescence staining patterns for actin and claudin‐11 indicate that the amount of junction present is greatly reduced over the time period that TBCs are known to be most prevalent during spermatogenesis. We use transfection techniques on cultured Sertoli cells to show that basal TBCs likely internalize ‘intact’ junctions. On the basis of our findings we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium. Supported by an NSERC Discovery Grant to AWV.

Grant Funding Source: NSERC Discovery Grant
Beta‐adrenergic receptors regulate cardiomyocyte proliferation via PI3K/AKT pathway and cell cycle kinases in neonatal mouse heart
Authors: XU Yanchun , Zhang Weizhi , Yano Naohiro , Mao Quanfu , Padbury James , Tseng Yi‐Tang
Published: June 2021

Objective

Chronic β‐adrenergic receptor (βAR) stimulation leads to cardiac hypertrophy. We have demonstrated a cardiac βAR‐PI3K crosstalk in adult mouse. Since both cardiac βAR and PI3K signaling activities are developmentally regulated, with high activities in fetal/early newborn stages, this study was set out to elucidate the biological consequence resulted from the βAR‐PI3K crosstalk in neonatal mouse heart.

Methods

Newborn (2.5 days old) C57BL/6 mice were treated with propranolol (10 mg/kg, ip) for 1 hr to block βAR. This was followed by treatment with EdU (40 mg/kg, ip) for 4 more hr to monitor cell proliferation. Heart cryosections were processed for double immunofluorescence staining to detect EdU, α‐actinin‐2 and MEF 2A. Cardiac tissue lysates were subject to in vitro lipid kinase assay for measurement of PI3K activity and to Western blotting to measure the expressions of PI3K pathway related proteins and cell cycle machinery proteins.

Results

βAR blockade significantly decreased cardiomyocyte proliferation (*p<0.05). βAR blockade also significantly reduced cardiac PI3K activity, and phosphorylation of its downstream effectors, including Akt (Ser473 and Thr308), GSK‐3α (Ser21), GSK‐3β (Ser9), FOXO1 (Thr24) and FOXO3a (Thr32, Ser253). However, acute βAR blockade did not affect the levels of mTOR/P70S6K/S6 axis and ERK1/2. βAR blockade also decreased the expression of cell cycle related proteins including p‐Chk1, p‐Chk2 and 14–3‐3 proteins, but increased the expression of Myt‐1 and p‐cdc2.

Conclusions

Our study suggests a novel mechanism regulating cardiomyocyte proliferation in newborn mouse. This involves the crosstalk from βAR to PI3K signaling pathway and results in alteration in cell cycle.
Visual correlation between iron, amyloid‐beta, and tau depositions in the medial temporal lobe of Alzheimer's disease post‐mortem brains
Authors: Stone Megan Alicia , Bridgewater James , Kavafyan Talar , Steed Kevin , Salin Megan , Saline Ashley , Barzee Brigham , Stark Elena , Dong Hongwei , Toga Arthur , Vinters Harry , Wisco Jonathan
Published: June 2021

Alzheimer's disease (AD) is becoming more prevalent in an aging society, and numerous efforts have been devoted to learning about the disease. Consecutive sections of the hippocampus, entorhinal cortex, and frontal lobe regions from brains diagnosed with AD were stained for non‐heme iron (Fe) using a peroxidase enhanced Perl's stain, and for amyloid beta‐40 (Aβ‐40) and tau protein (tau) using an immunohistochemistry stain. We compared the qualitative spatial correlation between Fe, Aβ‐40, and tau deposits from images of the tissue. We determined that in human AD temporal lobe sections, Fe corresponded with Aβ‐40 depositions and heavy tau protein depositions. In sections from the hippocampus and entorhinal cortex region we found a correlation between Fe and Aβ‐40, but little correlation between Fe and tau depositions. The correlations found in the temporal lobe section suggests that one potential cause of AD is related to the build‐up Fe, Aβ‐40, and tau as well as Fe and Aβ‐40 in the hippocampus and entorhinal cortex.

Grant Funding Source: NIH/NIA 1 R21 AG037843‐02, NIH/NCRR P41 RR013642‐12S1, Translational Research Fund (UCLA), and the McGinty Family Foundation
Alteration in the sphingolipid metabolism leads to activation of the apoptotic cascade in the MPTP induced mouse model of Parkinson's disease
Authors: Sivasubramanian Meenalochani , Dheen S T , Tay Samuel SW
Published: June 2021

Parkinson's disease is a degenerative disorder of the central nervous system. It results from the death of dopaminergic neurons in the substantia nigra of the mid brain. Sphingolipids are important for neuronal survival. The sphingolipid metabolic pathway is regulated by two enzymes, namely Sphk1 and Sphk2. Sphk1 phosphorylates sphingosine leading to the formation of S1P which is essential for the survival of the neurons. The present study has demonstrated that both SPHK1 and its isoform SPHK2 in dopaminergic neurons of the substantia nigra of the brain showed a significant decrease in the MPTP‐induced mouse model of Parkinson's disease. This decrease was confirmed in both tissue samples and in the MN9D cell line treated with MPP+. Furthermore inhibition of the isoforms using dimethylsphingosine (DMS) leads to apoptosis of the neurons which was analysed using flow cytometry. Moreover Caspase 3 seems to increase significantly in both the MPP+ and DMS treated groups. The present results suggest that, the alteration of sphingolipid metabolic pathway may be a cause for the death of dopaminergic neurons in the MPTP induced mouse model of Parkinson's disease. Supported by grant no: MOE 2009‐T2–1‐061 from the Ministry of Education, Singapore.
Biochemical Analysis of S. cerevisiae Msh2‐Msh3 Activity in Triplet Nucleotide Repeat Expansion
Authors: Yu Xiaoyu (Zoë) , Sawant Shreya , Hingorani Manju
Published: June 2021

Mismatch repair mechanisms improve DNA replication fidelity to only one base‐pairing error in 109 nucleotides. Msh2‐Msh6 (MutSα) is responsible for initiating repair of single base mismatches and small insertion/deletion loops and Msh2‐Msh3 (MutSβ) is responsible for insertion/deletion loops. However, Msh2‐Msh3 can also stabilize loops/hairpins formed by repeat nucleotide sequences and contribute to triplet nucleotide repeat (TNR) expansion. The goal of this study is to investigate the S.cerevisiae Msh2‐Msh3 mechanism of action by transient kinetics, in order to understand how it can accomplish both loop repair and expansion. Thus far, yMsh2‐Msh3 protein complex has been expressed in E.coli host cells and can be purified in large quantity for biochemical studies. I am optimizing the purification protocol by testing different ion exchange and affinity exchange chromatography resins. I am also developing different DNA substrates to assay the affinity and kinetics of yMsh2‐Msh3 binding to loops/hairpins and its coupled ATPase activity. This study is expected to yield kinetic parameters that distinguish between yMsh2‐Msh3 actions that trigger loop repair versus TNR expansion.
PCNA tyrosine phosphorylation by EGFR promotes genome instability
Authors: Ortega Janice , Li Jessie , Ensor Mark , Gu Liya
Published: June 2021

The proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and repair, including DNA mismatch repair (MMR), an essential mechanism that ensures replication fidelity. PCNA is required for MMR at both the initiation and re‐synthesis steps. Recent studies have shown that PCNA is phosphorylated at tyrosine 211 (Y211) by epidermal growth factor receptor (EGFR), whose overexpression is associated with during tumor progression. We therefore hypothesize that the EGFR tumor‐promotion function is through PCNA Y211 phosphorylation by altering the MMR function. To test this hypothesis, we directly examined the influence of PCNA Y211 phosphorylation on MMR activity using a functional in vitro repair assay. We demonstrate here that nuclear extracts derived from tumor cells with high levels of phosphorylated PCNA are defective in MMR, and that the deficiency can be restored by purified non‐phosphorylated PCNA, suggesting that PCNA Y211 phosphorylation inhibits MMR. The inhibition was due to the inability of the phosphorylated PCNA to interact with mismatch recognition proteins MutSα and MutSβ. We also show that the PCNA‐phosphorylated nuclear extracts have a delayed DNA resynthesis during MMR, which is coupled with nucleotide misincorporations, leading to an elevated mutation frequency. Our study therefore demonstrates that a posttranslational modification promotes genome instability and tumor progression by negatively regulating the MMR function and DNA synthesis. Thus, the work provides a potential new biomarker for cancer progression.
The DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase
Authors: Eapen Vinay Verghese , Dotiwala Farokh J , Harrison Jacob C , Eden Ayelet A , Ranade Vikram , Yoshida Satoshi , Haber James E
Published: June 2021

Budding yeast cells suffering a single, unrepaired DNA double strand break (DSB) arrest prior to anaphase for 12–15 h, in a Mec1 (ATR)‐dependent manner, following which checkpoint signaling is down‐regulated to allow resumption of cell cycle progression in a phenomenon known as adaptation. A similar, but not genetically equivalent process of recovery occurs after the DSB has been repaired. In this study, we found that inducing autophagy in wild‐type cells suffering DNA damage, either by the addition of rapamycin, by overexpression of a dominant‐active version of Atg13 (ATG13–8SA) or by deletion of genes required for Golgi‐Associated Retrograde Transport (GARP) prevented both adaptation and recovery. These effects were due to the DNA damage‐dependent mislocalization of the mitotic activator Esp1 (Separase) via the degradation of its chaperone/inhibitor Pds1 (Securin). Failure to adapt is dependent on vacuolar proteases and autophagy, as these defects were suppressed by 1) deleting the vacuolar protease genes or by 2) deleting Autophagy related genes (specifically those required for the Cytoplasm‐to‐Vacuole transport (CVT) pathway). Overexpression of ATG13–8SA also promoted the mislocalization of Pds1 and Esp1 after induction of a DSB. Arrest persisted even as Rad53 (Chk2) hyperphosphorylation diminished after 12–15 h. Finally we note that ATG13–8SA overexpressing cells were sensitive to a wide‐array of DNA damaging agents and autophagy mutants displayed a reduced duration of checkpoint arrest. Overall, our results suggest a role for DNA damage‐induced autophagy in maintaining robust checkpoint arrest by targeting mitotic activators for degradation and specifically implicate the CVT pathway as a target of the DNA damage checkpoint.
Regulation of NFkB‐dependent Interleukin‐8 in Human Macrophages By Nuclear IkBa and Proteasome Inhibition
Authors: Sanacora Shannon , Manna Subrata , Singha Bipradeb , Chang Tzu‐Pei , Ramaswami Sitharam , Vancurova Ivana
Published: June 2021

Transcription of NFkB‐dependent genes is regulated by the nuclear translocation and accumulation of IkBa. In the nucleus, IkBa suppresses transcription of NFkB‐dependent pro‐inflammatory and anti‐apoptotic genes providing potential therapeutic targets. Post‐induction repression in stimulated cells, inhibition of the CRM1‐ dependent nuclear IkBa export by leptomycin B (LMB), and the inhibition of the 26S proteasome have all been found to induce nuclear IkBa accumulation. However, studies from our laboratory have shown that the inhibition of NFkB‐dependent transcription by nuclear IkBa is promoter specific. Transcription is also dependent on the subunit composition of NFkB dimers and post‐translational modifications of the recruited NFkB proteins. In this study, we have investigated the mechanisms responsible for the regulation of gene expression of the NFkB‐dependent proinflammatory chemokine interleukin‐8 (IL‐8) by nuclear IkBa and proteasome inhibition in human macrophages. Our results show that a different mechanism regulates the NFkB‐dependent transcription of IL‐8 compared to other NFkB‐dependent proinflammatory and antiapoptotic cytokines, such as TNFa, IL‐1 and IL‐6. While both IL‐8 and TNFa promoters are occupied by p65/65 homodimers in LPS‐stimulated macrophages, TNFa expression is inhibited by the LMB‐induced nuclear IkBa and proteasome inhibitor bortezomib, yet the IL‐8 expression is not inhibited by the nuclear IkBa, and is significantly up‐regulated by bortezomib. In addition, while the p65 NFkB homodimers recruited to IL‐8 promoter are phosphorylated on S536, p65 NFkB recruited to TNFa promoter is not phosphorylated. Together, our data indicate that the S536 p65 phosphorylation plays a crucial role in the regulation of IL‐8 transcription by nuclear IkBa in human macrophages.

*Funded by NIH grants GM079581 and AI085497 to IV.
A His‐Rich Region in the Linker Region of PAM May Sense Lumenal pH
Authors: Vishwanatha Kurutihalli S , Mains Richard E , Eipper Betty A
Published: June 2021

PAM, a type I integral membrane protein, catalyzes sequential reactions that yield amidated peptides. When peptide secretion is stimulated, PAM deposited on the plasma membrane is endocytosed and recycled to secretory granules or degraded. Although expression of PAM alters cytoskeletal organization and limits secretogogue stimulated secretion, the underlying mechanism is poorly understood. Lumenal pH varies in the biosynthetic and endocytic pathways and we hypothesized that a His‐rich sequence in the linker connecting the enzymatic domains of PAM plays a role in this process. The effects of mutating 3 His residues to Ala (H3A) in PAM1 (PAM1/H3A) were evaluated by comparing AtT‐20 corticotrope tumor cell lines expressing PAM1 or PAM1/H3A to wildtype AtT‐20 cells. Cytoskeletal organization in PAM1/H3A cells resembled wildtype cells more than PAM1 cells. Based on POMC biosynthetic labeling, lumenal protein cleavage and granule accumulation at the tips of processes, the blockade of regulated secretion observed in PAM1 cells is absent in PAM1/H3A cells. Metabolic labeling revealed more rapid turnover of PAM1/H3A than PAM1. After endocytosis, PAM1/H3A failed to return to secretory granules as efficiently as PAM1. Whether the pH‐sensitive lumenal domain of PAM interacts with lumenal proteins or affects signaling through the soluble PAM cytosolic domain is not yet clear. NIH grant DK32949.
The ESCRT machinery regulates Akt signaling mediated by the G protein‐coupled receptor CXCR4
Authors: Verma Rita , Marchese Adriano
Published: June 2021

The chemokine receptor CXCR4/stromal cell‐derived factor (SDF)‐1 alpha signaling axis is important in health and disease. Dysregulated CXCR4 signaling has been linked to cardiovascular disease, WHIM syndrome and cancer. However, despite the role of aberrant CXCR4 signaling in disease, the molecular mechanisms that govern CXCR4 signaling are poorly understood. The objective of this study is to elucidate the molecular mechanisms that govern CXCR4 signaling. Activated CXCR4 is rapidly ubiquitinated and sorted for lysosomal degradation via the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT machinery (ESCRT‐0, I, II and III) act in a coordinated manner to target ubiquitinated membrane proteins into intraluminal vesicles of multivesicular bodies for subsequent lysosomal degradation. While the ESCRT machinery mediates downregulation of CXCR4, our data indicate that it also has a novel positive role in CXCR4 signaling. Here, we provide evidence that the ESCRT machinery mediates CXCR4‐induced activation of Akt. Our data indicate that depletion of ESCRT‐0, I and II by siRNA inhibits CXCR4‐induced phosphorylation of Akt on serine 473, which corresponds to a decrease in Akt activity as phosphorylation of Tsc2, an Akt substrate, is also reduced. We show that Tsg101 (an ESCRT‐I subunit) interacts directly with Akt and under basal conditions Tsg101 and Akt are constitutively associated, as assessed by co‐immunoprecipitation; CXCR4 activation disrupts this interaction. Our data also reveal that CXCR4 internalization is required for Akt activation. Therefore CXCR4‐induced Akt activation is mediated by CXCR4 internalization onto endosomes and by the ESCRT machinery. This work was supported by grants from the AHA and the NIGMS.
Structural and enzymatic study of GlgE, a validated anti‐tubercular drug target
Authors: Lindenberger Jared J , Veleti Sri Kumar , Sucheck Steven J , Ronning Donald R
Published: June 2021

The emergence of antibiotic resistance strains of Mycobacterium tuberculosis (M. tb) necessitates the identification of new drug targets and development of new therapeutics to combat this disease. The glgE gene that encodes a maltosyl transferase enzyme (GlgE) is essential for the viability of M. tb. GlgE possesses maltosyl transferase activity using maltose 1‐phosphate (M1P) as the donor substrate and is responsible for the synthesis of an α‐1,4 glucan in the bacterium. Inactivation of glgE leads to rapid killing of the bacterium via a toxic build up of M1P. Because of this rapid and novel method of killing, targeted inhibition of GlgE offers a new avenue for fighting this disease. We determined the crystal structure of GlgE to a resolution of 3.0 Å. Structural similarities between GlgE and other members of the α‐amylase family of enzymes indicate a probable enzymatic mechanism, using residue D418 as the catalytic nucleophile. However, active site electrostatics of GlgE suggests that residue D383 may be more important for catalysis. We tested mutants of both residues in order to ascertain their role in the catalytic activity of GlgE. These data provide insight into the enzymatic mechanism of GlgE and will allow for the future design and development of GlgE specific inhibitors.
A naturally variable residue in the S1 subsite of M1‐family aminopeptidases modulates catalytic properties and promotes functional specialization
Authors: Klemba Michael , Ragheb Daniel , Schubot Florian , Dalal Seema
Published: June 2021

The objective of this study was to identify principles governing the substrate specificity of M1‐family aminopeptidases at the S1 subsite, which interacts with the sidechain of the first residue of peptide substrates. By sequence comparison, we identified a residue in the S1 subsite that is highly variable and hypothesized that changes in this residue modulate S1 specificity. To test this hypothesis, we generated a large number of mutants at this position in the aminopeptidase PfA‐M1 from Plasmodium falciparum and in PepN from Escherichia coli. The kinetic properties of each mutant were characterized using a panel of natural dipeptide substrates. These studies revealed a complex interplay between the identity of the variable S1 residue and the kinetic properties of the enzyme. A dramatic shift in substrate specificity favoring the straight‐chain P1 sidechains Arg, Lys and Met could be effected by changing the S1 residue to proline. Structural analysis revealed that the proline residue induced a 1.1 Å shift of the polypeptide backbone, which resulted in constriction of the cylindrical S1 subsite. Together, the biochemical and structural data suggest that variation of a residue of the S1 subsite can modulate the catalytic properties of M1‐family aminopeptidases and ultimately contribute to functional specialization.

This work was supported by National Institutes of Health grant AI077638.
Decreased autophagic flux as a mechanism of glucose‐ and fatty acid‐induced dysfunction in human aortic endothelial cells
Authors: Weikel Karen Anne , Ido Yasuo , Ruderman Neil
Published: June 2021

The mechanisms by which cellular stress influences autophagic flux in cardiovascular disease vary between tissues and cellular contexts. The cellular response to glucose and fatty acids is regulated by adenosine monophosphate‐activated protein kinase (AMPK), but whether AMPK influences the effects of hyperglycemia and hyperlipidemia on autophagy in endothelial cells remains to be established. In human aortic endothelial cells (HAECs), we evaluated the effects of glucose and fatty acids on endothelial dysfunction by measuring transcription of endothelial nitric oxide synthase, adhesion of THP‐1 monocytes, and cellular ATP levels. Autophagic flux was assessed based upon expression levels of p62 and LC3‐II. Compared to HAECs in non‐stressed conditions, HAECs exposed to elevated concentrations of glucose and palmitate had a smaller ATP/ADP ratio and showed increased adhesion of monocytes. Under these hyperglycemic and hyperlipidemic conditions, autophagic flux was also decreased. Activation of AMPK attenuated glucose‐ and palmitate‐induced changes in these parameters, and chemical induction of autophagic flux diminished glucose‐ and palmitate‐induced changes in ATP/ADP and monocyte adhesion. Up‐regulation of autophagic flux may be one way in which AMPK can ameliorate glucose‐ and palmitate‐induced dysfunction in HAECs.

Research support was provided by NIH/NHLBI HL068758
Evaluation of a Parkinson's disease model in medaka fish
Authors: Ferng Shiana J. , Gonzalez David E. , Nguyen Mary N. , Sherman Scott J. , Falk Torsten , Rilo Horacio L.
Published: June 2021

Parkinson's disease (PD) is the 2nd most common neurodegenerative disease. Dopaminergic (DA) neurons in the substantia nigra of the brain are the most vulnerable to PD. 6‐ hydroxydopamine (6‐OHDA) is a neurotoxin structurally similar to dopamine that is transported inside the brain and destroys DA neurons. The resulting PD symptoms can be identified through behavioral analysis and verification of DA neuron loss through immunohistochemical analysis in medaka fish (Oryzias latipes). Medaka are well‐suited as a PD model because they are transparent during most stages of development, produce numerous offspring, and readily absorb toxicants through their skin. To establish a PD medaka model to time and cost‐efficiently screen drugs that may change the progression of PD, medaka were exposed to varying concentrations of 6‐OHDA through the water they swam in. Temporary acquired PD symptoms were evidenced by an initial decrease in total distance swam by 6‐OHDA‐treated medaka and changes in turn angle, but a complete behavioral recovery was evident at following time points. The results indicate that medaka exposure to 6‐OHDA in the water is not sufficient to induce loss of DA neurons, presumably due to a reduced blood‐brain barrier penetrance of 6‐OHDA in medaka as compared to the established zebrafish model. Support: Univ. of AZ (UA) from Howard Hughes Medical Inst. (#52006942); NIH NINDS 1R25NS076437; UA Dept. Neurology.
ICAM1 ligation‐induced transmigratory complex formation regulates leukocyte transendothelial migration
Authors: Williams Marcie R , Auerbach Scott D , Alcaide Pilar , Newton Gail , Luscinskas F W
Published: June 2021

ICAM1 expression is upregulated on activated endothelium and is important in leukocyte adhesion and transendothelial migration (TEM). ICAM1 engagement initiates several signaling events including cortactin phosphorylation and association with the ICAM1 cytoplasmic tail. We hypothesized that interactions between cortactin and a putative SH3 binding domain in the ICAM1 tail regulate leukocyte TEM. To test this we mutated 3 amino acids within the ICAM1 cytoplasmic tail such that the putative SH3 binding domain was no longer present (SH3Mut ICAM1). We used adenoviral infection to express WT ICAM1 or SH3Mut ICAM1 in human umbilical vein endothelial cells (HUVEC). Standard biochemical methods and transmigration assays were used to assess functional relevance of the SH3 mutation. ICAM1 engagement triggers cortactin association with ICAM1. Approximately 50% less cortactin associates with SH3Mut ICAM1 (n=3, p<0.05). Neutrophil TEM is also reduced by 43% in cells expressing SH3Mut ICAM1. Furthermore, ICAM1 ligation induces the formation of a large transmigratory complex which includes ICAM1, VE‐Cad, p120, pSrc, and cortactin. Formation of this complex is, in part, dependent on the SH3 binding domain. Our study identifies an SH3 binding motif in the ICAM‐1 cytoplasmic tail that is necessary for association with cortactin and provides evidence that transmigratory complex formation is important for TEM.
Ultra High‐throughput Screening Uncovers New Activities in Phosphatases of the Haloalkanoic Acid Dehalogenase Superfamily (HADSF)
Authors: Farelli Jeremiah D , Huang Hua , Liu Chunliang , Pandya Chetanya , Al Obaidi Nawar F , Hillerich Brandan S. , Love James D , Almo Steven C , Dunaway‐Mariano Debra , Allen Karen N
Published: June 2021

Since the first genomes were sequenced, there has been an exponential increase of protein sequences deposited into databases worldwide. This increase in sequence data has allowed for dramatic improvements in our understanding of the metabolism of organisms. Unfortunately, as the number of protein sequences grows, the number of definitive functional assignments diminishes. Current methods involve comparison of sequence identity between known proteins and newly sequenced ones. As the sequence identity decreases, proteins are annotated as “hypothetical,” and certainty in annotation shrinks. Thus a strategy for reliably determining protein function is necessary. The HADSF consists of Mg2+‐dependent enzymes that catalyze a wide range of reactions including dehalogenation, phosphoryltransfer and dephosphorylation. Here we present the role that high‐throughput screening (HTS) might play in functional discovery for families with members having common chemical function but divergence in physiological substrate (e.g., kinases). We highlight five areas of discovery enabled by the HTS‐guided substrate profiling: 1) uncovering new metabolic pathways, 2) tracking of orthologs, 3) distribution of promiscuous versus specific family members, 4) annotation of hypothetical proteins, 5) functional assignment of proteins with known structures. This research is supported by the NIGMS (U54 GM093342).
Synthesis, characterization and toxicological evaluation of maltodextrin capped cadmium sulfide nanoparticles in human cell lines and chicken embryos
Authors: Leon‐Buitimea Angel , Rodriguez‐Fragoso Patricia , Reyes‐Esparza Jorge Alberto , Rodriguez‐Fragoso Lourdes
Published: June 2021

Our aim was to synthesize, characterize and evaluate maltodextrin coated cadmium sulfide (CdS‐MD) semiconductor nanoparticles in human cell lines and chicken embryos. The particle size and morphology of the CdS‐MD nanoparticles were characterized using TEM. Toxicological evaluation was carried out by using human tumor cell lines. Fertile chicken eggs were used for stereoscopic evaluation of growth defects in CdS‐MD nanoparticles exposed embryos. CdS‐MD nanoparticles at 5ìg/mL induced cell death by apoptosis and necrosis in MDA‐MD‐231 cells in a dose response manner. The exposure of these cells to 7–9ìg/mL of CdS‐MD nanoparticles induced ROS production. CdS‐MD nanoparticles‐treated MDA‐MB‐231 cells at >;3ìg/mL increased cell proliferation in a dose response manner at 7 days. Exposures of chicken embryos to 6ìg/mL CdS‐MD nanoparticles resulted in a dose‐dependent increase in anomalies centered on the heart, central nervous system, placodes, neural tube and somites. Our results indicate that CdS‐MD nanoparticles induce cell death and alter cell proliferation in human cell lines at concentrations higher than 3ìg/mL, and they are embryotoxic at doses of 6ìg/mL and higher.
Amino‐functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages
Authors: Lunov Oleg , Syrovets Tatiana , Loos Cornelia , Nienhaus G. Ulrich , Mailänder Volker , Landfester Katharina , Simmet Thomas
Published: June 2021

Nanoparticles are increasingly used in different biological and biomedical applications. However, their impact on cell functions is not well understood. Here we demonstrate that aminofunctionalized polystyrene nanoparticles (PS‐NH2) of ~100 nm but not carboxy‐functionalized (PS‐COOH) or non‐functionalized (PS) particles of the same size induce NLRP3 inflammasome activation and subsequent release of IL‐1β by human macrophages. PS‐NH2 were found to induce time‐dependent lysosomal destabilization followed by release of lysosomal enzymes, which trigger mitochondrial damage and increased formation of reactive oxygen species (ROS). Accumulation of mitochondria‐derived ROS causes oxidation of thioredoxin and its dissociation from the thioredoxin‐interacting protein (TXNIP). Liberated TXNIP forms a complex with the inflammasome, which promotes recruitment and activation of caspase‐1 followed by processing of pro‐IL‐1β to IL‐ 1β. Treatment of macrophages with the ROS scavenger N‐acetyl‐ L‐cysteine abolishes both, the caspase‐1 activation and the subsequent release of IL‐1β. Using an in vitro knockdown approach, we showed that PS‐NH2 but not PS‐COOH or PS induce activation of specifically the NLRP3 inflammasome. Thus, aminofunctionalized nanoparticles may induce proinflammatory activation of human macrophages, an effect that can be antagonized by radical scavengers. Supported by the DFG.
Live‐cell imaging of the association of STAT6‐GFP with mitochondria
Authors: Khan Rasel , Sehgal Pravin B
Published: June 2021

Having failed to confirm previous reports of the association of STAT3 with mitochondria, especially using STAT3‐GFP localization assays, we addressed whether other STAT‐family proteins might associate with mitochondria. Strong anti‐STAT6‐ immunolocalization with mitochondria was apparent in fluorescence and electron microscopy assays of Hep3B and HPAECs first washed with a digitonin‐sucrose buffer to remove bulk soluble STAT proteins. In live‐cell imaging studies, STAT6‐ GFP, but not N1‐GFP, was observed to constitutively colocalize with MitoTracker‐ and TMRE‐positive mitochondria, and with mitochondrial F1‐ATPase when assayed by immunofluorescence after fixation. This association was Tyr‐phosphorylation independent in that a STAT6 truncated protein (STAT61–459‐GFP) which lacked the SH2 domain (517–632) and the Y641 phosphorylation site also accumulated in MitoTracker‐positive mitochondria. This was consistent with the discovery that anti‐ STAT6‐immunofluoresence also associated with mitochondria in MEFs from both wild‐type and the STAT6SH2−/SH2−mouse. MEFs from the latter mouse (deleted in the STAT6 SH2 domain ‐ 505‐ 584 aa) expressed a 50 kDa STAT6 truncated protein detectable in whole cell and mitochondria‐enriched fractions. These data provide the first definitive evidence of the association of any STAT‐protein family member with mitochondria ‐ that of STAT6.
Mitochondrial dynamics regulate brown adiopcyte energy expenditure
Authors: Mahdaviani Kiana , Wikstrom Jakob D , Sereda Samuel B , Si Yaguang , Liesa Marc , Las Guy , Twig Gilad , Corkey Barbara , Cannon Barbara , Nedergaard Jan , Shirihai Orian S.
Published: June 2021

Brown adipose tissue has gained much attention as a potential therapeutic approach for obesity and insulin resistance. This tissue is unique in its ability to go through hormonal mediated fast transition from efficient, ATP‐generating metabolism to inefficient, heat producing metabolism. As such, the brown adipocyte is a unique system in which the relationship between mitochondrial architecture and bioenergetics can be studied.

Brown pre‐adipocyes were harvested from C57BL6/J mice and differentiated in culture. Oxygen consumption (using Seahorse XF24) and mitochondrial membrane potential (using TMRE and Zeiss LSM 710 confocal microscope) were measured before and after activation with NE (1 uM) and FFA (Plamitate or Oleate 0.4 mM).

Immediately upon stimulation, mitochondrial networks underwent complete fragmentation and arrest of fusion. Fragmentation could only be achieved by beta3‐adrenergic stimulation and could not be mimicked by treatment with free fatty acids. Examination of the mitochondrial fusion proteins revealed that the long form of Opa1 was cleaved during the activation. Remarkably, within each cell, mitochondrial membrane depolarization and mitochondrial fragmentation propagated in a wave‐like fashion. To evaluate the role of mitochondrial fragmentation in energy expenditure, brown adipocytes were infected with a DRP1 dominant negative adenovirus. Inhibition of mitochondrial fission resulted in reduced levels of hormone stimulated oxygen consumption and membrane potential depolarization by 50%.

Together, these data reveals that mitochondrial fission is essential for the activation of uncoupled respiration in brown adipocytes and its transition from bioenergetic efficient to inefficient metabolism.
Para‐amino benzoic Acid (pABA) serves as ring precursors of Coenzyme Q in both yeast and E. Coli.
Authors: Xie Letian , Weng Emily , Clarke Catherine F.
Published: June 2021

Coenzyme Q (ubiquinone or Q) is a lipid electron carrier in the electron transport chain. In yeast Saccharomyces cerevisiae nine genes, designated COQ1 through COQ9, have been identified as being required for Q biosynthesis. Generally, 4‐hydroxybenzoic acid (4‐HB) serves as the aromatic ring precursor to Q biosynthesis. Recently, para‐aminobenzoic acid (pABA), a well‐known precursor of folate, has been discovered as an alternative ring precursor to Q biosynthesis in yeast. This was a surprising finding because folate is synthesized de novo from pABA in yeast, but is a vitamin for human. In this study, we find pABA is also an aromatic ring precursor in Q biosynthesis E.Coli. We utilize liquid chromatography and mass spectrometry (LC‐MS/MS), and show that E.Coli. HW272 cells labeled with 13C6‐pABA synthesize 13C6‐Q8. Further experimental results suggest that Q biosynthesis from pABA in yeast and E.Coli. differ at the initial stages. This work was supported by National Science Foundation Grant 0919609, National Institutes of Health (NIH) Grant GM45952, and Ruth L. Kirschstein National Research Service Award GM007185. The LC‐MS/MS determination of Q content was supported by Grant Number S10RR024605 from the National Center for Research Resources.