bluuebunny/crossref_metadata_2025 huggingface dataset:
Pilot study: quantification of motor unit stability, or “jiggle”, in amyotrophic lateral sclerosis (731.11)
Authors: Hourigan Maddison , Ives Colleen , Johnson Marjorie , Rice Charles , Doherty Timothy
Published: June 2021

Loss of motor units (MUs) have been found in aging, neuropathies, and motor neuron diseases. With MU loss, subsequent collateral reinnervation results in unstable motor unit potentials (MUPs) with variability in firing times and generation of muscle fiber action potentials. Jiggle is a measure of MUP stability, which can be calculated using Decomposition‐based Quantitative Electromyography (DQEMG). Two previous studies have attempted to measure jiggle, but both studies were limited by the EMG decomposition algorithms used. The purpose of this study is to measure jiggle in the upper trapezius muscle of 11 amyotrophic lateral sclerosis (ALS) patients compared to age‐matched healthy controls. Jiggle will be calculated using the normalized value of the median consecutive amplitude difference (CAD). Jiggle will also be compared to other MUP parameters including motor unit number estimation (MUNE), mean surface MUP (S‐MUP) size, MUP amplitude, and near fiber count. As a result of denervation and incomplete reinnervation, we expect to see significantly higher jiggle values in patients with ALS relative to controls. We also expect jiggle to be negatively correlated with MUNE, and positively correlated with mean S‐MUP size, MUP amplitude, and near fiber count. If successful, jiggle may be a valuable diagnostic parameter or outcome measure in age‐related sarcopenia, neuropathies, or motor neuron diseases such as ALS.
A quantitative analysis of skin collagen of Wistar rats submitted to painful procedures in the neonatal period (734.2)
Authors: Carmo Elisabete , Gonçalves Raphaela , Sanada Luciana , Machado Nathália , Violante Izilda , Fazan Valéria Paula
Published: June 2021

We aimed to quantify skin collagen fibers in animals exposed to painful stimulation after birth. Female Wistar rats were separated into pain group (N=6) and control group (N=6). Pain group animals were stimulated with a needle whereas for controls we used a cotton swab. Stimuli were done on the right paw, twice a day for 15 days since birth. The animals were weighed, anesthetized and killed. The skin of the right and left plantar area of the hindpaws were dissected and prepared for paraffin sections that were Masson trichrome stained. Histological quantification was performed with Image J software. Quantitative data were compared between groups and differences were considered significant when p<0.05. No differences between groups were found for body weight. There was a trend towards a higher collagen percentage on the stimulated paw of the pain group, but no significant difference was found. Also, no difference was found between paws on the control group. Our data suggest that painful stimulation on neonatal period can increase skin collagen fibers at the stimulated region. An increase in the number of specimens studied will confirm this hypothesis.

Grant Funding Source: CAPES, FAPESP, CNPq and FAEPA
QnrVC reducing fluoroquinolone susceptibility in epidemic strains of Vibrio cholerae (735.6)
Authors: Kumar Pramod , Yadav Priti , Biswal Sarmistha , Kumar Dhirendra , Sharma Naresh , Bhuyan Soubhagya , Joshi Pallavi , Jain Meenu , Goel Ajay , Kumari Abha , Santosh Baby , Mishra Deepak , Varshney Akhil , Jaiswal Rishi , Singh Lokendra , Yadava Pramod
Published: June 2021

Treatment of cholera still is a global challenge for health authorities across the world. It is important to use effective antimicrobials for curbing cholera and shortens the duration of illness. As per the WHO recommendations, ciprofloxacin and doxycycline are drugs of choice to be used in treatment of severe cholera. Single doses of ciprofloxacin have been reported to be better than single dose of doxycycline in eradication of cholera bacterium from stool. Earlier we have reported that reduced susceptibility or resistance to fluoroquinolones in Vibrio cholerae O1 is associated with either delay or failure in cholera treatment. V. cholerae harbours two related type II topoisomerases, which alter DNA topology through a DNA cleavage complex. Fluoroquinolones stabilizing the DNA breakage‐reunion complex inhibit DNA replication. Point mutations in gyraseA and ParC responsible for reduced susceptibility to fluoroquinolones are well known. Here, we discuss the QnrVC mediated acquired mechanism of resistance in epidemic strains of V. cholerae.

PCR analyses and DNA sequencing revealed presence of qnrVC gene (encoding QnrVC) in epidemic V. cholerae O1 in strains from central and Eastern parts of India. Higher order structural analyses confirmed that QnrVC exhibits right‐handed quadrilateral beta‐helical fold. The shape, size, and charge distribution on QnrVC is reminiscent of a 30‐bp long B‐form DNA. Docking studies revealed that it occupies the entire length of the G segment DNA binding site of type II topoisomerases, thus interfering with fluoroqunolone action. Strains harbouring the qnrVC elements were either resistant or less susceptible to ciprofloxacin as per CLSI, 2010 USA guidelines. Cloning of qnrVC in E. coli transformant mediated 4‐8 folds increment of MIC for ciprofloxacin. The use fluoroquinolones in cholera cases should be closely monitored and development of alternate chemotherapeutic agents targetting transition type II topoisomerases should be given due priority.

Grant Funding Source: UGC, DBT and DST, Govt. of India
ATM regulates glioma migration and invasion primarily via MEK‐ERK and not AKT signaling (735.8)
Authors: Allen Jasmine
Published: June 2021

Glioblastoma multiforme (GBM) is a lethal brain cancer with a life expectancy of only 12‐15 months. Current standard treatment for GBM is surgery and chemoradiation. However, there are many difficulties when it comes to treating this aggressive brain cancer due to inherent radio‐ and chemo‐resistance. Hence, there is a critical need for new therapeutic approaches to make radiation/chemotherapy more effective. Ataxia telangiectasia (A‐T) patients are extremely sensitive to ionizing radiation. The protein mutated in A‐T, ATM (A‐T mutated), controls the cells’ response to radiation and is referred to as the DNA damage response (DDR). ATM is a master protein kinase that targets numerous proteins during the DDR including regulators of cell cycle checkpoints, DNA repair, and apoptosis. In addition, we have shown that without any radiation, ATM might regulate glioma dispersal. Using a highly specific inhibitor of the ATM kinase (ATMi), we recently showed that ATM regulates both ERK and AKT signaling and controls glioma cell migration and invasion in vitro. In order to determine whether MEK‐ERK and/or AKT signaling regulate migration and invasion downstream of ATM, glioma cells were exposed to a MEKi (PD0325901) or AKTi (MK‐2266) in ‘scratch’ and invasion assays. We found that MEKi inhibited both migration and invasion whereas ATKi did not affect migration and only partially (□40%) inhibited invasion. Then, we used a genetic approach and knocked‐down ATM in human glioma cells. In vitro growth and survival studies showed that ATM KD cells were more radiosensitive and grew slower than control cells. Furthermore, we recapitulated the findings with the ATMi, MEKi, and AKTi and showed that ATM KD cells have reduced ability to migrate and invade in vitro suggesting that ATM regulates glioma migration and invasion primarily via MEK‐ERK and less so via AKT signaling. Ongoing studies are determining whether orthotopic ATM KD tumors grow slower and are less invasive than matched control tumors.

Grant Funding Source: Supported by NIH
Position‐dependent instability of ribosomal DNA repeats (736.1)
Authors: Hochwagen Andreas , Wang Danni , Mansisidor Andres
Published: June 2021

Repetitive DNA arrays are ubiquitous in eukaryotic genomes and represent a special challenge for genome integrity because of the risk of non‐allelic recombination. We used unique sequence tags marking individual repeats in the tandem ribosomal DNA (rDNA) array of budding yeast to determine the likelihood of non‐allelic recombination throughout the array. We found that rDNA repeat stability during meiotic recombination is strongly dependent on relative repeat position, with repeats near the rDNA border being more susceptible to meiotic DNA double‐strand breaks (DSBs) and thus non‐allelic recombination. The DSB position effect is promoted by rDNA heterochromatin and suppressed by the origin recognition complex component Orc1. Border‐dependent position effects in the rDNA also occur at the level of reporter gene expression in vegetative cells, suggesting that such effects are a reflection of a general feature of rDNA structure. Our results indicate that, despite their identity at the sequence level, rDNA repeats are functionally far from uniform. Position‐dependent epigenetic regulation may provide an opportunity to balance gene expression against the inherent instability of large repetitive DNA arrays.
Specialized human DNA polymerases can incorporate ribonucleotides during lesion bypass and DNA repair (736.5)
Authors: Maga Giovanni , Hübscher Ulrich , van Loon Barbara
Published: June 2021

During DNA replication, replicative DNA polymerases (pols) can incorporate up to 1,000,000 ribonucleotides (rNTPs) into a mammalian genome, which are subsequently removed by the specialized enzyme RNaseH2. The significance and consequences of the presence of rNTPs into the genome are still not well understood. In addition to replication, also DNA repair is a potential source of rNTPs incorporation, but very little is known on the ability of specialized repair DNA pols to incorporate rNTPs. Here we have analyzed the proficiency of rNTPs incorporation opposite normal and damaged bases by DNA pols λ, β, η and ι. Our results established a hierarchy with respect to deoxy‐ vs. ribo‐nucleotide discrimination among these DNA pols and support the notion that, especially in the presence of strongly unbalanced dNTPs/rNTPs pools, as in resting cells, DNA repair could be a major source or rNTPs incorporation.

Grant Funding Source: Supported by Italian Cancer Research Association AIRC
Investigation of lysine acetylation and autophagy in the protozoan parasite Toxoplasma gondii (737.3)
Authors: Varberg Joseph , Arrizabalaga Gustavo , Sullivan William
Published: June 2021

Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum Apicomplexa that chronically infects approximately one‐third of the world’s population. In particular, T. gondii is an opportunistic pathogen that causes severe comorbidities in AIDS patients and immunocompromised individuals. Despite the high disease burden, current drug therapy options are limited and are associated with high incidences of toxicity. Recent studies on the anti‐parasitic ionophore monensin suggest that this drug acts by inducing an autophagic cell death mechanism in the parasite. Interestingly, the T. gondii homologue of the autophagy protein Atg8 was recently identified as being acetylated in a global analysis of T. gondii protein lysine acetylation. As lysine acetylation has been shown to regulate autophagy in other species, we sought to identify the functional significance of the identified Atg8 acetylation mark in T. gondii. Parasites expressing ectopic forms of Atg8 in which the acetylated lysine was mutated to either arginine (non‐acetylateable mutation) or glutamine (an acetyl‐lysine mimic mutation) show altered Atg8 localization. Additionally, expression of the mutant forms of Atg8 led to morphological changes in the parasites reminiscent of those induced during starvation. Current studies are underway to identify if Atg8 mutants have impaired autophagic clearance, or altered ability to interact with other autophagy proteins.

Grant Funding Source: Supported by NIH AI077502 (to WJS) and NIH AI 89808‐01 (to GA)
Discrete histone marks are translated into degenerate readouts by reader proteins (739.11)
Authors: Wang Wei
Published: June 2021

The current paradigm to evaluate the combinatorial regulatory effect of histone modifications on gene transcription is to consider individual histone sites as the primary/basic functional units which are distinct from one another. However, numerous observations in chromatin biology cannot be explained by this paradigm. We propose a new framework to reconcile the evidences conflicting with the current paradigm: the individual histone sites can be functionally degenerate; thus, when considering combinatorial histone modifications, combination of these degenerate modifications but not that of the individual sites should be considered. This framework is based on our observation of significant degenerate binding between chromodomains and histone peptides, i.e. different histone sites can be recognized by the same set of chromodomains, and different chromodomains can share similar binding profiles to individual histone sites. Such degenerate binding is not dictated by amino acid sequence or PTM motif but rather rooted in the physiochemical properties defined by the PTMs on the histone peptides. This molecular mechanism is confirmed by the accurate prediction of the binding specificity by a computational model that captures the structural and energetic patterns of the domain‐peptide interaction. Our work represents the first but a crucial step towards elucidating the fundamental mechanism by which histone modifications correlate with their biological outputs.

Grant Funding Source: NIH
Phosphorylation of threonine 45 of histone H3 and the effects of alanine and glutamic acid mutations (739.5)
Authors: Yates Saiah , Campos Eric , Reinberg Danny
Published: June 2021

Epigenetics is the study of non‐genetic components that alter gene expression. One of the most notable examples is the chemical posttranslational modification of the DNA‐associated histone proteins. For my project, we focused on the effects of phosphorylation of threonine 45 on histone H3 by the STK38 kinase. In order to do this, we carried out a number of techniques: Western blotting of extracts from synchronized cells showed that the phosphorylation of H3 threonine 45 peaks at the G1/S transition of the cell cycle. Using Polymerase Chain Reaction (PCR)‐based Site‐directed Mutagenesis, we mutated a plasmid encoding wild‐type H3 into plasmids with Alanine (pET‐H3 T45A) and Glutamic Acid (pET‐H3 T45D) substitutions at threonine 45. Alanine eliminates phosphorylation while the negative charge on glutamic acid mimics the effects of threonine 45 when it is phosphorylated. We will now use these mutants to determine whether STK38 phosphorylates additional sites on histone H3 in vitro, and to immunoprecipitate histone H3 in its non‐phosphorylated and phospho‐mimic forms in vivo. This will allow us to determine whether phosphorylation at threonine 45 alters histone H3 binding proteins. From, current results we hypothesize that phosphorylation of threonine 45 plays an important role in the cell cycle, possibly having impact in the choice of whether or not the cell divides. In completing this study more will be known about how epigenetics can affect the cell cycle and more knowledge can be accessed about how histones and their modifications affect nucleosomes.

Grant Funding Source: Supported by Howard Hughes Medical Institute
Investigating the transcription initiation mechanism of E. coli RNAP by FRET (743.1)
Authors: Zhang Yurun , Sreenivasan Raashi , Heitkamp Sara , Record M.
Published: June 2021

E. coli RNA Polymerase (RNAP) recognizes the ‐10 and ‐35 upstream elements of the promoter DNA, which bends and wraps around RNAP on the back of the β subunit of the RNAP core. The recognition between RNAP and DNA promoter initiates the melting of about 13bp of DNA duplex surrounding the transcription start site in the active site of RNAP to form a promoter‐RNAP open complex (RPo). Even though transcription of DNA plays a critical role in gene expression and regulation, many important questions about the transcription initiation mechanism remains unanswered. To study the wrapping of DNA around RNAP, we have performed equilibrium FRET experiments on a strong promoter, λPr with Cy3 and Cy5 fluorophores on the upstream and downstream ends of the promoter DNA. We find that wrapping is present in closed complexes, as seen from complexes at 2°C and it persists in open complexes at 19°C. We further confirmed that the fluorophore‐labeled DNA complexed with RNAP is transcriptionally competent. Single‐labeled constructs also show permanganate reactivity, proving that fluorophore‐labeled promoter‐RNAP complexes are open. Further experiments are underway to study the kinetics of wrapping of promoter DNA around RNAP.

Grant Funding Source: NIH GM103061
Cataloging and comparing long noncoding RNAs generated at enhancers (eRNAs) in prostate, breast, and ovarian cancer cells (744.2)
Authors: Konovalov Sergiy , Mackintosh Carlos , Garcia‐Bassets Ivan
Published: June 2021

An important purpose of sequencing the human genome was to determine the number of human genes and to catalog them for future study. The final gene count matched the most conservative predictions, with gene sequences representing only 1.5% of the entire human genome. However, more recent transcriptomic analyses have revealed that the cumulative coverage of transcribed regions is at least 75‐85% of the human genome. Moreover, since these analyses did not investigate the process of transcription itself, but only the accumulation of RNAs, the range of cumulative transcribed genome is likely close to the full human genome. The question is how much of the human transcriptome (i.e. the transcribed genome) is functional and how much of the human transcriptome is transcriptional noise. Thus, we return to the question of how many genes (i.e. sequences coding for functional transcripts) are there in the human genome, and to the challenge of cataloging them.

In this study, we catalog the full repertoire of long noncoding (lnc)RNAs (length >200 nucleotides) generated at enhancers, also known as enhancer (e)RNAs, in prostate (LNCaP), breast (MCF7), and ovarian (A2780) cancer cells. We use profiles of intergenic DNaseI hypersensitive sites (DNase‐seq) and H3K4me1/3 and H3K27ac regions (ChIP‐seq) to identify enhancers, combined with global run‐on next‐generation sequencing (GRO‐seq) and RNA‐seq to identify, characterize, and compare the eRNA transcriptomes in these three cancer lines. We expect that our analyses will facilitate the study of the functionality of the eRNA transcriptome, and will contribute to distinguishing the fraction of the human transcriptome that is functional from the fraction that could be regarded as noise.
Inhibiting HIV‐1 with chimeric tRNA‐miRNA mimics (749.8)
Authors: Gao Eva , Haushalter Karl
Published: June 2021

Gene therapy utilizing RNA interference (RNAi) has the potential to repress HIV‐1 replication in patients living with HIV. This study investigates the effectiveness of microRNA (miRNA) mimics fused to tRNA in inhibiting the synthesis of proteins important to HIV‐1’s lifecycle. Expression vectors coding for chimeric RNA molecules consisting of tRNA(Arg) fused to miRNA mimics directed against either Gag‐Pol or CCR5 were designed and constructed. Using a dual‐luciferase assay in cultured cells, the effectiveness of these tRNA‐miRNA mimic constructs will be compared with the effectiveness of analogous tRNA‐short hairpin (shRNA) constructs. Furthermore, the miRNA mimic constructs will be tested to determine whether they interfere with endogenous miRNA.
Characterization of eukaryotic translation initiation factor 5A isoform A (752.13)
Authors: Tamborlin Letícia , Pereira Karina , Almeida Isadora , Meneguello Letícia , Cambiaghi Tavane , Luchessi Augusto
Published: June 2021

The eukaryotic translation initiation factor 5A (EIF5A) protein is highly conserved in eukaryotes. Some studies shown that eIF5A has important roles in cell proliferation control and HIV‐1 replication. EIF5A possesses a particular amino acid residue named hypusine that is not found in others proteins. The hypusine is essential for its activity and is produced by a post‐translational modification using spermidine as substrate. The EIF5A protein has been involved with the protein synthesis process and recently it was shown that it acts on translation of polyproline motifs. In humans, four transcript variants for EIF5A are known (variants A, B, C and D), derived from alternative splicing associated with the first exon. The variants B, C and D encode for the canonical isoform B, the main studied isoform to date. It is predicted that the variant A is responsible for the establishment of an upstream start codon in frame to the start codon of the others variants. In HeLa cells, we confirmed that variant A encodes an isoform containing an additional sequence of 30 amino acids at the N‐terminus of EIF5A (isoform A). The subcellular localizations of EIF5A isoforms were also compared. In addition, it was observed that the start codon of isoform A has a weak Kozak consensus, showing that the variant A encodes for both isoforms. The isoform A is not predicted for mice, however it was detected in different tissues. The results obtained so far contribute to the characterization of EIF5A isoform A in mammals.

Grant Funding Source: Supported by FAPESP 2010/18095‐0
The alpha‐tocopherol transfer protein regulates vitamin E transport across placental cell monolayers (758.1)
Authors: Thakur varsha , Cotton Calvin , Manor Danny
Published: June 2021

Oxidative stress is thought to cause/ play critical causative role in the etiology of pregnancy disorders such as pre‐eclampsia, intrauterine growth restriction and repeated miscarriages. Vitamin E is a major lipid soluble chain breaking antioxidant that is essential for fetal development. The alpha‐tocopherol transfer protein (TTP) is the only specific regulator of vitamin E homeostasis in vertebrates, which is expressed in liver, brain and placenta. We hypothesized that mother‐fetus transport of vitamin E across the placenta is mediated and regulated by TTP. To test this hypothesis, we used the human choriocarcinoma cell‐line BeWo cultured as monolayers on transwells. Monolayer formation was confirmed by histological evaluation and electric resistance measurements. Trans‐epithelial resistance ranged between 90‐120 Ohms, indicating functional tight junctions throughout the monolayer. Anti‐ E cadherin staining indicated the manolayers converted to a syncytiotrophoblast layer. Media containing serum‐complexed [14C]‐α tocopherol was delivered to the apical side of the cells for 48 hrs and the replaced with serum free media. Cells that express TTP displayed significant (ca. 2‐fold) increase in the extent of tocopherol secretion by the monolayer. These findings show that placental TTP is necessary for trans‐placental transport of vitamin E. The functional consequences of heritable mutations in the TTPA gene on trans‐placental vitamin E transport are being addressed in ongoing experiments.
ATP signaling complex is altered in muscular dystrophy and was partly recovered after nifedipine treatment (762.3)
Authors: Valladares Denisse , Altamirano Francisco , Henríquez‐Olguín Carlos , Díaz‐Vegas Alexis , Intriago Giomar , Contreras‐Ferrat Ariel , Buvinic Sonja , López José , Allen Paul , Jaimovich Enrique
Published: June 2021

Duchenne muscular dystrophy (DMD) is a common genetic disorder characterized by a severe muscle wasting caused by the absence of dystrophin. In mdx muscle fibers, we have shown that basal ATP release is increased and that high extracellular ATP is a pro‐apoptotic stimuli. We also have shown that Dihydropyridine receptors (DHPR) are needed for ATP release through pannexin‐1(Pnx1) channels. The aim of this work was to study the potential therapeutic effect of DHPR blockage by nifedipine in DMD. We used muscle fibers isolated from six‐week‐old normal and mdx mice that were treated with daily intraperitoneal injections of 1mg/Kg nifedipine for 1‐week. We studied differences in the interaction between DHPR and Pnx1by proximity ligation assay. In mdx fibers there was an increase in the number and volume of positive particles but not in the intensity of the positive reaction when compared to normal fibers. After the treatment with nifedipine, both number and volume of the positive reaction particles was normalized. This result correlates well with a decrease in both ATP release and intracellular resting Ca2+ concentration in mdx fibers after the treatment. Moreover, ATP signaling was no longer a pro‐apoptotic stimuli. Finally, nifedipine treatment increased muscle strength assessed by both the inverted grip‐hanging and forced swimming test. These data suggest that dihydropyridines can be used as a therapeutical tool to reduce muscle damage observed in dystrophic muscles.

Grant Funding Source: Fondecyt 1110467, ACT1111, AFM14562, AT‐24110211
PGC1α‐1 nucleosome position in skeletal muscle of overweight individuals may determine mRNA splicing and be predictive of adiposity and hyperlipidemia (763.1)
Authors: Henagan Tara , Stewart Laura , Johannsen Neil , Forney Laura , Sparks Lauren , Church Timothy
Published: June 2021

Epigenetic alterations, leading to aberrant metabolism and dyslipidemia, partially determine obesity and associated CVD. PGC1α is a transcriptional coactivator whose expression in skeletal muscle is decreased during obesity and CVD. Several splice variants of PGC1α are known to have similar effects on metabolism as the full length mRNA isoform (FL) and may compensate for decreased FL expression. Here, we employed scanning PCR to determine if FL and N‐truncated PGC1α (NT) mRNA splice variant expression may be epigenetically regulated through ‐1 nucleosome positioning (‐1N) in the skeletal muscle of overweight/obese, diabetic individuals. The ‐1N tended to determine splice variant mRNA expression, with a ‐1N upstream (‐1NU) in the promoter (N=9) tending to have higher FL (P=0.175) and those with a ‐1N further downstream (‐1ND; N=4) exhibited higher NT (P=0.032). The ‐1ND group had a significantly higher body fat percentage (P=0.046) and cholesterol (P=0.025) and a higher trend in LDL cholesterol (P=0.055) compared to the ‐1NU group. Importantly, ‐1N occurs upstream of changes in mRNA expression and may occur before changes in gene expression or lipids are observed. These preliminary data include a small sample size yet reveal that PGC1α ‐1N may determine mRNA splicing and serve as a predictive biomarker for the onset of obesity and adverse cardiometabolic outcomes.

Grant Funding Source: Supported by NIH 5T32‐DK064584‐09, Cola‐cola Company and NIDDK R01‐068298.
Adverse effect of high and low fat diet on reproductive cycle (765.2)
Authors: Gomes Victoria , Donthireddy Laxminarasimha , Chakraborty Tandra
Published: June 2021

The prevalence of obesity is on its increase and diet‐induced weight loss has been used as a treatment. Although obesity causes insulin resistance, earlier puberty, menarche in girls and infertility, low fat diet can change the metabolic parameters causing infertility in women as well. This study is aimed to study the effect of high and low fat diet (HFD and LFD) which could change the hormonal milieu affecting the reproductive cycles in mice model. Results indicate that there was no significant difference in food intake between VHFD and ND. However, the caloric intake for animals on VHFD was significantly higher than ND indicating that it is not the amount of food but the caloric intake that was responsible for the differences in the weight increase between the two groups. Mice fed in VHFD showed elongation and slight acyclicity in estrous cycle within 3 months and were completely acyclic within 6 months. Obesity associated hyperleptinemia and hyperinsulinemia was observed which is probably the cause for complete disruption in the reproductive cycle in the VHFD animals. Preliminary results with LFD showed slight decrease in body weight within the first two weeks, which probably would affect the estrous cycle. It seems that high and low fat both cause changes in the hormonal milieu, which has a direct role in the acyclicity of estrous cycle in mice. Overall these results indicate the importance of a balanced diet for a healthy life.
S1P receptor‐1 and USP4 induced by microRNA‐148a deregulation facilitate liver cancer progression (766.1)
Authors: Heo Mi Jeong , Kim Young Mi , Koo Ja Hyun , Kim Sang Geon
Published: June 2021

Hepatocellular carcinoma (HCC) is frequently aggressive. MicroRNAs (miRNAs) emerge as key contributors to tumor progression. In the present study, we report that deregulation of miR‐148a causes the induction of sphingosine 1‐phosphate receptor 1 (S1P1) and ubiquitin specific protease 4 (USP4) in association with HCC progression. In patients with HCC, S1P1 and USP4 were both up‐regulated with miR‐148a deregulation. Moreover, decrease of miR‐148a discriminated microvascular invasion of HCC, the overall survival and recurrence free survival rates of the patients. In cell models, miR‐148a was repressed in mesenchymal‐typed liver‐tumor cells, which facilitated S1P1 and USP4 induction, as shown by the assays using mimic, antisense oligonucleotide and 3’‐UTR reporter. S1P1 and USP4 levels were markedly increased in liver‐tumor cells having a mesenchymal phenotype. S1P1 over‐expression enhanced ERK1/2 activation, increasing proliferation and migration of liver‐tumor cells, which was attenuated by miR‐148a mimic transfection. Decrease of miR‐148a additionally caused USP4 induction, which may account for TGFβ1‐dependent epithelial‐mesenchymal transition (EMT) in HCC. In summary, S1P1 and USP4 induced by miR‐148a deregulation may facilitate HCC growth and migration in microenvironments, and contribute to cancer progression.
Knockdown of UDP‐glucose dehydrogenase in INS‐1 832/13 cells inhibits insulin release (767.12)
Authors: Perez Jaritza , MacDonald Michael
Published: June 2021

Introduction: Our unpublished mass spectrometry studies of pancreatic islet insulin‐producing cells, as well as a published study by others, showed that several nucleotide‐sugar compounds increased with glucose stimulation. These compounds included ADP‐glucose (not known to be present in mammals), UDP‐glucose and GDP‐mannose. That these compounds increased with glucose stimulation suggests they might play a role in insulin secretion. We focused our study on two enzymes that use UDP‐glucose as a substrate: UDP‐glucose Dehydrogenase (UGDH) and UDP‐glucose glycoprotein glucosyltransferase (UGGT1).

Results: Transcripts of both genes were detected in human pancreatic islets and in the glucose‐responsive INS‐1 832/13 cell line. Human and rat islets showed six‐fold more UGDH mRNA than liver. Enzyme activity of UGDH was detected in rat liver, but was below the level of detection in the INS‐1 832/13 cell line. Two shRNAs targeting the UGDH gene significantly knocked down the mRNA of the gene in INS‐1 832/13‐derived cell lines. Knockdown of the UGDH mRNA was associated with 38% and 22% decrease in glucose‐stimulated insulin release in two UGDH shRNA / INS‐1 832/13‐derived cell lines. Cell lines with knockdown of UGGT1 are currently being generated.

Conclusions: Based on enzyme activity assay, the concentration of UGDH in INS‐1 832/13 cells is very low. However an inhibition of insulin release was observed in two separate UGDH knockdown cell lines, and it is unlikely that the inhibition of insulin release was due to an off target shRNA effect. Further studies are needed to establish whether UGDH and UGGT1 play a role in insulin release in INS‐1 832/13 cells.

Acknowledgements: NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM.

Grant Funding Source: Supported by NIH / NIDDK STEP‐UP Program award to JMP and NIH Grant DK28348 to MJM
Effect of ketones on ROS, NADPH oxidases, and monocyte adhesion in endothelial cells (767.8)
Authors: Kanikarla Marie Preeti , Jain Sushil
Published: June 2021

Cardiovascular disease (CVD) incidence is higher in Type 1 diabetic (T1D) patients. Along with hyperglycemia, T1D is associated with hyperketonemia, a condition with elevated blood levels of acetoacetate (AA) and 3‐β‐hydroxybutyrate (BHB). It is not known whether hyperketonemia per se contributes to the excess CVD in T1D. This study investigates the hypothesis that ketones upregulate NADPH oxidases (NOX) and ROS and therby increase monocyte‐endothelial adhesion. HUVEC were treated with AA or BHB (0‐8 mM) ± high‐glucose (25 mM) for 24 h. Results show that AA, but not BHB, increased ROS production, NOX activity, and NOX4 expression in HUVEC. Pretreatment with DPI (20 μM , inhibitor of NOX) abolished the effect of AA on ROS production. This suggests that AA effect on ROS production is mediated through the NOX. To further examine the role of NOX, we determined the effect of ketones in NOX4 knockdown (KD) HUVEC. There were significantly lower levels of ROS (65% vs. 20%), ICAM‐1 (42% vs. 23%), and monocyte adhesion (22% vs. 1%) in AA (4 mM) treated NOX4 KD cells compared with control cells. Effect of AA on ROS and ICAM‐1 was higher in cells knocked down for GCLC (that induces GSH deficiency). This suggests that ketone‐induced increase in ICAM‐1 and adhesion is mediated by the upregulation of NOX and ROS production. This study provides evidence for a biochemical mechanism for the role of hyperketonemia in the development of CVD in T1D.

Grant Funding Source: Supported by NIH and Malcolm Feist Chair in Diabetes
Construction of Escherichia coli plasmid vectors encoding site‐specific methylenetetrahydrofolate reductase mutants (769.12)
Authors: Seng John , Trimmer Elizabeth
Published: June 2021

Flavoenzyme methylenetetrahydrofolate reductase (MTHFR) is a critical component of folate metabolism in humans and Escherichia coli. Deleterious mutations to the MTHFR gene can cause mild and severe chronic ailments. To study the consequences of specific mutations, we can construct DNA vectors encoding the mutant protein of interest. Since the catalytic domain of MTHFR is highly conserved between E. coli and humans, E. coli is an ideal model organism for this sort of study. Here we have begun to produce a collection of new plasmid vectors encoding eMTHFR containing substitution mutations at sites hypothesized to be important in MTHFR’s enzymatic activity. Our methods are based on Stratagene’s protocol for their QuikChange II site‐directed mutagenesis kit. To date we have successfully produced and isolated plasmid vector pJUS‐1D, encoding eMTHFR substitution mutant Glu28Asp. Sequencing results have confirmed the plasmid’s desired identity. We are now in the process of producing additional vectors encoding eMTHFR mutants Ser26Thr and Ser26Ala.
Determination of hydrolases, oxidases and synthases in hypertensive patients (771.1)
Authors: Osman Mohamed Yousry , Osman Hassan , Moharem Dalia , Elsaiedy Ahmed
Published: June 2021

Hypertension (HT) affects about 20 percent of the adult population placing a high burden on health care systems. Cholinestrase is responsible of the hydrolysis of the vasodilator acetylcholine. Monoamine oxidase (MAO) is responsible for degradation of the vasoconstrictor biogenic amines. Nitric oxide synthase (eNOS) is responsible for the production of the vasodilator nitric oxide (NO). The study aimed to find the relationship between these enzymes and essential hypertension in recently diagnosed patients. The results indicated a significant decrease in the activity of pseudo ChE in HT patients compared to control resulting in increase of ACh content and also indicated a significant decrease in plasma MAO‐A in HT patients saving the biogenic amines. Similarly; there is a significant decrease in the plasma nitrites + nitrates [NOx] of HT patients due to decrease in NOS activity. Eventually; it could be concluded that the investigated enzymes played a role in the regulation of essential HT. As the study showed; increase of 5‐hydroxytryptamine and adrenaline (due to decrease in MAO) that exceeded the decrease of pseudo ChE (increasing ACh content) and NOS (decreasing NO) resulting in vasoconstriction exceeding vasodilatation and the blood pressure consequently rises. Reversal of the previous mechanism could be helpful in treating essential HT.

Grant Funding Source: Alexandria University
Lipoprotein‐associated phospholipase A2 structure and function (774.3)
Authors: Gonzalez Tara , Bahnson Brian
Published: June 2021

Lipoprotein‐associated phospholipase A2 (LpPLA2) or plasma platelet‐activating factor acetylhydrolase (pPAFAH) is a serine hydrolase that acts on platelet‐activating factor (PAF) and oxidized phospholipids. The enzyme has a classic α/β hydrolase fold and predicted membrane binding regions. Plasma PAFAH binds readily to both high density lipoproteins (HDL) and low density lipoproteins (LDL) and is believed to play a pro‐atherosclerotic role. The purpose of this study is to further elucidate the structure and function of LpPLA2 through the use of specific inhibitors. It was necessary to develop and refine an expression method in E. coli to produce high quantities of protein of high purity for a truncated form of LpPLA2. Successful expression and purification of this LpPLA2 construct have been achieved and initial trials of crystallization were positive. The next step in this study is to obtain the crystal structure of LpPLA2 along with its selective inhibitors.

Grant Funding Source: Supported by NIHNHLBI grant #RO1HL084366
Genetic variability in CRHR1 and its association with participant response to glucocorticoid receptor‐mediated signaling (776.2)
Authors: Knox Rachel , Biette Kelly , Fox Olivia , Murphy Patrick
Published: June 2021

The goal of this study was to identify correlations between interpersonal genetic variability and alterations in the intracellular glucocorticoid receptor (GR)‐mediated signaling pathway. Salivary cortisol concentrations were measured in healthy volunteers (n=66) in order to determine differences in circulating cortisol concentrations and cortisol response to HPA stimulation. Statistical analysis indicated inter‐participant cortisol variability was not correlated to participant demographics, health history, or SNPs in genes encoding the GR (NR3C1) or the essential GR chaperone protein hsp90 (HSP90AA1). Two participants with highly similar demographic profiles and opposing responses to HPA stimulation were selected for matched exome sequencing. Filtering analysis identified candidate SNPs in approximately 30 genes involved in mediating the cortisol response and GR signaling. Interestingly, corticotropin releasing hormone receptor 1 (CRHR1) showed a difference of over 20 SNPs between the matched exome pair. CRHR1 regions were sequenced from the larger participant group, and participants with average cortisol concentrations that slightly decreased following stimulation most commonly exhibited a unique SNP profile. We speculate participants with SNP profiles associated with altered cortisol response may share a corresponding sensitivity to corticosteroid‐based drug therapies.

Grant Funding Source: Supported by NIH AREA grant GM086822
A metagenomic analysis of triclosan resistant bacteria in Alum Creek, Westerville OH (776.5)
Authors: Agyepong Nana , Agyepong Abena , Bennett Jennifer
Published: June 2021

Triclosan (TCS) is a widely used biocidal agent that is found in various watersheds. The objective of this study is to analyze how the occurrence of TCS impacts bacterial resistance and to gather metagenomic data of resistant strains. Water samples were collected in duplicate from six specific sites. Four samples were collected from the Alum Creek watershed and two samples were collected from the nearby Big Walnut watershed. These sites were compared to pristine watersheds in Pennsylvania. Triclosan resistance of bacteria was determined for all six sample sites and the pristine sites. DNA of the resistant bacteria was prepared for PCR for the amplification of the 16S gene and subsequent metagenomic analysis. Resistance levels for Alum creek sites ranged from 17%‐47%. Resistance levels for Big Walnut sites ranged from 29%‐65%. Resistance levels for the pristine sites ranged from 13%‐74%. UPLC‐MS/MS data showed that resistance levels were not necessarily tied to TCS levels in Alum Creek and Big Walnut watersheds. Metagenomic data of the resistant strains has been obtained through PCR and is being sequenced.
Nanotopographic cues increase epithelial paracellular permeability through cytoskeletal reorganization (780.6)
Authors: Hutto Tarianna , Ollerenshaw Jeremy , Swarnakar Snehasikta , Bock Suzanne , Ross Russell , Lillard James , Koval Michael
Published: June 2021

Paracellular permeability between epithelial cells is regulated by tight junctions (TJs) which act as a barrier to drug delivery across the epithelium. Previous studies (Kam et al., Nano Lett. (2013)) have found that nanostructured films (NSFs) imprinted on polypropylene (PP) with pitch spacing of 300 nm, average height of 300 nm and average diameter of 200 nm (P(1.5);DN2) increase paracellular permeability of CaCo2 colonic epithelial cells to macromolecule sized agents, independently from changes to transepithelial resistance. To define a mechanism for the effects of NSFs on CaCo2 cells, we examined changes to F‐actin by immunofluorescence (IF). Contact of CaCo2 cells with DN2 NSFs disrupted cortical actin at the plasma membrane, in contrast to non‐imprinted PP which had little effect. To determine whether matrix metalloproteases (MMPs) may be involved in this response, we examined the effect of DN2 films on expression and function of MMP2 and MMP9. By IF and in‐gel zymography, DN2 induced changes in MMP9 expression relative to MMP2. MMP9 inhibitors are being assessed to determine whether they inhibit changes to paracellular flux and actin organization induced by DN2 as a strategy to modulate NSF‐facilitated drug delivery.
Regulated axonal transport of APP and alcadein by kinesin‐1 (783.1)
Authors: Suzuki Toshiharu , Chiba Kyoko , Okumura Yuka , Shiraki Yuzuha , Sobu Yuriko , Kinjo Masataka , Hata Saori , Taru Hidenori
Published: June 2021

Alzheimer’s β‐amyloid precursor protein (APP) and Alcadein (Alc) are cargo of kinesin‐1. APP associates to kinesin‐1 by JIP1b mediation, while Alc directly binds to kinesin‐1. APP is subject to axonal transport faster than Alc in independent cargos, even if both APP and Alc are transported kinesin‐1 in axon. To understand molecular mechanisms how both cargos are transported in different velocities, we revealed molecular interactions biochemically among APP, JIP1b and kinesin light chain (KLC), and between Alc and KLC.

Methods: Detailed interaction between Alc and KLC, and JIP1b and KLC were analyzed biochemically by co‐immunoprecipitaion and GST pull‐down assays. We generated many deletion mutants for Alcα (one of Alcadein family) and JIP1b (JIP1 family which can bind APP strongly), and analyzed the interaction with KLC1 (neuron‐specific isoform of KLC) mutants. Function was investigated with analysis of APP‐GFP and Alcα‐GFP transport in neural axon with TRIF microscopy.

Results and Discussion: Direct binding between Alcα and KLC shows a simple but strong affinity, while JIP1b‐mediated interaction between APP and KLC is more complex. The mediation of JIP1b may regulate fast velocity of anterograde cargo transport by kinesin‐1.

Grant Funding Source: Supported by Grants‐in‐Aid for Scientific Research from JSPS, Japan.
investigating the mechanisms involved in aberrant neurosphere migration in schizophrenia (784.2)
Authors: Patruno Anna , Tran Ngoc , Abdelrahim Mohammed , Brennand Kristen
Published: June 2021

Schizophrenia (SCZD) is a chronic, debilitating disorder with a prevalence of 1% and typically appears in the late adolescent and early adulthood years. Postmortem studies have been pivotal in understanding the neuropathology of schizophrenic brains by revealing characteristics such as reduced cell size, brain volume and neuronal connections; however, the cellular and molecular mechanisms involved in the disease as well as its development remain unclear. To investigate disease progression, we modeled genetic predisposition to SCZD by reprogramming fibroblasts from SCZD patients into human induced pluripotent stems cells (hiPSCs) and differentiating these cells into neural progenitor cells (NPCs) and neurons. Using a neurosphere migration assay, we observed aberrant migration of SCZD NPCs in vitro. To investigate the mechanism responsible for this aberrant migration, we manipulated the RHO‐ROCK (Rho‐associated kinase) signaling pathway in SCZD NPCs. In addition, we investigated whether treatment with antipsychotics such as Loxapine and Clozapine was sufficient to ameliorate aberrant migration. Neurosphere migration assays are a useful model to study molecular mechanisms in SCZD as well as disease predisposition, which may contribute in developing potential treatments.

Grant Funding Source: Supported by NYSCF and NIH/NIMH, R01 MH101464
Uniquely human changes in expression and binding specificity of Siglec‐11: implications for human brain evolution (787.2)
Authors: Schwarz Flavio , Deng Lingquan , Landig Corinna , Varki Nissi , Varki Ajit
Published: June 2021

We are exploring the role of Siglec‐11, a member of the sialic acid‐recognizing immunoglobulin‐like lectin family, in the modulation of microglial functions in the human brain. Human SIGLEC11 underwent two tandem and likely simultaneous gene conversions by the adjacent pseudogene SIGLEC16P (Wang et al., Mol. Biol. Evol., 29:2073‐2086, 2012). These genes conversions preserved an intact open reading frame, but changed 5' untranslated sequences, likely altering transcription of SIGLEC11. Using specific antibodies, we confirmed the resulting uniquely human expression of Siglec‐11 in brain microglia, and next wished to define its ligands. Surprisingly, we did not observe robust binding of human Siglec‐11 to any sialic acid‐containing glycan in multiple assay platforms. In contrast chimpanzee Siglec‐11 strongly recognized several glycans terminating with sialic acid, with a preference for the non‐human sialic acid N‐glycolylneuraminic acid (Neu5Gc). Accordingly, chimp Siglec‐11 bound to cells proportionally to their Neu5Gc content, but human Siglec‐11 exhibited minimal binding to the same cells. We also found that human Siglec‐11 binds to a microglial cell line independently of sialic acid, and are characterizing the nature of this ligand. Alterations in the expression pattern and ligand‐binding specificity of human Siglec‐11 may have evolved to generate novel, human‐specific functions in the brain.

Grant Funding Source: Supported by P01 HL057345
Hyperglycemia affects the glycophenotype and the growth of colon carcinoma cells in vivo (789.2)
Authors: Vasconcelos‐dos‐Santos Andreia , Mantuano Natalia , Loponte Hector , Oliveira Isadora , Moll Fernanda , Dias Wagner , Todeschini Adriane
Published: June 2021

Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. Recent work suggests that the metabolite availability to the hexosamine and Golgi O‐glycosylation pathways exerts control over cell signaling, gene expression and cell migration (Alisson‐Silva etal., Plos One, 8: e60471, 2013). Indeed, high glucose concentration increases O‐glycosylation of FN (onFN), which generates the onfFN, and modulates tumorogenesis. Herein we implicate hiperglycemia in facilitating cancer progression and increasing onFN expression in vivo. Cells from colon carcinoma of C57BL/6 mice (MC‐38 cell line) were subcutaneously inoculated in streptozotocin (STZ)‐treated or untreated C57BL/6 mice. Subcutaneous tumors from STZ‐treated group were higher and more vascularized when compared with vehicle group. Tumors from STZ‐treated mice presented high expression of alpha2‐6‐Neu5Ac and alpha1‐3‐/ alpha1‐6‐Fucose (Fuc) containing glycoconjugates as well as increased expression of onFN. These data suggest that metabolite availability modulates cell surface glycosylation contributing to tumor formation and progression.

Grant Funding Source: Supported by CNPq, FAPERJ and CAPES