Dissecting the Spatiotemporal Regulation of Yeast Kinetochore Interactions

Kinetochores are conserved protein complexes that bind the centromeres in replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we investigated the phospho-regulated dynamic interaction between the conserved kinetochore protein Cnn1CENP-T, the centromere region and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization is unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain that associates with centromere region, and a N-terminal Spc24/25-interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that a previously established Ndc80 binding site in the N-terminus of Cnn1, Cnn160-84, should be extended to include flanking residues, Cnn125-91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74 based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that in G1 through metaphase, Cnn1 localizes via the histone-fold domain or a N-terminal Spc24/25

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn160–84, should be extended with flanking residues, Cnn125–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase

Effects of chronic intermittent ethanol exposure and withdrawal on neuroblastoma cell transcriptome

Cycles of heavy drinking and abstinence can lead to ethanol abuse disorder. We studied the effects of chronic intermittent ethanol exposure (CIE) over three weeks on neuroblastoma cells, using an ethanol concentration frequently attained in binge drinking (40 mM, 184 mg/dl). There were many changes in gene expression but most were small. CIE affected pathways instrumental in the development or plasticity of neurons, including axonal guidance, reelin signaling and synaptogenesis. Genes involved in dopamine and serotonin signaling were also affected. Changes in transporters and receptors could dampen both NMDA and norepinephrine transmissions. Decreased expression of the GABA transporter SLC6A11 could increase GABA transmission and has been associated with a switch from sweet drinking to ethanol consumption in rats. Ethanol increased stress responses such as unfolded protein response. TGF-β and NFκB signaling were increased. Most of the genes involved in cholesterol biosynthesis were decreased in expression. Withdrawal for 24 h after CIE caused most of the CIE-induced expression changes to move back toward unexposed levels

38766 Massively Parallel Reporter Assay Reveals Functional Impact of 3™-UTR SNPs Associated with Neurological and Psychiatric Disorders

ABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD

Dissecting the Spatiotemporal Regulation of Kinetochore Interactions

Kinetochores are conserved protein complexes that bind the centromeres in replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we investigated the phospho-regulated dynamic interaction between the conserved kinetochore protein Cnn1CENP-T, the centromere region and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization is unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain that associates with centromere region, and a N-terminal Spc24/25-interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that a previously established Ndc80 binding site in the N-terminus of Cnn1, Cnn160-84, should be extended to include flanking residues, Cnn125-91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74 based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that in G1 through metaphase, Cnn1 localizes via the histone-fold domain or a N-terminal Spc24/25- interaction sequence because deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. Endogenous expression of the N-terminal region is sufficient to localize to the kinetochore demonstrating the availability to bind to Cnn1 and indicating the presence of the Cnn1-kinetchore linkages throughout the cell cycle. At anaphase onset (when Mps1 kinase activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/25-interaction sequence confirming previous studies using full-length Cnn1. In sum, we provide the first in vivo evidence of Cnn1 pre-anaphase linkages with the kinetochore and enrichment of the linkages during anaphase with this interaction sequence. Cse4 is a centromere-specific nucleosomal protein that has a similar motif organization to Cnn1 in that it has a histone-fold and a N-terminal tail sequence. Shugoshin (Sgo1) is also reported to localize to the centromere and pericentromere and is a key component in tension-sensing as a result of bi-polar attachment. We demonstrate a novel interaction between Sgo1 and Cse4 (yeast ortholog of CENP-A) at the centromere using yeast two-hybrid, live cell imaging and co-immunoprecipitation assays. We mapped this interaction to the first 132 residues of Sgo1 and the Cse4 N-terminal tail. Using mutational analysis, we identified several regions including a basic patch, S105 site and a putative coiled-coil region in Cse4 involved in the Sgo1 interaction. In sum, the identification of the Cse4-Sgo1 interaction is a key determinant in recruiting Sgo1 to the centromere and controlling the tension-sensing mechanism

Post‐Coronavirus disease syndrome (post‐COVID‐19) syndrome: A case report

Abstract Post‐COVID syndrome, a cluster of symptoms that develops or persists even after the recovery from COVID‐19 or viral clearance, can have multi‐system manifestations. This entity should be considered in patients who recently tested positive for COVID‐19 after ruling out other possible obvious causes. Its management should involve a multidisciplinary approach

Loading Dose only versus Standard Dose Magnesium Sulfate Seizure Prophylaxis in Severe Pre-eclamptic Women

Introduction: Magnesium sulfate is the drug of choice for prevention of seizures in the pre-eclamptic woman. There is no agreement in the published randomized trials regarding the optimal time to initiate magnesium sulfate, the dose to use (both loading and maintenance) as well as the duration of therapy. The objective of this study is to determine whether magnesium sulfate (MgSO4) prophylaxis is needed for up to 24 hours postpartum in all patients with severe pre-eclampsia for the prevention of seizure. Methods: It is a randomized controlled trial done on 60 pregnant women with severe preeclampsia randomized into standard dose regimen and loading dose only regimen. Results: Out of 30 cases in each group 1 (3.3%) patient in standard regimen and 2 (6.7%) patients in loading dose only developed seizure. The occurrence of seizure is not significant statistically. In both regimens there was no maternal mortality. Total of 3 patients needed MICU care and 12 patient developed maternal complications. MgSO4 toxicities were seen only in standard dose regimen that is in 17 (56.7%) of the patients. The median number of IM injections of MgSO4 received in standard dose regimen was 8±2.176. In standard dose regimen 73.3 percent baby were alive whereas in case of loading dose only regimen 93.3 percent of baby were alive after 48 hours of delivery. Conclusions: Single dose of magnesium sulfate is equally effective as standard dose regimen in terms of seizure prophylaxis in severe pre eclamptic women, with added advantage of reduced maternal toxicity and better neonatal outcome. Keywords:  severe preeclampsia; MgSO4; magnesium sulfate; loading dose; eclampsia

Pattern of Mobile Phone Use among Students of An Institution

  Introduction: The charm of mobile phone is more among young generation and the increasing use can lead to various harmful effects and may result in dependence. The objective of the study is to evaluate the pattern of mobile phone use among medical students. Methods: A descriptive cross sectional study was conducted on a total of 229 medical students aged between 18 and 25 years who were using mobile phones for more than 1 year by using simple random sampling technique. Students were requested to complete a pre-tested self-administered questionnaire which comprised their socio-demographic characteristics and pattern of mobile phone usage. Results: Among the total 229 students, all of them had their own smart phones. Major purpose of using mobile was for calling, listening music, taking photos/videos and internet use. Most of the students 97 (42.4%) have owned mobile phones since 4-6 years. The median time spent by the students on the mobile phone using different features of mobile phone was 3.5 hours. Majority of the students i.e. 157 (68.5%) use mobile phones at night. The median amount of money spent on recharge per month was 500. Age of student was significantly (P <0.05) associated with calls per day. There was no significant association of pattern of mobile phone use with other selected socio-demographic variables Conclusions: Students were found to use mobile phone excessively. The pattern of mobile phone usage among the students in this study may signal the evolution of mobile phone use from a habit to an addiction

Reviving the lost art of scleral buckling surgery for rhegmatogenous retinal detachment: evaluation of risk factors of detachments, poor physiological outcomes, and perspective from a developing country

Purpose: To evaluate the primary anatomic and physiological success of scleral buckling surgery for rhegmatogenous retinal detachment and factors influencing its outcomes. Methods: This is a prospective analytical study of 92 eyes that underwent scleral buckling at the Lumbini Eye Institute and Research Center, in Lumbini, Nepal. Parameters evaluated which could influence the outcome of the surgery included the lens status, duration of symptoms, locations of breaks, the extent of retinal detachment, and preoperative proliferative vitreoretinopathy. Results: A total of 92 eyes from 88 patients with rhegmatogenous retinal detachment were evaluated; 68 (74%) eyes were of male and 24 (26%) were of female. The mean time of presentation was 4.71 ± 8.45 months. The overall primary anatomical and physiological success was achieved in 79 (84.9%) and 68 (73.9%) of the cases at 6 months. Sixteen cases developed re-detachment (mean duration of 2.8 ± 1.8 months). Eleven of the cases had a successful anatomical outcome and five of the patients had persistent detachment despite second surgery. In phakic patients, the primary success rate was 92.7% whereas in pseudophakic it was 71.4%. Proliferative vitreoretinopathy 10 (63%) was the most common cause of surgical failure. Bilateral buckling at the same setting was done to two patients—both achieving primary success. Conclusion: Scleral buckling is a very good surgical option for rhegmatogenous retinal detachment and represents a surgical technique worth being trained, performed, practiced, and continued despite advancements in modern vitreoretinal surgical devices and preference for vitrectomy and tamponade agents. It may also be successfully tried in cases of bilateral rhegmatogenous retinal detachment if a doubt regarding compliance for follow-up and surgery for the fellow eye exists