A role for the ubiquitin-proteasome system in activity-dependent presynaptic silencing

Chronic changes in electrical excitability profoundly affect synaptic transmission throughout the lifetime of a neuron. We have previously explored persistent presynaptic silencing, a form of synaptic depression at glutamate synapses produced by ongoing neuronal activity and by strong depolarization. Here we investigate the involvement of the ubiquitin-proteasome system (UPS) in the modulation of presynaptic function. We found that proteasome inhibition prevented the induction of persistent presynaptic silencing. Specifically, application of the proteasome inhibitor, MG-132, prevented decreases in the size of the readily releasable pool of vesicles and in the percentage of active synapses. Presynaptic silencing was accompanied by decreases in levels of the priming proteins, Munc13-1 and Rim1. Importantly, overexpression of Rim1α prevented the induction of persistent presynaptic silencing. Furthermore, strong depolarization itself increased proteasome enzymatic activity measured in cell lysates. These results suggest that modulation of the UPS by electrical activity contributes to persistent presynaptic silencing by promoting the degradation of key presynaptic proteins

Stereotypes of salespeople in Taiwan:an empirical examination and research agenda

Stereotypes of salespeople are common currency in US media outlets, and research suggests that these stereotypes are uniformly negative. However there is no reason to expect that stereotypes will be consistent across cultures. The present paper provides the first empirical examination of salesperson stereotypes in an Asian country, specifically Taiwan. Using accepted psychological methods, Taiwanese salesperson stereotypes are found to be twofold, with a negative stereotype being quite congruent with existing US stereotypes, but also a positive stereotype, which may be related to the specific culture of Taiwan

The Effect of Student-Run Vision Screenings on Ophthalmic Education and Recognition of Visual Impairment

Objective: For many in the United States, standard health insurance does not cover eyecare, leading to lapses in care and exacerbations of pre-existing conditions. Touro College of Osteopathic Medicine (TouroCOM) recognizes the importance of ocular health and offers the opportunity to engage the community through student-run vision screenings. This study aims to assess the effect of medical student-run vision screenings in supplementing pre-clinical education and to review health fair data on common vision pathologies seen in Harlem, New York.Methods: Pre- and post-surveys were administered to medical student volunteers to assess their comfort in performing a basic vision screening. Training was given in regards to screening protocols and applied at health fairs.Results: 90% of medical students (n=20) indicated discomfort in performing a basic vision screening when solely relying on their preclinical coursework. In comparison, after a training session and use during a health fair, 100% (n=20) indicated that they were comfortable with performing a vision screening.  60% of health fair participants (n=193) met referral criteria in requiring further testing or follow-up care. 100% of participants had some degree of refractive error, with 6% (n=7) having concomitant color vision abnormality and 9% (n=11) with macular abnormalities.Conclusion: Osteopathic medical students are better equipped to perform basic vision screenings and recognize visual disease with additional training and practice at health fairs. This engagement allows for early clinical experience, osteopathic outreach, and interprofessionalism. Furthermore, this provides an opportunity for community members to receive information that may guide future health decisions

A Standardized, Pragmatic Approach to Knee Ultrasound for Clinical Research in Osteoarthritis: The Johnston County Osteoarthritis Project

OBJECTIVE: This study sought to develop and employ a comprehensive and standardized ultrasound (US) protocol and scoring atlas for the evaluation of features relevant to knee osteoarthritis (KOA) in a community-based cohort in the United States, with the goals of demonstrating feasibility, reliability, and validity. METHODS: We utilized data from the fourth follow-up (2016-2018) of the Johnston County OA Project, which includes individuals with (~50%) and without radiographic KOA. All participants underwent standardized knee radiography and completed standard questionnaires including the Knee Injury and Osteoarthritis Outcome Score (KOOS). Bilateral knee US images were obtained by a trained sonographer using a standardized protocol and scored by trained rheumatologists using an atlas developed for this study. A total of 396 knees were each scored by two readers according to the atlas. Associations between US features, radiographic findings (graded by an expert radiologist), and KOOS scores were assessed. RESULTS: Overall interreader reliability for US scoring was fair to moderate. The strongest correlations between US and radiographic features were seen for osteophytes, and similarly strong correlations were seen between US osteophytes and overall radiographic Kellgren-Lawrence Grade, demonstrating criterion validity. Features of effusion/synovitis and osteophytes were most associated with KOOS pain and impaired function. CONCLUSION: US is a feasible, reliable, and valid method to assess features relevant to KOA in clinical and research settings. The protocol and atlas developed in this study can be utilized to evaluate KOA in a standardized fashion in future clinical studies, enabling greater utilization of this valuable modality in osteoarthritis

Gastrin Induces Nuclear Export and Proteasomal Degradation of Menin in Enteric Glial Cells

Background & aims: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner\u27s glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Methods: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Results: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. Conclusions: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae

Intermittent Moderate Energy Restriction Improves Weight Loss Efficiency in Diet-Induced Obese Mice

BACKGROUND:Intermittent severe energy restriction is popular for weight management. To investigate whether intermittent moderate energy restriction may improve this approach by enhancing weight loss efficiency, we conducted a study in mice, where energy intake can be controlled. METHODS:Male C57/Bl6 mice that had been rendered obese by an ad libitum diet high in fat and sugar for 22 weeks were then fed one of two energy-restricted normal chow diets for a 12-week weight loss phase. The continuous diet (CD) provided 82% of the energy intake of age-matched ad libitum chow-fed controls. The intermittent diet (ID) provided cycles of 82% of control intake for 5-6 consecutive days, and ad libitum intake for 1-3 days. Weight loss efficiency during this phase was calculated as (total weight change) ÷ [(total energy intake of mice on CD or ID)-(total average energy intake of controls)]. Subsets of mice then underwent a 3-week weight regain phase involving ad libitum re-feeding. RESULTS:Mice on the ID showed transient hyperphagia relative to controls during each 1-3-day ad libitum feeding period, and overall ate significantly more than CD mice (91.1±1.0 versus 82.2±0.5% of control intake respectively, n = 10, P<0.05). There were no significant differences between CD and ID groups at the end of the weight loss or weight regain phases with respect to body weight, fat mass, circulating glucose or insulin concentrations, or the insulin resistance index. Weight loss efficiency was significantly greater with ID than with CD (0.042±0.007 versus 0.018±0.001 g/kJ, n = 10, P<0.01). Mice on the CD exhibited significantly greater hypothalamic mRNA expression of proopiomelanocortin (POMC) relative to ID and control mice, with no differences in neuropeptide Y or agouti-related peptide mRNA expression between energy-restricted groups. CONCLUSION:Intermittent moderate energy restriction may offer an advantage over continuous moderate energy restriction, because it induces significantly greater weight loss relative to energy deficit in mice

A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action

Dynamic two-axis curvature measurement using multicore fiber Bragg gratings interrogated by arrayed waveguide gratings

We describe the use of arrayed waveguide gratings (AWGs) in the interrogation of fiber Bragg gratings (FBGs) for dynamic strain measurement. The ratiometric AWG output was calibrated in a static deflection experiment over a ±200 με range. Dynamic strain measurement was demonstrated with a FBG in a conventional single-mode fiber mounted on the surface of a vibrating cantilever and on a piezoelectric actuator, giving a resolution of 0.5 με at 2.4 kHz. We present results of this technique extended to measure the dynamic differential strain between two FBG pairs within a multicore fiber. An arbitrary cantilever oscillation of the multicore fiber was determined from curvature measurements in two orthogonal axes at 1125 Hz with a resolution of 0.05 m-1. © 2006 Optical Society of America

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes

L-5-methyltetrahydrofolate supplementation increases blood folate concentrations to a greater extent than folic acid supplementation in Malaysian women

Background: Folic acid fortification of grains is mandated in many countries to prevent neural tube defects. Concerns regarding excessive intakes of folic acid have been raised. A synthetic analog of the circulating form of folate, l-5-methyltetrahydrofolate (l-5-MTHF), may be a potential alternative. Objective: The objective of this study was to determine the effects of folic acid or l-5-MTHF supplementation on blood folate concentrations, methyl nutrient metabolites, and DNA methylation in women living in Malaysia, where there is no mandatory fortification policy. Methods: In a 12-wk, randomized, placebo-controlled intervention trial, healthy Malaysian women (n = 142, aged 20–45 y) were randomly assigned to receive 1 of the following supplements daily: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF, or a placebo. The primary outcomes were plasma and RBC folate and vitamin B-12 concentrations. Secondary outcomes included plasma total homocysteine, total cysteine, methionine, betaine, and choline concentrations and monocyte long interspersed nuclear element-1 (LINE-1) methylation. Results: The folic acid and l-5-MTHF groups had higher (P < 0.001) RBC folate (mean ± SD: 1498 ± 580 and 1951 ± 496 nmol/L, respectively) and plasma folate [median (25th, 75th percentiles): 40.1 nmol/L (24.9, 52.7 nmol/L) and 52.0 nmol/L (42.7, 73.1 nmol/L), respectively] concentrations compared with RBC folate (958 ± 345 nmol/L) and plasma folate [12.6 nmol/L (8.80, 17.0 nmol/L)] concentrations in the placebo group at 12 wk. The l-5-MTHF group had higher RBC folate (1951 ± 496 nmol/L; P = 0.003) and plasma folate [52.0 nmol/L (42.7, 73.1 nmol/L); P = 0.023] at 12 wk than did the folic acid group [RBC folate, 1498 ± 580 nmol/L; plasma folate, 40.1 nmol/L (24.9, 52.7 nmol/L)]. The folic acid and l-5-MTHF groups had 17% and 15%, respectively, lower (P < 0.001) plasma total homocysteine concentrations than did the placebo group at 12 wk; there were no differences between the folic acid and l-5-MTHF groups. No differences in plasma vitamin B-12, total cysteine, methionine, betaine, and choline and monocyte LINE-1 methylation were observed. Conclusion: These findings suggest differential effects of l-5-MTHF compared with folic acid supplementation on blood folate concentrations but no differences on plasma total homocysteine lowering in Malaysian women. This trial was registered at clinicaltrials.gov as NCT01584050