Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Background

Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications.Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia.The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation.

Methods

Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered.

Results

Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p =?0.22), erythrocyte GALT activity (p =?0.87), urinary galactose (p =?0.52) and urinary galactitol levels (p =?0.41). Patients’ fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect.

Conclusions

This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

     

Do Canadian female surgeons feel discriminated against as women?

OBJECTIVE: To describe female surgeons'' perceptions of discrimination against them as women during the selection and training process and in career development and advancement, and to describe trends over time. DESIGN: Population survey of practising Canadian female surgeons. SETTING: Canada. PARTICIPANTS: All 459 female members in good standing of the Royal College of Physicians and Surgeons of Canada or the Corporation professionnelle des médecins du Québec, or both, practising in Canada as of March 1990. Participants completed a survey between March 1990 and May 1992, the response rate was 91% (419/459). OUTCOME MEASURES: Reported levels of discrimination during selection and training and in career development and advancement, institutional policies on maternity leave and job sharing, and the existence of female role models or mentors. RESULTS: Discrimination during the process of selection for residency was reported by 15% (63/413) of the respondents. Just over half of the respondents (206/405) reported male attending staff as being discriminatory during training, and 41% (168/407) reported nursing staff as being discriminatory. Almost half of the respondents (199/408) indicated that discrimination did not hinder their career development or advancement at all, and 29% (118) indicated that it had little effect. Almost two thirds (245/381) reported no maternity leave policies during residency or practice, and 78% (296/379) reported having no job-sharing opportunities. Although 82% (338/413) agreed that female medical students need female role models, 80% (330/415) reported they did not have a female mentor. CONCLUSIONS: Although most of our respondents perceived no discrimination in their selection for residency and reported that discrimination did not hinder their career development or advancement, the perception of discrimination during surgical training suggests that there needs to be a concentrated effort to identify and address problems. Moreover, since few respondents reported having institutional policies on maternity leave and job-sharing or female mentors, these issues need to be examined.     

Regulation of bile acid synthesis in cultured rat hepatocytes: stimulation by apoE-rich high density lipoproteins

Cultured rat hepatocytes obtained by liver perfusion with collagenase in the presence of soybean trypsin inhibitor were used to examine the role of high density lipoproteins (HDL) in supplying cholesterol to the hepatocyte for bile acid synthesis. Within 6 hr of adding HDL (d 1.07-1.21 g/ml) obtained from rat serum there was a significant stimulation of bile acid synthesis and secretion that reached 2-fold after 24 hr. The stimulation by HDL occurred at normal plasma concentrations (i.e., 500 micrograms/ml) and showed further stimulation in a dose-dependent manner reaching a maximum stimulation of 2- to 2.5-fold. The stimulation of bile acid synthesis was dependent on the cholesteryl ester content of the HDL. Several lines of evidence show that the HDL is taken up by a receptor-mediated process dependent on apoE. These include: 1) at the same concentration (500 micrograms/ml) apoE-poor HDL (not retained by heparin affinity chromatography of HDL isolated from the plasma of rats fasted for 72 hr stimulated bile acid synthesis by 48%, whereas apoE-rich HDL stimulated bile acid synthesis by 110%; 2) reductive methylation totally blocked the stimulation of bile acid synthesis by HDL; 3) HDLC, which contained apoE as its major protein component, also maximally stimulated bile acid synthesis; and 4) human HDL, which contained no detectable apoE, failed to stimulate bile acid synthesis. Additional studies showed that apoE-enriched HDL and HDLC both inhibited cholesterol synthesis (determined by the incorporation of 3H2O) and caused a net accumulation of cholesteryl esters in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical examination of the reproductive organs of range beef bulls in Mozambique

Physical examinations were performed on 3991 bulls reared in subtropical (75%) and tropical (25%) Mozambique. A culling rate of 16.3% (651 sires) was found. The range of culling rate between farms varied from 3 to 44%. The main culling reasons were a) epididymitis, b) atrophy and/or hypoplasia, and c) testicular fibrosis (contributing to 30.4, 20.9, and 15.2%, respectively, of the culled bulls). Epididymitis-vaginitis (epivag) syndrome was considered the main reason for the high incidence of epididymitis and testicular fibrosis. A highly significant difference (P<0.001) in culling rate for Simmental (37.7%) and Brahman (13.1%) bulls was found. A more comprehensive investigation of culling rate of different breeds used in the country is needed, as well as a program for controlling epivag in the more affected areas. Examining the reproductive organs of the beef bulls in Mozambique before the breeding season is very important to improve fertility in the beef herds.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.