Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.     

Malaria vectors and transmission dynamics in coastal south-western Cameroon

Background

Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.

Methods

A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.

Results

In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.

Conclusion

Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

     

An analysis of cancer prevention by selenium

The nutritional functions of selenium (Se) are recognized as being due to a number of Se-containing proteins. It is not clear, however, whether any of these function in the anti-tumorigenic effects of Se most of which have been demonstrated for Se exposures greater than those required for selenoprotein expression. Indeed, other anti-tumorigenic mechanisms have been demonstrated for certain Se-metabolites. The Nutritional Prevention of Cancer Trial found supplemental Se (200 microg/day, as Se-enriched yeast) to be associated with significant reductions in cancer risks in subjects with pre-treatment plasma Se concentrations below ca. 120 ng/ml (1.5 nmoles/ml), which level would appear to require food-Se intakes of ca. 1.5 microg/kg body weight/day. However, the putative anti-carcinogenic Se-metabolite(s) should be more relevant than total plasma Se as a supplementation target for cancer prevention. These may be components of the non-protein-bound fraction of Se in plasma, which constitutes 2-4% of total plasma Se.     

Determination of Quinidine and its major metabolites by high-performance liquid chromatography

A specific and precise assay, capable of quantitating in human plasma simultaneously but separately quinidine, dihydroquinidine and the quinidine metabolites 2′-quinidinone, 3-OH-quinidine and a third metabolite found — tentatively identified as the product formed by rearrangement of quinidine-N-oxide — is reported. The assay uses a normal phase high-performance liquid chromatographic (HPLC) system with a variable-wavelength UV detector at 235 nm and has a limit of sensitivity at approximately 20 ng/ml. The mobile phase consists of hexanes—ethanol—ethanolamine (91.5:8.47:0.03). A 2-ml plasma sample is worked up by adding primaquine base as an internal standard and extracting with ether—dichloromethane—isopropanol (6:4:1). The organic extract is evaporated and the residue reconstituted in 100—600 μl of mobile phase and an aliquot injected onto the column.Comparison of this procedure with the Edgar and Sokolow (dichloroethane) extraction—fluorescence procedure and with the Cramer and Isaksson (benzene) double extraction—fluorescence assay indicates that both fluorescence procedures give quinidine concentrations up to 2.3 times those determined by HPLC. These discrepancies were shown to be due to carry-over of metabolites and some extraneous background fluorescence.     

Expression of ras oncogene p21 during human fetal development as determined by monoclonal antibodies RAP-5, Y13-259, and DWP

In this report we describe the expression of the ras proto-oncogene p21 protein in various tissues during normal fetal development. Conventional, formalin fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging 9 to 42 weeks of gestation. Immunohistochemical localization of ras p21 was accomplished using the broadly reactive, mouse monoclonal antibodies RAP-5 and Y13-259. The monoclonal antibody DWP, which is specific for a mutated form of ras p21 having a valine/cysteine at amino acid position 12, was also used. Detectable expression of the p21 protein was seen at different time periods during fetal development depending on the tissue. The expression of ras p21 (as detected by RAP-5 and Y13-259) was noted in a wide range of cell types and tissues; intense immunostaining was noted in epithelial cells of the gastrointestinal tract, exocrine and endocrine pancreas, renal tubules and transitional urotheliem, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances ras p21 expression, when present, occurred during periods of rapid growth in given organ systems. However, some actively proliferating fetal tissues such as thymus and spleen, failed to express detectable ras p21 suggesting that factors other than cell cycle may influence its expression. No reactivity with DWP was noted in any of the tissues, suggesting that the mutated forms detected by this monoclonal antibody are not expressed during normal human embryogenesis. These data show that there is regulated expression, and broad distribution of this gene product in normal developing human fetal tissue.     

Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein

Protein disulfide isomerase (PDI) is a component of the microsomal triglyceride transfer protein (MTP) complex. This study was initiated to help elucidate the role of PDI in MTP. The 88-kDa polypeptide of MTP (88K) was dissociated from PDI by using chaotropic agents (NaClO4 and KSCN), low concentrations of a denaturant (guanidine hydrochloride) or a nondenaturing detergent (octyl glucoside). As assessed by fluorescence and circular dichroism spectroscopy, these three different approaches appeared to dissociate the components of MTP under mild, nondenaturing conditions. The dissociating agents were diluted or removed by dialysis, and the free PDI and 88K were further characterized. In all cases, the dissociation coincided with the loss of triglyceride transfer activity. The free 88-kDa polypeptide readily aggregated, suggesting that it is a hydrophobic peptide. Even in the presence of chaotropic agents, when 88K was not aggregated, transfer activity was not expressed. These results suggest that the association of PDI with 88K is necessary to maintain the catalytically active form of the triglyceride transfer protein and prevent the aggregation of 88K.     

Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis

Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae , the agent of swine dysentery, and S. pilosicoli , the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 10³–10⁴ cells of either organism seeded into 0·2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli , both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation.