Validation of the human T-lymphocyte cloning assay — ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.     

Identification of three oligosaccharide binding sites in ricin.

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.     

A direct method for the formation of peptide and carbohydrate dendrimers.

Two new methods for the modification of PAMAM dendrimers have been developed which allow the covergent synthesis of either peptide or carbohydrate-bearing dendrimer molecules. Both methods involve condensation between hydroxylamino nucleophiles and appropriate carbonyl-bearing reaction partners.     

“We know about schistosomiasis but we know nothing about FGS”: A qualitative assessment of knowledge gaps about female genital schistosomiasis among communities living in Schistosoma haematobium endemic districts of Zanzibar and Northwestern Tanzania

BackgroundSchistosoma haematobium causes urogenital schistosomiasis and is widely distributed in Tanzania. In girls and women, the parasite can cause Female Genital Schistosomiasis (FGS), a gynecological manifestation of schistosomiasis that is highly neglected and overlooked by public health professionals and policy makers. This study explored community members’ knowledge, attitudes and perceptions (KAP) on and health seeking behavior for FGS.Methods/Principal findingsUsing qualitative research methods—including 40 Focus Group Discussions (FGDs) and 37 Key Informant Interviews (KIIs)—we collected data from 414 participants (Males n = 204 [49.3%] and Females n = 210 [50.7%]). The study engaged 153 participants from Zanzibar and 261 participants from northwestern Tanzania and was conducted in twelve (12) purposively selected districts (7 districts in Zanzibar and 5 districts in northwestern Tanzania). Most participants were aware of urogenital schistosomiasis. Children were reported as the most affected group and blood in urine was noted as a common symptom especially in boys. Adults were also noted as a risk group due to their involvement in activities like paddy farming that expose them to infection. Most participants lacked knowledge of FGS and acknowledged having no knowledge that urogenital schistosomiasis can affect the female reproductive system. A number of misconceptions on the symptoms of FGS and how it is transmitted were noted. Adolescent girls and women presenting with FGS related symptoms were reported to be stigmatized, perceived as having a sexually transmitted infection (STI), and sometimes labeled as “prostitutes”. Health seeking behavior for FGS included a combination of traditional medicine, self-treatment and modern medicine.Conclusion/SignificanceCommunity members living in two very different areas of Tanzania exhibited major, similar gaps in knowledge about FGS. Our data illustrate a critical need for the national control program to integrate public health education about FGS during the implementation of school- and community-based mass drug administration (MDA) programs and the improvement of water, sanitation and hygiene (WASH) facilities.     

Irreproducibility in searches of scientific literature: A comparative analysis

1. Repeatability is the cornerstone of science, and it is particularly important for systematic reviews. However, little is known on how researchers’ choice of database, and search platform influence the repeatability of systematic reviews. Here, we aim to unveil how the computer environment and the location where the search was initiated from influence hit results.
2. We present a comparative analysis of time‐synchronized searches at different institutional locations in the world and evaluate the consistency of hits obtained within each of the search terms using different search platforms.
3. We revealed a large variation among search platforms and showed that PubMed and Scopus returned consistent results to identical search strings from different locations. Google Scholar and Web of Science''s Core Collection varied substantially both in the number of returned hits and in the list of individual articles depending on the search location and computing environment. Inconsistency in Web of Science results has most likely emerged from the different licensing packages at different institutions.
4. To maintain scientific integrity and consistency, especially in systematic reviews, action is needed from both the scientific community and scientific search platforms to increase search consistency. Researchers are encouraged to report the search location and the databases used for systematic reviews, and database providers should make search algorithms transparent and revise access rules to titles behind paywalls. Additional options for increasing the repeatability and transparency of systematic reviews are storing both search metadata and hit results in open repositories and using Application Programming Interfaces (APIs) to retrieve standardized, machine‐readable search metadata.

     

Tyrosine phosphorylation of DEPTOR functions as a molecular switch to activate mTOR signaling

Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor–dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2.     

Primate digestion: Interactions among anatomy,physiology, and feeding ecology

Food is vital for life. It provides nutrients for growth, maintenance, and reproduction, and is the source of energy that drives the chemical reactions occurring in every cell.^1,2 However, most food, as it is initially procured, is not in a form suitable for use; it must first be broken down so that it can be transported through cell membranes.¹ The breaking down of food molecules via a system of both mechanical and chemical processes so that they are of use to the body is called digestion.^2,3 © 1998 Wiley-Liss, Inc.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface.

Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415-420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.