Use of SSCP analysis to identify germline mutations in HNPCC families fulfilling the Amsterdam criteria

Hereditary non-polyposis colorectal cancer (HNPCC) is a clinical syndrome characterised by an inherited predisposition to early onset colorectal and uterine cancers and an increased incidence of other cancers. It is caused by germline defects in the human mismatch repair genes. Defects in two of the known mismatch repair genes (namely hMSH2 and hMLH1) account for over 90% of mutations found in HNPCC families. In this study we have identified 14 families that fulfilled the clinical criteria for HNPCC and screened the hMSH2 and hMLH1 genes for germline mutations using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Seven mutations were identified. Of these, there were five frameshifts, one missense mutation and a further novel mutation that involved separate transition and transversion changes in successive amino acid residues. Three of the mutations were in hMSH2 and four in hMLH1. The identification of germ-line mutations in an HNPCC family enables targeted surveillance and the possibility of early curative intervention. SSCP is a simple and effective method for identifying most mutations in the human mismatch repair genes using DNA from fresh, frozen or archival material. Received: 24 July 1996 / Revised: 26 September 1996     

Effects of an experimental Trypanosoma congolense infection on the reproductive performance of West African Dwarf goats

Thirty-six West African Dwarf (WAD) goats were used to assess the effects of an experimental Trypanosoma congolense infection on their reproductive system. Estrous cycles were synchronised and when confirmed pregnant (n = 31), the does were randomly allocated into control and trypanosome-infected groups. After infection, the animals were carefully observed till parturition. Trypanosome infection caused an increase of rectal temperature, a significant drop in PCV (infected: 23.3 +/- 0.3%; control: 28.5 +/- 0.4%; P < 0.0001) and abortions in 27.8% of the infected does. Kids born from infected does had a lower birth weight than kids born from control goats (0.9 +/- 0.1 kg versus 1.6 +/- 0.1 kg; P < 0.0001). Eight out of 13 kids (61.5%) that were born alive from infected does died during their first week of life. Plasma pregnancy-associated glycoprotein (PAG) and progesterone concentrations were lower in the infected animals than in the controls. In general, PAG concentration in does which aborted dropped before abortion. Our results revealed that artificial T. congolense infection affected reproductive performance of WAD goats with abortions, premature births and perinatal losses being observed. Neither transplacental transmission of T. congolense nor histopathological lesions of the placenta could be demonstrated.     

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Cwl0971, a novel peptidoglycan hydrolase,plays pleiotropic roles in Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.     

By-passing selection: direct screening for antibody-antigen interactions using protein arrays

We have developed a system to identify highly specific antibody-antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody-antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this 'naive' screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.     

Transducing properties of Drosophila Frizzled proteins.

In Drosophila, two closely related serpentine receptors, Frizzled (Fz) and D-Frizzled2 (Fz2) are able to act as receptors for the secreted Wnt peptide, Wingless (Wg). In addition to transducing the Wg signal, Fz (but not Fz2) is able to transduce a second, unidentified signal that mediates planar polarity. Much attention has been focused on the structure of the N-termini of the Fz-class receptors and their role in ligand binding. Experiments using techniques of high-level expression have suggested a role for the C-termini in specifying which of the two second messenger systems the receptors are able to activate (M. Boutros, J. Mihaly, T. Bouwmeeste and M. Mlodzik (2000). Science 288, 1825-1828). We argue here that experiments involving high level expression of the receptors cannot be adequately interpreted and we have tested the ability of the receptors and chimeric forms when driven at moderate levels to rescue loss of function of the fz and fz2 genes. Under these conditions we find that all receptors tested will function as Wg receptors, but only a subset show the ability to rescue the polarity pathway. The presence of this subset implies that the N terminus is necessary but not sufficient and suggests that the ability to transduce the polarity signal is widely distributed throughout the protein.     

Predicting low testosterone in aging men: a systematic review

Background:Physicians diagnose and treat suspected hypogonadism in older men by extrapolating from the defined clinical entity of hypogonadism found in younger men. We conducted a systematic review to estimate the accuracy of clinical symptoms and signs for predicting low testosterone among aging men.Methods:We searched the MEDLINE and Embase databases (January 1966 to July 2014) for studies that compared clinical features with a measurement of serum testosterone in men. Three of the authors independently reviewed articles for inclusion, assessed quality and extracted data.Results:Among 6053 articles identified, 40 met the inclusion criteria. The prevalence of low testosterone ranged between 2% and 77%. Threshold testosterone levels used for reference standards also varied substantially. The summary likelihood ratio associated with decreased libido was 1.6 (95% confidence interval [CI] 1.3–1.9), and the likelihood ratio for absence of this finding was 0.72 (95% CI 0.58–0.85). The likelihood ratio associated with the presence of erectile dysfunction was 1.5 (95% CI 1.3–1.8) and with absence of erectile dysfunction was 0.83 (95% CI 0.76–0.91). Of the multiple-item instruments, the ANDROTEST showed both the most favourable positive likelihood ratio (range 1.9–2.2) and the most favourable negative likelihood ratio (range 0.37–0.49).Interpretation:We found weak correlation between signs, symptoms and testosterone levels, uncertainty about what threshold testosterone levels should be considered low for aging men and wide variation in estimated prevalence of the condition. It is therefore difficult to extrapolate the method of diagnosing pathologic hypogonadism in younger men to clinical decisions regarding age-related testosterone decline in aging men.Male hypogonadism is defined as the presence of low serum testosterone and spermatozoa levels, accompanied by clinical signs and symptoms.¹ The Endocrine Society divides the symptoms and signs of androgen deficiency into 2 groups, based on expert consensus.¹ The first group, which is considered more specific, includes incomplete or delayed sexual development; eunuchoidism; reduced sexual desire (libido); erectile dysfunction; gynecomastia; decreased axillary, facial and pubic hair; small testes (i.e., volume < 5 mL); infertility: low-trauma fracture; low bone mineral density; and hot flushes.¹ The second group includes less specific signs and symptoms, such as decreased energy and motivation, depressed mood, poor concentration and memory, sleep disturbance, mild anemia, reduced muscle bulk and strength, increased body fat or body mass index, and diminished physical performance. ¹ Similar definitions have recently been developed by the Canadian Men’s Health Foundation Multidisciplinary Guidelines Task Force on Testosterone Deficiency.²In young men, hypogonadism is more commonly characterized by signs and symptoms from the first group, such as reduced libido and erectile dysfunction. This condition is most often caused by testicular or pituitary pathology, including hyperprolactinemia, pituitary or hypothalamic disorders, testicular disease, radiation exposure or genetic diseases such as Klinefelter syndrome.³ Testosterone replacement is indicated in these cases of “classic hypogonadism,” as it ameliorates the clinical symptoms.⁴In contrast, although these entities exist in older men too, they are less frequent causes of low testosterone than age-related changes. There is evidence that testosterone levels decline with age in all men, regardless of symptoms, at an estimated rate of 1%–3% per year.^5,6 One study found that serum testosterone levels were below the normal range in 20% of men in their 60s and in close to 50% of men in their 80s.⁷ However, the prevalence of symptomatic low testosterone (hypogonadism) is estimated by some to be much lower in this population, at about 2%.⁸ Given the high prevalence of low testosterone and more limited correlation with symptoms in aging men, it is uncertain to what extent this represents a physiologic or pathologic event.^6,7 Moreover, symptoms typically associated with low testosterone are less specific in older men and may be caused by other comorbidities. For example, erectile dysfunction can be the result of vascular insufficiency, neurologic impairment, psychogenic causes or substance use.⁹ Conditions such as diabetes mellitus and atherosclerosis are more common in older men, with up to 40% of men over 50 years of age having evidence of vascular insufficiency as the primary cause of their erectile dysfunction.¹⁰ Low libido similarly can result from psychiatric or medical conditions that are more common in older men.¹¹Currently, many clinicians diagnose hypogonadism in older men on the basis of low serum testosterone levels, with or without symptoms, largely on the assumption that this is a pathologic condition requiring treatment. The purpose of this study was to systematically review the available literature to estimate the accuracy and operating characteristics of signs and symptoms for predicting low testosterone in aging men.