Solution structure of the FCS zinc finger domain of the human polycomb group protein L(3)mbt‐like 2

Polycomb group proteins are epigenetic regulators that maintain patterns of gene expression over multiple rounds of cell division. Many of these proteins, including polyhomeotic and the MBT repeat containing proteins SCM and dSfmbt, contain an atypical C2C2 zinc finger with a characteristic phenylalanine–cysteine–serine sequence motif. The reoccurrence of this so‐called FCS zinc finger in a variety of polycomb group proteins suggests that it has an important regulatory function. We have determined the solution structure of the FCS zinc finger of the human dSfmbt homologue L(3)mbt‐like 2 (L3MBTL2). The structure consists of a β‐hairpin followed by an α‐helix. The zinc ligands are situated in the β‐hairpin and at the N‐terminus of the α‐helix an arrangement typical of the treble clef class of zinc fingers. The structure is consistent with the proposal that FCS zinc fingers bind to regulatory RNAs.     

4FISH-IF,a Four-Color Dual-Gene FISH Combined with p63 Immunofluorescence to Evaluate NKX3.1 and MYC Status in Prostate Cancer

NKX3.1 allelic loss and MYC amplification are common events during prostate cancer progression and have been recognized as potential prognostic factors in prostate cancer after radical prostatectomy or precision radiotherapy. We have developed a 4FISH-IF assay (a dual-gene fluorescence in situ hybridization combined with immunofluorescence) to measure both NKX3.1 and MYC status on the same slide. The 4FISH-IF assay contains four probes complementary to chromosome 8 centromere, 8p telomere, 8p21, and 8q24, as well as an antibody targeting the basal cell marker p63 visualized by immunofluorescence. The major advantages of the 4FISH-IF include the distinction between benign and malignant glands directly on the 4FISH-IF slide and the control of truncation artifact. Importantly, this specialized and innovative combined multiprobe and immunofluorescence technique can be performed on diagnostic biopsy specimens, increasing its clinical relevance. Moreover, the assay can be easily performed in a standard clinical molecular pathology laboratory. Globally, the use of 4FISH-IF decreases analytic time, increases confidence in obtained results, and maintains the tissue morphology of the diagnostic specimen.     

Determinants of non-vaccination against pandemic 2009 H1N1 influenza in pregnant women: a prospective cohort study

Background

In October 2009, the French government organized a national-wide, free of charge vaccination campaign against pandemic H1N1 influenza virus, especially targeting pregnant women, a high risk group for severe illness. The study objective was to evaluate pandemic flu vaccine uptake and factors associated with non-vaccination in a population of pregnant women.

Methodology/Principal Findings

In a prospective cohort conducted in 3 maternity hospitals in Paris, 882 pregnant women were randomly included between October 12, 2009 and February 3, 2010, with the aim to study characteristics of pandemic influenza during pregnancy. At inclusion, socio-demographic, medical, obstetrical factors and those associated with a higher risk of flu exposition and disease-spreading were systematically collected. Pandemic flu vaccine uptake was checked until delivery. 555 (62.9%) women did not get vaccinated. Determinants associated with non-vaccination in a multivariate logistic regression were: geographic origin (Sub-Saharan African origin, adjusted Odd Ratio aOR = 5.4[2.3–12.7], North African origin, aOR = 2.5[1.3–4.7] and Asian origin, aOR = 2.1[1.7–2.6] compared to French and European origin) and socio-professional categories (farmers, craftsmen and tradesmen, aOR = 2.3[2.0–2.6], intermediate professionals, aOR = 1.3[1.0–1.6], employees and manual workers, aOR = 2.5[1.4–4.4] compared to managers and intellectual professionals). The probability of not receiving pandemic flu vaccine was lower among women vaccinated against seasonal flu in the previous 5 years (aOR = 0.6[0.4–0.8]) and among those who stopped smoking before or early during pregnancy (aOR = 0.6[0.4–0.8]). Number of children less than 18 years old living at home, work in contact with children or in healthcare area, or professional contact with the public, were not associated with a higher vaccine uptake.

Conclusions/Significance

In this cohort of pregnant women, vaccine coverage against pandemic 2009 A/H1N1 flu was low, particularly in immigrant women and those having a low socio-economic status. To improve its effectiveness, future vaccination campaign for pregnant women should be more specifically tailored for these populations.     

Quantifying the Association between Bovine and Human Trypanosomiasis in Newly Affected Sleeping Sickness Areas of Uganda

Background

Uganda has active foci of both chronic and acute HAT with the acute zoonotic form of disease classically considered to be restricted to southeast Uganda, while the focus of the chronic form of HAT was confined to the northwest of the country. Acute HAT has however been migrating from its traditional disease focus, spreading rapidly to new districts, a spread linked to movement of infected cattle following restocking. Cattle act as long-term reservoirs of human infective T. b. rhodesiense showing few signs of morbidity, yet posing a significant risk to human health. It is important to understand the relationship between infected cattle and infected individuals so that an appropriate response can be made to the risk posed to the community from animals infected with human pathogens in a village setting.

Methodology/Principal Findings

This paper examines the relationship between human T. b. rhodesiense infection and human infective and non-human T. brucei s.l. circulating in cattle at village level in Kaberamaido and Dokolo Districts, Uganda. The study was undertaken in villages that had reported a case of sleeping sickness in the six months prior to sample collection and those villages that had never reported a case of sleeping sickness.

Conclusions and Significance

The sleeping sickness status of the villages had a significant effect with higher odds of infection in cattle from case than from non-case villages for T. brucei s.l. (OR: 2.94, 95%CI: 1.38–6.24). Cattle age had a significant effect (p<0.001) on the likelihood of T. brucei s.l. infection within cattle: cattle between 18–36 months (OR: 3.51, 95%CI: 1.63–7.51) and cattle over 36 months (OR: 4.20, 95%CI: 2.08–8.67) had significantly higher odds of T. brucei s. l. infection than cattle under 18 months of age. Furthermore, village human sleeping sickness status had a significant effect (p<0.05) on the detection of T. b. rhodesiense in the village cattle herd, with significantly higher likelihood of T. b. rhodesiense in the village cattle of case villages (OR: 25, 95%CI: 1.2–520.71). Overall a higher than average T. brucei s.l. prevalence (>16.3%) in a village herd over was associated with significantly higher likelihood of T. b. rhodesiense being detected in a herd (OR: 25, 95%CI: 1.2–520.71).     

Characterization of RNA polymerase type II from human term placenta

RNA polymerase type II from human term placenta has been isolated and characterized with respect to its template, ammonium sulfate, divalent cation, and buffer preferences. In addition, the apparent Michaelis constants for AMP and UMP incorporation have been determined. The enzyme was also analyzed by native and denaturing polyacrylamide gel electrophoresis, and evidence is presented that a single polypeptide is radiolabeled with azido purine nucleoside triphosphate photoprobes.     

Comparison of Effects of Trichuris muris and Spontaneous Colitis on the Proximal Colon Microbiota in C3H/HeJ and C3Bir IL10–/– Mice

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host''s microbial community are not fully understood. We hypothesized that Trichuris muris-infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4^−/− IL10^−/− and C3H/HeJ TLR4^−/− IL10^+/+ mice) with and without T. muris infection, in uninfected C3BirIL10^−/− mice with and without spontaneous colitis, and in normal C3H/HeJ mice. T. muris-infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Approximately 80% of infected IL10^−/− mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10^−/− and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10^−/− and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10^−/− mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10^−/− mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10^−/− mice or noncolitic C3H/HeJ mice. Therefore, Trichuris-infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.roup method with arithmetic averaging; OTU, operational taxonomic unit; qPCR, quantitative real-time PCR; SIMPER, similarity percentage; T-RFLP, terminal restriction fragment length polymorphism

Trichuris spp. are gastrointestinal nematodes that dwell in close association with a complex bacterial community in the host''s colon. After ingestion, embryonated eggs hatch in the cecum or colon releasing first-stage larvae that penetrate the epithelium and undergo 4 molts before becoming sexually mature. Both larval and adult Trichuris form syncytial tunnels in the colonic epithelium^21,30 that anchor the organisms in the proximal colon, where females produce eggs that pass in feces and embryonate in the environment.T. suis excretory secretory products (ESP) condition the colonic environment for enhanced worm survival, including effects on intestinal bacteria. Previous work demonstrated that T. suis ESP had dose-dependent effects on the tight junctions of epithelial cells.¹ The ESP fraction below a molecular weight of 10,000 kDa was mainly composed of an antimicrobial moiety² with bactericidal activity against gram-negative (Campylobacter jejuni, C. coli, and Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. In addition, due to several enzymatic activities, T. suis ESP have been demonstrated to aid the worms in burrowing into the host''s colonic epithelium and in feeding.^1,10,12 In addition to a 20-kDa diagnostic antigen,^10,11 higher molecular-weight fractions of ESP harbored a 42-kDa zinc metalloprotease that likely functions to provide nutrition for the worms through collagenase and elastase activities.¹⁰ Furthermore, a serine protease inhibitor (TsCEI) was purified from adult-stage T. suis by using acid precipitation, affinity chromatography, and reverse-phase HPLC.³³ This 6.43-kDa TsCEI inhibited chymotrypsin, pancreatic elastase, neutrophil elastase, and cathepsin G and was suggested to function as a parasite defense mechanism by modulating host immune responses. Indeed, exposure of cultured epithelial cells to T. suis ESP elicited IL6 and IL10 cytokine responses.³¹Trichuris has also been reported to interact with bacteria in vivo. Early studies demonstrated development of diarrhea in weaning age pigs concurrently harboring T. suis and various bacteria.³⁵ A mixed inoculum of T. suis and cecal scrapings containing Brachyspira, Campylobacter spp., or Salmonella spp. were implicated in this diarrhea by means of passive transfer to SPF pigs.³⁵ Interactions between this helminth and enteric bacteria were also explored by antibiotic treatment of T. suis-infected pigs.^20,27 Results of both passive transfer and antibiotic treatment experiments in pigs showed that Trichuris and various bacterial strains were necessary to produce the type of diarrhea and colonic lesions seen in weaning aged pigs in production, but did not implicate a single bacterial agent. In 2003, synergism between T. suis and C. jejuni was proven to cause mucohemorrhagic colitis in that germ-free piglets inoculated with both agents developed disease, whereas those infected with a single agent did not.²⁵ Recent studies in T. suis-infected pigs show changes in the microbial community of the colon with some accompanying metabolic changes.^22,45 Similar interactions have been found in extensive studies of captive rhesus monkeys with chronic enterocolitis. In these monkeys, severe disease was associated with presence of Trichuris trichiura and several enteric pathogens including C. coli, C. jejuni, Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni.³⁸ Therefore, Trichuris interacts with and may demonstrate synergy in disease production with the host''s colonic microflora.Interactions between Trichuris and bacteria have also been studied in mice.^9,20,36 One study found 100% morbidity in C57BL/6 IL10^−/− and congenic IL10^−/− IL4^−/− mice after challenge with T. muris.³⁶ The authors hypothesized that this high morbidity was due to an overgrowth of opportunistic invasive bacteria that use the mechanical damage caused by T. muris larvae to breach the intestinal tract. Adding the broad-spectrum antibiotic neomycin sulfate to the drinking water of IL10^−/− IL4^−/− mice and then infecting them with T. muris resulted in a statistically significant increase in the percentage of mice that survived infection.³⁶ The authors concluded that growth of opportunistic bacteria may have contributed to the previously observed morbidity and mortality. Most recently, another group⁹ found that increased levels of colonic microflora favor increased numbers of T. muris and chronic infections. The group also demonstrated that T. muris eggs hatched more efficiently in vitro when incubated with explants of mouse cecum containing 5 isolates of bacteria (E. coli, Staphylococcus aureus, Salmonella typhimurium, or Pseudomonas aeruginosa) and the yeast Saccharomyces cerevisiae, with the greatest effects seen at 37 °C. Similarly, work from our laboratory²⁰ demonstrated that treatment of T. muris-infected C57BL/6 IL10^−/− mice with metronidazole but not prednisolone increased survival.²⁰ Most recently, chronic infections with T. muris in C57BL/6 mice have been shown to decrease the diversity of intestinal microbiota,¹³ increase the abundance of Lactobacillus spp., and alter the metabolome.¹⁴Taken together, these data suggest an important microbial component to the pathogenesis of Trichuris infections in a variety of species. Given that Trichuris suis has been administered to patients with inflammatory bowel disease (IBD), and in some studies appeared to diminish IBD symptoms^42,43 we sought to understand the community-wide interactions of this worm with enteric bacteria in a mouse model of colitis. We hypothesized that the microbiota of the proximal colon would differ significantly in mice infected with T. muris as compared with uninfected mice. We theorized that these effects would occur due to the worm''s immunomodulatory properties in the host and may contribute to the successful outcomes of Trichuris treatment in patients with IBD.     

A method for the cryopreservation of thrombocytes

By using dimethylsulfoxide (0.7 mol/l) a technique of cryoconservation of thrombocyte concentrates was elaborated, with own material-technological possibilities being taken into consideration. In comparison to fresh thrombocytes the results of parameters tested in vitro reveal a mean functional ability of 50%. The survival time of cells stored in this way in vivo may be compared with fresh cells. On this basis HLA-typed single-donor pools could be established for clinical application.     

Epidemiology and reporting characteristics of preclinical systematic reviews

In an effort to better utilize published evidence obtained from animal experiments, systematic reviews of preclinical studies are increasingly more common—along with the methods and tools to appraise them (e.g., SYstematic Review Center for Laboratory animal Experimentation [SYRCLE’s] risk of bias tool). We performed a cross-sectional study of a sample of recent preclinical systematic reviews (2015–2018) and examined a range of epidemiological characteristics and used a 46-item checklist to assess reporting details. We identified 442 reviews published across 43 countries in 23 different disease domains that used 26 animal species. Reporting of key details to ensure transparency and reproducibility was inconsistent across reviews and within article sections. Items were most completely reported in the title, introduction, and results sections of the reviews, while least reported in the methods and discussion sections. Less than half of reviews reported that a risk of bias assessment for internal and external validity was undertaken, and none reported methods for evaluating construct validity. Our results demonstrate that a considerable number of preclinical systematic reviews investigating diverse topics have been conducted; however, their quality of reporting is inconsistent. Our study provides the justification and evidence to inform the development of guidelines for conducting and reporting preclinical systematic reviews.

A cross sectional study of a sample of recent preclinical systematic reviews reveals deficiencies in reporting and provides the justification and evidence to inform the development of specific guidelines for conducting and reporting preclinical systematic reviews.