Rapid interrogation of cancer cell of origin through CRISPR editing

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9–sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

Models to study the earliest stages in cancer progression must, by definition, start with normal cells to assess the consequences of a suspected oncogenic perturbation. Inbred mouse strains and genetically engineered mouse models (GEMMs) have, for decades, served as gold standards for such studies but require significant time (years) to generate, breed, and age mice. CRISPR technology has greatly accelerated the pace of generating GEMMs through delivery of sgRNAs (guide RNAs) and/or Cas9 to tissues such as lung and liver (1–3). The ability to grow primary tissues ex vivo as organoids and introduce precise genetic changes into these cultures provides an alternative platform to model genomic alterations with speed and efficiency, as reported for intestine and prostate (4–7). Here we demonstrate highly efficient (50 to 90%) editing of primary prostate epithelial organoid cultures, including multigenic or intrachromosomal (>2 Mb) deletions, through transient electroporation of Cas9–sgRNA ribonucleoprotein (cRNP) complexes. We also show cRNP-based CRISPR editing can be performed on freshly isolated prostate epithelial cells and then transplanted orthotopically into the prostates of recipient mice in a single day, enabling extremely rapid generation of in vivo cancer models in immunodeficient as well as immunocompetent settings. Finally, we show that this approach can be used to address cancer cell-of-origin questions by multigenic editing selectively in luminal versus basal epithelial cells.     

Additional Diagnostic Yield of Adding Serology to PCR in Diagnosing Viral Acute Respiratory Infections in Kenyan Patients 5 Years of Age and Older

The role of serology in the setting of PCR-based diagnosis of acute respiratory infections (ARIs) is unclear. We found that acute- and convalescent-phase paired-sample serologic testing increased the diagnostic yield of naso/oropharyngeal swabs for influenza virus, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, and parainfluenza viruses beyond PCR by 0.4% to 10.7%. Although still limited for clinical use, serology, along with PCR, can maximize etiologic diagnosis in epidemiologic studies.     

Risk factors for active trachoma and Chlamydia trachomatis infection in rural Ethiopia after mass treatment with azithromycin

Objectives To investigate risk factors for ocular Chlamydia trachomatis infection and active trachoma, comparing communities receiving or not receiving an intervention programme of community‐wide azithromycin treatment and health education. Methods In a 3‐year post‐intervention follow‐up survey, 1722 children aged 3–9 years, from randomly selected households in 37 communities, were examined for signs of active trachoma and had samples taken to test for ocular C. trachomatis by polymerase chain reaction. Multivariate random effects logistic regression analyses considered interventions at community level, adjusting for other independent risk factors as appropriate. Results Younger age, ocular discharge and flies on eyes were risk factors for active trachoma in communities with and without antibiotic treatment. After azithromycin treatment, odds of active trachoma were lower in children aged 6–9 years than in children aged 3–5 years (OR 0.48, 95% CI: 0.36–0.66) and higher for children with ocular discharge (OR 4.5, 95% CI: 2.6–7.7) or flies on their eyes (OR 2.5, 95% CI: 1.6–3.7). Odds of C. trachomatis infection were lower in children aged 6–9 years than in younger children (OR 0.47, 95% CI: 0.23–0.96); and in children who received 2 or 3 doses rather than 1 (OR 0.26, 95% CI: 0.08–0.88). Conclusions In communities that received or did not receive the mass antibiotic treatment, the same risk factors for C. trachomatis and active trachoma were identified. Education and environmental improvements need to supplement antibiotic campaigns in order to positively impact on these remaining child level risk factors.     

Microvascular function is impaired in children with morbid obesity

Children's obesity is a growing problem in Western societies. We hypothesized that morbid obesity (body mass index [BMI] > 99.5th percentile) might affect microvascular function at an early stage. Therefore, we assessed the microvascular function of 41 obese children (13.2 ± 2.8 years, BMI 32.9 ± 6.6) in comparison to 91 healthy controls (12.7 ± 2.1 years, BMI 18.2 ± 2.5) by post-occlusive reactive hyperemia measured by a laser Doppler: baseline perfusion, biological zero (defined as 'no-flow' laser Doppler signal during supracystolic occlusion), peak perfusion (following occlusion), time to peak perfusion and recovery time (time until resuming baseline perfusion) were recorded and compared between both groups. Peak perfusion was higher in children with morbid obesity than in controls (1.67 ± 0.76 AU [arbitrary units] vs 1.26 ± 0.5 AU, p < 0.001). Consecutively, recovery time was longer in children with morbid obesity (118.21 ± 34.64 seconds) than in healthy children (83.18 ± 35.08 seconds, p < 0.001). In conclusion, higher peak perfusion and prolonged recovery time in children with morbid obesity seem to reflect microvascular dysfunction due to an impaired vasoconstrictive ability of precapillary sphincters.     

Assessment of vector microfilarial uptake as a comparatively non-invasive technique for monitoring onchocerciasis treatment campaigns in the Americas

Summary Since 1992, efforts have been made to combat onchocerciasis in Guatemala through mass distribution of ivermectin. The impact of the campaign is assessed by taking skin-snips from sentinel groups within selected communities. This method gives an estimate of the prevalence and intensity of infection, and thus the efficacy of the treatment. In some communities people are becoming reluctant to volunteer for skin-snipping, and so there is a need for an alternative technique that will give quantitative results. In most hyperendemic communities in Guatemala, biting blackflies are so ubiquitous that few people object to allowing 10 to 20 flies to engorge upon them. We examined data on the quantitative uptake of microfilariae by Simulium ochraceum before and after ivermectin distribution to see whether results similar to skin-snip data could be obtained. Counts of microfilariae ingested by S. ochraceum are compared to the numbers found in skin-snips from the same volunteers. In a group of 31 untreated infected persons, a skin-snip survey detected 64.5% positive, while feeding flies (vector microfilarial uptake, VmfU) detected 96.8%. Post-treatment, in a sample of 58 of whom 52 (89.7%) had a history of infection, both skin-snips and VmfU detected 54.2%. Vector blood meals contained more microfilariae than a mg of skin before treatment, but both recorded about equal numbers after treatment. When the data set was subdivided to compare samples taken at 2–3, 6–8 and 14–17 months post-treatment, the effect of ivermectin was still apparent at 6–8 months, but had virtually disappeared by 14 months post-treatment. A surprising observation was that the flies ingested fewer microfilariae from treated persons than was expected from the skin densities as estimated by skin-snip. This effect lasted for over 8 months, and could indicate that ivermectin has a greater effect on transmission than previously suspected. We conclude that VmfU could be used as an alternative to skin-snipping, and discuss the ethical implications.     

The effects of three nonoxynol-9 preparations on vaginal flora and epithelium.

To evaluate the effects of nonoxynol-9 (N-9) on the vaginal flora and epithelium, 48 women (16 in each group) were evaluated by use of quantitative vaginal cultures and colposcopy. at baseline and at 0.5, 4, 24, 48, and 72 h after insertion of one of three N-9 preparations (4% gel [Conceptrol], 3.5% gel [Advantage-24], or a 28% vaginal contraceptive film). The proportion positive for H2O2+ or H2O2- lactobacilli did not change significantly with any of the preparations, but lactobacilli concentrations decreased transiently. Both the proportion of women with Gardnerella vaginalis and the concentration of G. vaginalis decreased transiently. The proportion of women with Escherichia coli increased with the 4% gel, and the concentration increased with all preparations. The number with anaerobic gram-negative rods increased, although the concentrations decreased. Symptoms and colposcopic abnormalities were rare. Changes in levels of vaginal bacteria were transient after single applications of N-9, but adverse effects may be enhanced with frequent, chronic use.     

Morphological measurements of anatomic landmarks in pulp chambers of human anterior teeth

This in vitro study measured key morphological features of pulp chambers in anterior teeth and tested the hypothesis that the distance from the lingual surface (midpoint from the cusp tip to the lingual CEJ) to the pulp chamber (the midpoint from the buccal to the lingual CEJ) was similar for different tooth types. Extracted human teeth were sorted and 100 samples of each of the following tooth types were chosen: maxillary central incisor (UCI), lateral incisor (ULI), and canine (UC), as well as mandibular central incisor (LCI), lateral incisor (LLI), and canine (LC). All teeth were digitally radiographed on a 1-mm X-ray grid. The mean values of measurement C, the distance from the lingual surface to the pulp chamber, varied significantly between tooth types (p<0.001). The mean values and SNK rankings were as follows: LC (5.9+/-0.5 mm)>UC (5.5+/-0.5)=UCI (5.4+/-0.4)>ULI (5.0+/-0.4)>LLI (4.8+/-0.5)>LCI (4.4+/-0.4).     

Immunoassay and molecular methods to investigate DNA methylation changes in peripheral blood mononuclear cells in HIV infected patients on cART

This study aimed to investigate the influence of antiretroviral therapy on methylation markers, in a group of HIV infected, heavily treated patients. Immune and molecular methods were used to investigate potential changes in methylation profile in DNA isolated from peripheral blood mononuclear cells collected from antiretroviral-experienced HIV infected patients and healthy controls. The percentage of 5-methylcytosine was inversely correlated with proviral DNA and active replication while DNMT1 (p = 0.01) and DNMT3A (p = 0.004) independently correlated with active viral replication. DNMT3A expression increased with total treatment duration (p = 0.03), number of antiretroviral drugs ever used (p = 0.003), and cumulative exposure to protease inhibitors (p = 0.02) even in currently HIV undetectable patients.