High-throughput, microscope-based sorting to dissect cellular heterogeneity.

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.

PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting Trypanosoma cruzi kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect T. cruzi in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown T. cruzi infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; p = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; p > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized T. cruzi DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

A novel simian retrovirus subtype discovered in cynomolgus monkeys (Macaca fascicularis).

A new simian retrovirus (SRV) subtype was discovered in China and the USA from Cambodian-origin cynomolgus monkeys. Histopathological examination from necropsied animals showed multifocal lymphoplasmacystic and histocytic inflammation. The complete genome sequences demonstrated that the US virus isolates were nearly identical (99.91-99.93 %) and differed only slightly (99.13-99.16 % identical) from the China isolate. Phylogenetic analysis showed that the new virus isolates formed a distinct branch of SRV-1 through -7, and therefore were named this subtype, SRV-8. This SRV-8 variant was also phylogenetically and serologically more closely related to SRV-4 than any other SRV subtype.

Fatal atypical O:3 Yersinia pseudotuberculosis infection in cynomolgus macaques.

Fatal Yersinia pseudotuberculosis infection in cynomolgus macaques was diagnosed based upon pathology, microbiology and PCR for this study. Pathological findings included acute, erosive to ulcerative, necrohemorrhagic enterocolitis. Genotyping by PCR showed an O:3 pattern (gmd-fcl(+), ddhC-prt(+), manB(+), ddhA-B(+)), but an additional gene, wbyK, was detected. This is the second report to identify wbyK+ O:3 genotype as the cause of fatal yersiniosis. The first case was reported in 2008, and involved farm deer in the U.S. As the frequency of wbyK+ O:3 genotype is found more often in different carriers, O:3 genotype is proposed to be divided into two subtypes: O:3a without wbyK and O:3b with wbyK. Virulence gene analysis showed the presence of inv, ypmC, irp1, ybtP-ybtQ, yadA, yopB, yopH, lcrF, and suggested that this O:3b isolate could be a highly pathogenic strain to cynomolgus macaques.

Evolution of a health navigator model of care within a primary care setting: a case study.

OBJECTIVE: Patient navigation originated as an approach for reducing disparities in cancer care and consequent health outcomes. Over time navigator models have evolved and been used to address various health issues in differing contexts. This case study outlines the evolution, purpose and effects of a lay-led health navigator model in a deprived, sparsely populated, New Zealand rural setting, where primary care services are frequently understaffed and routinely overstretched. METHODS: Routinely collected service utilisation data, survey results and health navigator interview data were utilised to illustrate the client group the service works with, why primary care refer to the service, as well as lessons learned from implementation to ongoing service provision. RESULTS: Those referred to the navigator service generally represented the most vulnerable in the community. Survey respondents, overall, were highly satisfied with the service. Navigators identified barriers and facilitators to implementation, as well as ongoing obstacles and enablers to service provision. CONCLUSIONS: This lay-led navigator service provided support to a group of unwell individuals, with few resources and multiple barriers to negotiate, and has effectively engaged with health and social care services, while overcoming various barriers and obstacles to its establishment and ongoing operation.

Virological and serological characterization of SRV-4 infection in cynomolgus macaques.

The nature of SRV-4 infection in cynomolgus macaques remains unclear to date. Here, we report the monitoring of 24 cynomolgus monkeys that were naturally infected with SRV-4 for virus isolation, proviral load and antibody. The results indicated that the SRV-4 antibody status was statistically correlated to environmental temperature.

The complete genome and genetic characteristics of SRV-4 isolated from cynomolgus monkeys (Macaca fascicularis).

At least 5 serotypes of exogenous simian retrovirus type D (SRV/D) have been found in nonhuman primates, but only SRV-1, 2 and 3 have been completely sequenced. SRV-4 was recovered once from cynomolgus macaques in California in 1984, but its genome sequences are unknown. Here we report the second identification of SRV-4 and its complete genome from infected cynomolgus macaques with Indochinese and Indonesian/Indochinese mixed ancestry. Phylogenetic analysis demonstrated that SRV-4 was distantly related to SRV-1, 2, 3, 5, 6 and 7. SRV/D-T, a new SRV/D recovered in 2005 from cynomolgus monkeys at Tsukuba Primate Center in Japan, clustered with the SRV-4 isolates from California and Texas and was shown to be another occurrence of SRV-4 infection. The repeated occurrence of SRV-4 in cynomolgus monkeys in different areas of the world and across 25years suggests that this species is the natural host of SRV-4.