Applications and potential uses of fish gill cell lines: examples with RTgill-W1

Gills are unique structures involved in respiration and osmoregulation in piscinids as well as in many aquatic invertebrates. The availability of the trout-derived gill cell line, RTgill-W1, is beginning to make impacts in fish health and toxicology. These cells are available from the American Type Culture Collection as ATCC CRL 2523. The cells have an epithelioid morphology and form tight monolayer sheets that can be used for testing epithelial resistance. The cells can be grown in regular tissue culture surfaces or in transwell membranes in direct contact with water on their apical surfaces. The ability of RTgill-W1 to withstand hypo- and hyper-osmotic conditions and their optimal growth capacity at room temperature, make these cells ideal sentinel models for in vitro aquatic toxicology as well as model systems to study fish gill function and gill diseases. RTgill-W1 support growth of paramyxoviruses and orthomyxoviruses like salmon anemia virus. RTgill-W1 also support growth of Neoparamoeba pemaquidensis, the causative agent of amoebic gill disease. The cells have been used to understand mechanisms of toxicity, ranking the potencies of toxicants, and evaluating the toxicity of environmental samples. These cells are also valuable for high throughput toxicogenomic and toxicoproteomic studies which are easier to achieve with cell lines than with whole organisms. RTgill-W1 cell line could become a valuable complement to whole animal studies and in some cases as gill replacements in aquatic toxicology.     

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.     

Profile of an HIV Testing and Counseling Unit in Bangladesh: Majority of New Diagnoses among Returning Migrant Workers and Spouses

Introduction

Analysis of data from HIV testing and counseling (HTC) services provides an opportunity to identify important populations for targeting of HIV prevention efforts. Our primary aim was to describe the demographics of clients presenting to HTC in Bangladesh, a low HIV prevalence country. Our secondary aim was to determine the risk factors for HIV positivity among returning migrant workers who were tested.

Methods

We performed a cross-sectional study of data collected between 2002 and 2010 from the first HTC service established in Bangladesh, located in three large cities.

Results

8973 individuals attended HTC services, with 558 (6.2%) of clients testing positive for HIV, including 33 children. The majority of those who tested positive were aged 25–44 (71%), male (70%), and married (68%). Key populations considered at increased risk of HIV, such as female sex workers, people who inject drugs, and males who have sex with males accounted for only 11% of adults who tested positive. Notably, 75% of adults testing positive had a history of migrant work or was the spouse of a migrant worker. In multivariable logistic regression of those with a migrant work history presenting for HTC, we found rural residence, working in the Middle East, and longer duration of migrant work to be independently associated with testing positive, and female gender and higher level of education to be negatively associated.

Conclusions

These data suggest that in Bangladesh, in addition to targeting traditional key populations, HIV prevention efforts should also focus on migrant workers and their spouses.     

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.     

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.     

Evolutionary history and global spread of the emerging g12 human rotaviruses

G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.     

Generation of easily accessible human kidney tubules on two‐dimensional surfaces in vitro

The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β₁, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.     

Invitromatics,invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18–22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.     

Isolation of Shigella dysenteriae Type 1 and S. flexneri Strains from Surface Waters in Bangladesh: Comparative Molecular Analysis of Environmental Shigella Isolates versus Clinical Strains

Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh. Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined. We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples. Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes. Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples. Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD. However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH. This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes. Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh.