Multiplex PCR assay to detect high risk lineages of Salmonella Typhi and Paratyphi A.

Enteric fever infections remain a significant public health issue, with up to 20 million infections per year. Increasing rates of antibiotic resistant strains have rendered many first-line antibiotics potentially ineffective. Genotype 4.3.1 (H58) is the main circulating lineage of S. Typhi in many South Asian countries and is associated with high levels of antibiotic resistance. The emergence and spread of extensively drug resistant (XDR) typhoid strains has increased the need for a rapid molecular test to identify and track these high-risk lineages for surveillance and vaccine prioritisation. Current methods require samples to be cultured for several days, followed by DNA extraction and sequencing to determine the specific lineage. We designed and evaluated the performance of a new multiplex PCR assay, targeting S. Paratyphi A as well as the H58 and XDR lineages of S. Typhi on a collection of bacterial strains. Our assay was 100% specific for the identification of lineage specific S. Typhi and S. Paratyphi A, when tested with a mix of non-Typhi Salmonella and non-Salmonella strains. With additional testing on clinical and environmental samples, this assay will allow rapid lineage level detection of typhoid of clinical significance, at a significantly lower cost to whole-genome sequencing. To our knowledge, this is the first report of a SNP-based multiplex PCR assay for the detection of lineage specific serovars of Salmonella Typhi.

Investigating interactions between epicardial adipose tissue and cardiac myocytes: what can we learn from different approaches?

Heart disease is a major cause of morbidity and mortality throughout the world. Some cardiovascular conditions can be modulated by lifestyle factors such as increased exercise or a healthier diet, but many require surgical or pharmacological interventions for their management. More targeted and less invasive therapies would be beneficial. Recently, it has become apparent that epicardial adipose tissue plays an important role in normal and pathological cardiac function, and it is now the focus of considerable research. Epicardial adipose tissue can be studied by imaging of various kinds, and these approaches have yielded much useful information. However, at a molecular level, it is more difficult to study as it is relatively scarce in animal models and, for practical and ethical reasons, not always available in sufficient quantities from patients. What is needed is a robust model system in which the interactions between epicardial adipocytes and cardiac myocytes can be studied, and physiologically relevant manipulations performed. There are drawbacks to conventional culture methods, not least the difficulty of culturing both cardiac myocytes and adipocytes, each of which has special requirements. We discuss the benefits of a three-dimensional co-culture model in which in vivo interactions can be replicated. LINKED ARTICLES: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Culture and differentiation of preadipocytes in two-dimensional and three-dimensional in vitro systems.

Adipogenesis is a complex process that involves the differentiation of preadipocytes into mature adipocytes. We have developed two-dimensional (2D) and three-dimensional (3D) cell culture systems for the purpose of culturing and differentiating primary preadipocytes in vitro. Differentiating preadipocytes show multiple lipid droplet accumulation and comparable protein expression patterns to mature adipocytes in vivo. We report that in both in vitro systems terminally differentiated adipocytes show characteristics similar to those of mature adipocytes in vivo, assessed by the expression of the S100alpha/beta protein, insulin receptor and caveolin-1, and receptors for inflammatory mediators, namely tumor necrosis factor-alpha receptors I and II (TNFRI and TNFRII) and chemokine receptor 5 (CCR5). Our results demonstrate that the S100 protein, caveolin-1, and insulin receptor are expressed and up-regulated in differentiating and terminally differentiated cells. In addition, the receptors for TNFalpha are not present in preadipocytes but are expressed in differentiating preadipocytes and in differentiated adipocytes. Similarly, CCR5 was exclusively expressed in differentiating preadipocytes and terminally differentiated adipocytes, but not in preadipocytes. Both 2D and 3D culture models are highly robust and reproducible and offer the potential to study adipogenesis and cellular interactions closely resembling and comparable to those in vivo. Our 3D collagen system offers a distinct advantage over the 2D system in that the adipocytes remain confined within the matrix and remain intact during biochemical analysis. Moreover, the collagen matrix allows adipocytes to closely simulate morphological characteristics and behavior as in vivo whilst permitting manipulation of the microenvironment in vitro to study adipogenesis.