Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques.

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype.

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix.

A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP) M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease.

Molecular diagnosis and analysis of Chikungunya virus.

BACKGROUND: In March 2005 a Chikungunya fever outbreak began on the islands of the Indian Ocean. The number of cases of this disease dramatically rose amongst these islands before affecting over a million people in India. Travellers to these regions have returned to the UK with the disease leading to a greater than 15-fold increase in the annual number of Chikungunya virus (CHIKV) sero-positive samples in 2006. OBJECTIVES: A real-time RT-PCR test was developed for CHIKV and designed to detect currently circulating strains of virus as well as other genotypes. Its sensitivity was compared with an existing standard RT-PCR assay and a previously published real-time assay. STUDY DESIGN: A real-time RT-PCR assay was optimised and evaluated using a panel of 55 clinical serum samples and a synthetic RNA transcript as a positive control. Nucleotide sequencing of part of the E1 gene of CHIKV was used to investigate the relatedness of the samples. RESULTS: The real-time RT-PCR was 10-fold more sensitive than a conventional block-based RT-PCR and could detect as low as 20 copies of RNA transcript. The assay also had 10-fold improved sensitivity in detecting the outbreak strain of virus when compared to another published TaqMan assay. Analysis of sequences from patients that had travelled to India, Mauritius or the Seychelles showed high similarity with published sequences from the Indian Ocean island of Réunion. CONCLUSIONS: A sensitive and rapid real-time RT-PCR assay has been developed for CHIKV and tested against current isolates.

Co-evolutionary patterns of variation in small and large RNA segments of Crimean-Congo hemorrhagic fever virus.

The genus Nairovirus of the family Bunyaviridae includes the Crimean-Congo haemorrhagic fever (CCHF) species group. The species is predominated by the hazard-group 4 pathogens, from which the name and majority of strain entries are derived. Additionally, the species embraces hazard-group 2 viruses that are classified as members by antigenic cross-reactivity. CCHF viruses have a tripartite RNA genome consisting of large (L), medium (M) and small (S) segments. Here, the sequence characterization of previously undescribed L and S segments from novel strains originating in the Middle East and Africa is reported. Further scrutiny of this data with phylogenetic tools, in the context of other publicly available sequence information, reveals analogous grouping patterns between the L and S segments. These groups correlate with the geographical distribution of strain isolation and indicate that the L and S segments of CCHF viruses have evolved together.

Dengue hemorrhagic fever with fulminant hepatic failure in an immigrant returning to Bangladesh.

An immigrant from Bangladesh living in the United Kingdom presented with a nonspecific febrile illness after visiting his homeland and subsequently developed fulminant hepatic failure accompanied by hypotension, ascites, a generalized coagulopathy, and thrombocytopenia. Serology and detection of dengue virus serotype 3 by PCR established a postmortem diagnosis of hepatic failure secondary to dengue hemorrhagic fever.