Evolution of bacterial and fungal growth media

Microbial media has undergone several changes since its inception but some key challenges remain. In recent years, there has been exploration of several alternative nutrient sources, both to cater to the specificity in requirement of growth of “fussy microorganisms” and also to reduce costs for large-scale fermentation that is required for biotechnology. Our mini-review explores these developments and also points at lacunas in the present areas of exploration, such as a lack of concerted effort in pH and osmolarity regulation. We hope that our commentary provides direction for future research in microbial media.     

Heterologous Prime-Boost Vaccination with MF59-Adjuvanted H5 Vaccines Promotes Antibody Affinity Maturation towards the Hemagglutinin HA1 Domain and Broad H5N1 Cross-Clade Neutralization

In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.     

Pan-genome dynamics of Pseudomonas gene complements enriched across hexachlorocyclohexane dumpsite

Background

Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g⁻¹ sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient.

Results

Although strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g⁻¹ of soil).

Conclusions

Unlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1488-2) contains supplementary material, which is available to authorized users.     

Drug discovery for Duchenne muscular dystrophy via utrophin promoter activation screening

Background

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.

Methodology/Principal Findings

We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.

Conclusions/Significance

We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.     

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination

Background

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Methods and Findings

To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.

Conclusion

The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.     

A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells

Background

Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.

Methodology/Principal Findings

We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.

Conclusions/Significance

Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains.     

Differences in candidate gene association between European ancestry and African American asthmatic children

Background

Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry.

Methodology/Principal Findings

Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls.

Conclusions/Significance

We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.     

Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30 μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.     

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2^P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2^+/− mice. The Phd2^P294R/+-associated erythrocytosis is reversed in a Hif2a^+/−, but not a Hif1a^+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.     

Differential Effects of Rapamycin and Dexamethasone in Mouse Models of Established Allergic Asthma

The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4–6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4⁺ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4⁺ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established.