Diversity and dynamics of free-living and particle-associated Betaproteobacteria and Actinobacteria in relation to phytoplankton and zooplankton communities

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

Comparison of WTC dust size on macrophage inflammatory cytokine release in vivo and in vitro

Background

The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to ∼50 mm were recovered from rescue workers’ lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways.

Methodology/Principal Findings

Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM_10–53 or WTC-PM_2.5 at concentrations of 10, 50 or 100 µg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM_10–53 and PM_2.5. GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM_10–53 and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM_10–53 consistently induced more cytokine release than WTC-PM_2.5 at 100 µg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC.

Conclusions

WTC-PM_10–53 induced a stronger inflammatory response by human AM than WTC-PM_2.5. This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.     

Biochemical analysis and investigation on the prospective applications of alkaline protease from a <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain

Proteases have prospective financial and environment-friendly applications; hence attention is focused currently on the finding of new protease producing microorganism so as to meet the requirements of industry. A thermophilic bacterial strain producing extracellular protease activity was isolated from soil and identified as Bacillus cereus by analysis of 16S rRNA. Protease production by the microorganism was improved by studying the impact of the type of nitrogen and carbon source, fermentation period, growth temperature and initial pH of the culture medium in cultivation optimization experiments. The enzyme was purified to homogeneity in two step procedure involving Sephadex G-75 and Q-Sepharose chromatography. The molecular weight of purified enzyme was found to be 58 kDa by SDS-PAGE. Protease exhibited a pH and temperature optima of 7.5 and 60°, respectively. The enzyme was active in the pH range of 6.0–9.0 and stable up to 70°C. Histological analysis of protease treated goat and cow skin pelts showed complete removal of non leather forming structures such as hair shaft, hair follicles and glandular structures. The protease showed the stain removing property from blood stained cotton cloth and found to be compatible with six commercially available detergents. The protease could release peptides from natural proteins after digestion of coagulated egg albumin and blood clot.     

The isolation, purification and biological activity of a novel antibacterial compound produced by Pseudomonas stutzeri

A novel compound designated zafrin [4beta-methyl-5, 6, 7, 8 tetrahydro-1 (4beta-H)-phenanthrenone] was isolated from a crude extract of a marine bacterium identified as Pseudomonas stutzeri. Zafrin showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. The compound was purified and its structure was elucidated by spectroscopic methods including 1H-nuclear magnetic resonance (NMR), 13C-NMR, 1D-NMR and 2D-NMR spectroscopy. It could be demonstrated that a purified solution of zafrin was active against several human pathogens, including Staphylococcus aureus, and Salmonella typhi. By contrast, zafrin did not inhibit the growth of eukaryotic organisms Candida albicans and Schizosaccharomyces pombe. The minimal inhibitory concentration for Gram-positive bacteria ranged from 50 to 75 microg mL(-1) and varied between 75 and 125 microg mL(-1) for Gram-negative bacteria. Zafrin lysed Bacillus subtilis cells grown in an osmotically protected medium, suggesting that it does not act upon the cell wall. Further investigation using B. subtilis indicated that the compound is bactericidal and is likely to target the cell membrane.     

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

Drosophila photoreceptors respond to oscillating light of high frequency (～100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca²⁺-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trp^S936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trp^S936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trp^S936A flies reveal light-induced current (LIC) with short latency relative to trp^WT or trp^S936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.     

Osteopontin as a biomarker for COVID-19 severity and multisystem inflammatory syndrome in children: A pilot study

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0–21 years of age) admitted to Children’s Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention''s case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.