Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.     

3CL hydrolase-based multiepitope peptide vaccine against SARS-CoV-2 using immunoinformatics

The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.     

Identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays

Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.     

A technique for temporary control of hemorrhage

Traumatic injury to blood vessels is a common presentation in the Emergency Department. Control of hemorrhage is generally attempted using tight absorbent compression dressings. This may be a cause of inefficient control of hemorrhage and also contributes to tissue ischemia. We present a simple technique to control hemorrhage that does not cause tissue ischemia, which frequently accompanies the tight compression bandages applied for vascular injuries.     

Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay

The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9mg/ml; SPLUNC1 concentrations ranged from 34.7ng/ml to 13.8μg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7ng/ml to 5.3μg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.     

Effect of anti-mosquito hemolymph antibodies on fecundity and on the infectivity of malarial parasite Plasmodium vivax to Anopheles stephensi (Diptera:Insecta)

Rabbit antibodies to hemolymph antigens (102.5, 101, 100, 96, 88, 80, 64, 55, 43, 29, and 23 kDa) of Anopheles stephensi reduced fecundity as well as viability in An. stephensi. However, ingestion of these antibodies was not associated with a marked effect on the engorgement of mosquitoes but egg laying was significantly delayed. Antisera raised against hemolymph proteins were also used to identify cross reactive antigens/epitopes present in other tissues by Western blotting, as well as by in vivo ELISA. In addition, a significant reduction in oocyst development was also observed in An. stephensi mosquitoes that ingested anti-hemolymph antibodies along with Plasmodium vivax. The results confirmed the feasibility of targeting mosquito antigens as a novel anti-mosquito strategy, as well as confirmed the usefulness of such antigens for the development of a transmission-blocking vaccine.     

Extracorporeal treatment of cast nephropathy

Acute kidney injury (AKI) is common in patients with multiple myeloma (MM), most commonly caused by cast nephropathy resulting from precipitation of free light chains (FLC) in renal tubules. AKI may be irreversible and require dialysis and predicts a poor prognosis. Reduction in serum FLC concentration is thought to be associated with improved likelihood of kidney function recovery in MM patients with AKI. Plasma exchange (PE), by removing circulating FLC, has been used as a treatment modality to improve kidney function in MM, although its efficacy remains uncertain. Extracorporeal treatment with extended high cutoff hemodialysis (HCO-HD) has also recently been studied as a potentially more effective means of FLC removal. Both PE and HCO-HD may be beneficial in some patients, but only when used as adjuncts to successful chemotherapy. Further research is necessary to establish the specific efficacy of each of these extracorporeal methods in the treatment of cast nephropathy.