Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

Influence of Lactobacillus reuteri, Bifidobacterium animalis subsp. lactis, and prebiotic inulin on dysbiotic dental biofilm composition ex vivo.

BACKGROUND: Probiotic bacterial supplementation has shown promising results in the treatment of periodontitis and the maintenance of periodontal health. The purpose of this investigation was to evaluate the influence of Lactobacillus reuteri or Bifidobacterium animalis subsp. lactis supplementation with and without prebiotic inulin on biofilm composition using an ex vivo biofilm model. METHODS: Subgingival plaque specimens from three periodontitis-affected human donors were used to grow biofilms on hydroxyapatite disks in media supplemented with varying combinations of prebiotic inulin, Lactobacillus reuteri, and Bifidobacterium animalis subsp. lactis. Relative abundances of bacterial genera present in mature biofilms were evaluated using 16S rRNA next-generation sequencing. Diversity metrics of microbial communities were evaluated using a next-generation microbiome bioinformatics platform. RESULTS: Inulin supplementation produced statistically significant dose-dependent increases in relative abundances of Lactobacillus and Bifidobacterium species (p < 0.001) with concomitant decreases in relative abundances of Streptococcus, Veillonella, Fusobacterium, Parvimonas, and Prevotella species (p < 0.001). Inoculation with L. reuteri or B. animalis subsp. lactis increased the relative abundance of only the supplemented probiotic genera (p < 0.05). Supplemental inulin led to a statistically significant decrease in biofilm alpha diversity (p < 0.001). CONCLUSIONS: The described ex vivo model appears suitable for investigating the effects of probiotic bacteria, prebiotic oligosaccharides, and combinations thereof on biofilm composition and complexity. Within the limitations imposed by this model, results from the present study underscore the potential for prebiotic inulin to modify biofilm composition favorably. Additional research further elucidating biologic rationale and controlled clinical research defining therapeutic benefits is warranted.

Prevalence of Intestinal Parasitic Infections, Genotypes, and Drug Susceptibility of Giardia lamblia among Preschool and School-Aged Children: A Cross-Sectional Study in Thailand.

This study aimed to estimate the prevalence of intestinal parasitic infections in children and assess the drug susceptibility and genotypes/assemblages of Giardia lamblia in Thailand. This cross-sectional study was conducted among children aged 3-12 years in Sangkhlaburi District, Kanchanaburi Province, Thailand, between 25 September 2017 and 12 January 2018. Parasites were identified by stool microscopic examination, cultivation of intestinal parasitic protozoa, and enzyme-linked immunosorbent assay (ELISA). Drug susceptibility and genotype of G. lamblia were performed, respectively, by a resazurin assay and Triosephosphate Isomerase A and B genes using modified primers and probes. Among the 661 participants, 445 had an intestinal parasitic infection, resulting in a prevalence of 67.32% (95% CI: 63.60-70.89%). Blastocystis hominis was the most prevalent protozoa infection (49.32%; 95% CI: 45.44-53.22%), while Ascaris lumbricoides was the most prevalent helminth infection (0.91%; 95% CI: 0.33-1.97%). The prevalence of G. lamblia was 17.40%, with genotype B being the most common. According to our study, intestinal parasitic infections were commonly found in Thai children. G. lamblia was the most common pathogenic protozoa infection identified and exhibited less susceptibility to metronidazole compared to furazolidone and mebendazole.

Time- and Species-Dependent Bacterial Adhesion to Titanium over Short Exposure Periods: An In Vitro Study.

A considerable percentage of dental implant patients experience biofilm-mediated peri-implant disease following transmucosal abutment application. Bacterial adhesion is an early step in biofilm development. Our purpose was to assess adhesion of specific bacterial species to titanium over short exposure periods. Eight bacterial species were selected for this analysis: Streptococcus oralis, Streptococcus mitis, Gemella haemolysans, Streptococcus gordonii, Streptococcus sanguinis, Neisseria flavescens, Streptococcus salivarius, and Pseudomonas aeruginosa. We cultured each species with appropriate media and exposed titanium foil discs to the bacteria for 60, 15, 5, 1, or 0.25 minutes. Optical density at 600-nm wavelength (OD600) was assessed for the baseline inoculum and each species/exposure combination. The proportion of bacteria adherent to titanium was determined for each experimental condition. Striking titanium adhesion was noted for all evaluated species even when exposure time was limited to 15 seconds. Strategies to limit bacterial adhesion at dental implant surfaces may offer potential for improved treatment outcomes and preservation of peri-implant health.

Absence of "Cytokine Storm" in Hospitalized COVID-19 Patients: A Retrospective Cohort Study.

Background: A rapidly growing number of publications cite "cytokine storm" as a contributing factor in coronavirus disease 2019 (COVID-19) pathology. However, a few recent reports led to questioning of "cytokine storm" theory in COVID-19. This study's primary goal is to determine if exaggerated cytokine response in the range of a "cytokine storm" develops during the initial weeks of hospitalization in COVID-19 patients. Methods: Five proinflammatory cytokines reported to be involved in "cytokine storm" and elevated in COVID-19 (IL-6, IL-8, TNF-α, MCP-1, and IP-10) were analyzed in COVID-19, influenza (with "cytokine storm": CS), and burn injury patients. The effect of dexamethasone use on cytokine response in COVID-19 was also analyzed. Results: None of the five cytokines in COVID-19 patients reached the lower threshold (95% CI) of the influenza (CS) group at any point during the study period. Furthermore, mean concentrations of all five cytokines in the influenza (CS) group and IL-6, IL-8, TNF-α in the burn group were significantly greater than in COVID-19 patients (p < 0.01). Dexamethasone treatment did not significantly alter the concentrations of any of the cytokines analyzed. Conclusions: Exaggerated cytokine response similar to "cytokine storm" was not observed in COVID-19 patients during two weeks of hospitalization.

Combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against HIV Type 1, HIV Type 2, and selected carbohydrate-binding agent-resistant HIV Type 1 strains.

Carbohydrate-binding agents (CBAs) are potential HIV microbicidal agents with a high genetic barrier to resistance. We wanted to evaluate whether two mannose-specific CBAs, recognizing multiple and often distinct glycan structures on the HIV envelope gp120, can interact synergistically against HIV-1, HIV-2, and HIV-1 strains that were selected for resistance against particular CBAs [i.e., 2G12 mAb and microvirin (MVN)]. Paired CBA/CBA combinations mainly showed synergistic activity against both wild-type HIV-1 and HIV-2 but also 2G12 mAb- and MVN-resistant HIV-1 strains as based on the median effect principle with combination indices (CIs) ranging between 0.29 and 0.97. Upon combination, an increase in antiviral potency of griffithsin (GRFT) up to ∼12-fold (against HIV-1), ∼8-fold (against HIV-2), and ∼6-fold (against CBA-resistant HIV-1) was observed. In contrast, HHA/GNA combinations showed additive activity against wild-type HIV-1 and HIV-2 strains, but remarkable synergy with HHA and GNA was observed against 2G12 mAb- and MVN-resistant HIV-1 strains (CI, 0.64 and 0.49, respectively). Overall, combinations of GRFT and other CBAs showed synergistic activity against HIV-1, HIV-2, and even against certain CBA-resistant HIV-1 strains. The CBAs tested appear to have distinct binding patterns on the gp120 envelope and therefore do not necessarily compete with each other's glycan binding sites on gp120. As a result, there might be no steric hindrance between two different CBAs in their competition for glycan binding (except for the HHA/GNA combination). These data are encouraging for the use of paired CBA combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent HIV transmission.

The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers.

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.

NMR structure of the N-terminal coiled coil domain of the Andes hantavirus nucleocapsid protein.

The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal domain (residues 1-74, called N1-74) of the Andes hantavirus N protein. N1-74 forms two long helices (alpha1 and alpha2) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix alpha1-alpha2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg22 or Lys26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg22 and Lys26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.