Internet gaming disorder: The prevalence and associated gaming behavior, anxiety, and depression among 8-12-year-old children of private schools in Salem city, India.

BACKGROUND: The escalating concern over Internet gaming disorder (IGD) among children underscores the urgency of comprehending its determinants and links to mental health, particularly for interventions targeting school-aged children. AIM: This study aimed to evaluate the prevalence and determinants of IGD and its association with depression, anxiety, and behavior among 8-12-year-old children attending private schools in Salem city. SETTINGS AND DESIGN: A cross-sectional study involving 780 children aged 8-12 years from Salem district was conducted. Schools were randomly sampled, and data were collected through a self-administered questionnaire. MATERIALS AND METHODS: Data were gathered from children without genetic, systemic, or mental disorders and brain trauma. The questionnaire, adapted from Alhamoud M A et al. (2022), encompassed sections on sociodemographic characteristics, gaming behavior, and scales for assessing IGD, depression, and anxiety. Administration occurred during school hours with a 30-35 min completion time. STATISTICAL ANALYSIS USED: Data analysis utilized SPSS v23.0, including descriptive statistics, ANOVA, Chi-square tests for intergroup comparisons, and Pearson's correlation coefficient to determine associations. RESULTS: The prevalence of IGD in Salem district was 1.2%, with higher rates of anxiety and depression observed among older children, particularly males. CONCLUSIONS: A positive correlation was evident between IGD, anxiety, and depression. Urgent preventive measures have to be warranted to curb the rising trend of IGD, such as limiting screen time and promoting outdoor activities to enhance children's overall health.

Enterobacterial common antigen biosynthesis in Yersinia pestis is tied to antimicrobial peptide resistance.

Resistance to antimicrobial peptides (AMPs) plays an important role in allowing Yersinia pestis to maintain a successful infection in the flea vector Xenopsylla cheopis . Mutants that are unable to modify lipid A in their outer membrane with aminoarabinose (Ara4N), showed increased sensitivity to AMPs such as polymyxin B (PB), as well as decreased survival in fleas. A deletion mutant of wecE , a gene involved in biosynthesis of enterobacterial common antigen (ECA), also displayed hypersusceptibility to PB in vitro. Additional mutants in the ECA biosynthetic pathway were generated, some designed to cause accumulation of intermediate products that sequester undecaprenyl phosphate (Und-P), a lipid carrier that is also used in numerous other pathways, including for peptidoglycan, O-antigen, and Ara4N biosynthesis. Mutants that accumulate Und-PP-linked intermediates (ECA-lipid II) showed increased susceptibility to PB, reduced Ara4N-modified lipid A, altered cell morphology, and decreased ability to maintain flea infections. These effects are consistent with a model where Y. pestis has a sufficiently limited free Und-P pool such that sequestration of Und-P as ECA-lipid II prevents adequate Ara4N biosynthesis, ultimately resulting in AMP hypersusceptibility.

Antibacterial activity of Xenopsylla cheopis attacins against Yersinia pestis.

Antimicrobial peptide resistance has been proposed to play a major role in the flea-borne transmission of Yersinia pestis . However, the antimicrobial peptide response in fleas and their interaction with Y. pestis is largely unknown. Attacins are one of the most abundantly expressed antimicrobial peptides within the first hours after Y. pestis infection of Xenopsylla cheopis , a major vector of plague. In this study, we report the cloning, expression, and purification of two X. cheopis attacin peptides and describe their interactions with and antimicrobial activities against Y. pestis . These flea attacins were shown to bind lipopolysaccharides and have potent activity against Y. pestis , however the mechanism of killing does not involve extensive membrane damage. Treatment with attacins rapidly inhibits Y. pestis colony formation and results in oxidative stress, yet live-cell imaging revealed that bacteria continue to grow and divide for several hours in the presence of attacins before undergoing morphological changes and subsequent lysis. This data provides insights into an early battle between vector and pathogen that may impact transmission of one of the most virulent diseases known to man.

Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.

Yersinia pestis Lipopolysaccharide Remodeling Confers Resistance to a Xenopsylla cheopis Cecropin.

Fleas are major vectors of Yersinia pestis, the causative agent of plague. It has been proposed that Y. pestis has developed the ability to overcome the innate immune responses of fleas. Despite the fact that they transmit a number of bacterial infections, very little is known about the immune responses in fleas. In this study, we describe the antimicrobial activities of a cecropin from Xenopsylla cheopis (cheopin), an efficient vector for Y. pestis in the wild. This is the first cecropin-class antimicrobial peptide described from Siphonaptera insects. Cheopin showed potent activity against Gram-negative bacteria but little activity against wild-type Y. pestis KIM6+. Deletion of the aminoarabinose operon, which is responsible for the 4-amino-4-deoxy-l-arabinose (Ara4N) modification of LPS, rendered Y. pestis highly susceptible to cheopin. Confocal microscopy and whole cell binding assays indicated that Ara4N modification reduces the affinity of cheopin for Y. pestis. Further, cheopin only permeabilized bacterial membranes in the absence of Ara4N-modified LPS, which was correlated with bacterial killing. This study provides insights into innate immunity of the flea and evidence for the crucial role of Ara4N modification of Y. pestis LPS in conferring resistance against flea antimicrobial peptides.

Comparative evaluation of shear bond strength of two adhesive materials on demineralized dentin treated with silver diamine fluoride: An in-vitro study.

CONTEXT: Early childhood caries (ECC) is one of the most common diseases in the children of developing countries, affecting their oral health-related quality of life. From an economic perspective, silver diamine fluoride (SDF) can limit the progression of active carious lesions. AIMS: To estimate and compare the loss of shear bond strength among two adhesive materials on SDF-treated demineralized primary teeth dentin. SETTINGS AND DESIGN: Laboratory setting and in-vitro study design. METHODS AND MATERIAL: For the in-vitro study, 40 primary teeth indicated for extraction were selected. The demineralization solution was 5% nitric acid. The samples were divided into four groups, Group 1 - GIC Type IX, Group 2 - Self-etch (SE) adhesive + G-Aenial Universal Flo, Group 3 - SDF + GIC Type IX, and Group 4 - SDF + SE adhesive + G-Aenial Universal Flo. STATISTICAL ANALYSIS USED: Descriptive analysis and analysis of variance with Tukey's Post hoc test. RESULTS: The shear bond strength of GC G-Aenial Universal Flo(18.8165 ± 13.0448 MPa) found to be superior to GIC (5.7845 ± 1.8968 MPa). However, bond strength was significantly reduced with GC G-Aenial Universal Flo(34.0441 ± 14.1949/18.8165 ± 13.0448 MPa) compared to GIC (7.7956 ± 2.2804/5.7845 ± 1.8968 MPa) following the application of SDF. CONCLUSIONS: It was concluded that SE adhesive + GC G-Aenial Universal Flois the material with better shear bond strength compared to GIC Type IX with and without SDF. Considering the severity and prevalence of ECC, socioeconomic strata of Indian population, the choice of material for masking the discoloration, and prevention of disease, GIC can be suggested as an alternative over GC G-Aenial Universal Flo.

Heat-shrinking DNA nanoparticles for in vivo gene delivery.

The particle size of a PEG-peptide DNA nanoparticle is a key determinant of biodistribution following i.v. dosing. DNA nanoparticles of <100 nm in diameter are sufficiently small to cross through fenestrated endothelial cells to target hepatocytes in the liver. In addition, DNA nanoparticles must be close to charge-neutral to avoid recognition and binding to scavenger receptors found on Kupffer cells and endothelial cells in the liver. In the present study, we demonstrate an approach to heat shrink DNA nanoparticles to reduce their size to <100 nm to target hepatocytes. An optimized protocol heated plasmid DNA at 100 °C for 10 min resulting in partial denaturation. The immediate addition of a polyacridine PEG-peptide followed by cooling to room temperature resulted in heat-shrunken DNA nanoparticles that were ~70 nm in diameter compared with 170 nm when heating was omitted. Heat shrinking resulted in the conversion of supercoiled DNA into open circular to remove strain during compaction. Heat-shrunken DNA nanoparticles were stable to freeze-drying and reconstitution in saline. Hydrodynamic dosing established that 70 nm heat-shrunken DNA nanoparticles efficiently expressed luciferase in mouse liver. Biodistribution studies revealed that 70 nm DNA nanoparticles are rapidly and transiently taken up by liver whereas 170 nm DNA nanoparticles avoid liver uptake due to their larger size. The results provide a new approach to decrease the size of polyacridine PEG-peptide DNA nanoparticles to allow penetration of the fenestrated endothelium of the liver for the purpose of transfecting hepatocytes in vivo.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

PEG-Peptide Inhibition of Scavenger Receptor Uptake of Nanoparticles by the Liver.

PEGylated polylysine peptides represent a new class of scavenger receptor inhibitors that may find utility at inhibiting DNA nanoparticle uptake by Kupffer cells in the liver. PEG-peptides inhibit scavenger receptors in the liver by a novel mechanism involving in situ formation of albumin nanoparticles. The present study developed a new in vivo assay used to explore the structure-activity-relationships of PEG-peptides to find potent scavenger receptor inhibitors. Radio-iodinated PEG-peptides were dosed i.v. in mice and shown to saturate liver uptake in a dose-dependent fashion. The inhibition potency (IC50) was dependent on both the length of a polylysine repeat and PEG molecular weight. PEG30kda-Cys-Tyr-Lys25 was confirmed to be a high molecular weight (33.5 kDa) scavenger receptor inhibitor with an IC50 of 18 µM. Incorporation of multiple Leu residues improved potency, allowing a decrease in PEG MW and Lys repeat, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys that inhibited scavenger receptors with an IC50 = 20 µM. A further decrease in PEG MW to 2 kDa increased potency further, resulting in a low molecular weight (4403 g/mol) PEG-peptide with an IC50 of 3 µM. Optimized low molecular weight PEG-peptides also demonstrated potency when inhibiting the uptake of radio-iodinated DNA nanoparticles by the liver. This study demonstrates an approach to discover low molecular weight PEG-peptides that serve as potent scavenger receptor inhibitors to block nanoparticle uptake by the liver.

Chimeric analogs of human ß-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms.

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human ß-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.

Variations in the interaction of human defensins with Escherichia coli: Possible implications in bacterial killing.

Human α and ß-defensins are cationic antimicrobial peptides characterized by three disulfide bonds with a triple stranded ß-sheet motif. It is presumed that interaction with the bacterial cell surface and membrane permeabilization by defensins is an important step in the killing process. In this study, we have compared interactions of three human α-defensins HNP3, HNP4, HD5 and human ß-defensins HBD1-4 that are active against Escherichia coli, with its cell surface and inner membrane as well as negatively charged model membranes. We have also included the inactive α-defensin HD6 in the study. Among the α-defensins, HNP4, HD5 and HD6 were more effective in increasing the zeta potential as compared to HNP3. Among the ß-defensins, HBD1 was the least effective in increasing the zeta potential. The zeta potential modulation data indicate variations in the surface charge neutralizing ability of α- and ß-defensins. Comparison of E. coli inner membrane and model membrane permeabilizing abilities indicated that HD5, HD6 and HBD1 do not permeabilize membranes. Although HBD4 does not permeabilize model membranes, considerable damage to the inner membrane of E. coli is observed. Our data indicate that mammalian defensins do not kill E. coli by a simple mechanism involving membrane permeabilization though their antibacterial potencies are very similar.

Antimicrobial activity of human α-defensin 6 analogs: insights into the physico-chemical reasons behind weak bactericidal activity of HD6 in vitro.

Human α-defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α-defensins in the crystalline state. However, the physico-chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α-defensins, shows broad-spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N-terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N-terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full-length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria.

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs.

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.

Engineering of a linear inactive analog of human ß-defensin 4 to generate peptides with potent antimicrobial activity.

Human ß-defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N-terminus of HBD4. Our results show that l-arginine to d-arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l-arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d-arginine. Substitution of cysteine with the hydrophobic helix-promoting amino acid α-aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d-arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d-amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents.