Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid.

A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P<0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.

Quality and composition of archived nucleic acids after use in SARS-CoV-2 molecular testing.

BACKGROUND: Reverse transcription real-time PCR (rRT-PCR) has been a gold-standard method to detect SARS-CoV-2, for which quality assessment of nucleic acids (NAs) is not needed. In order to prepare for future use, we evaluated NA quality from archived SARS-CoV-2 rRT-PCR samples. METHODS: NA samples were collected in February 2021 and extracted using the QIAamp DSP Virus Spin Kit, (53 SARS-CoV-2-positive and 100 SARS-CoV-2-negative). Quality, quantity, and purity of NA were measured spectrophotometrically or fluorescently. Droplet digital PCR was used to characterize the double strand DNA (dsDNA) origin and composition by quantifying 16S rDNA and RPP30. RESULTS: The RIN and purity were not significantly different between groups (p = 0.3828). RNA quantity was significantly higher than dsDNA in both groups (p < 0.0001); both dsDNA and RNA quantity were significantly higher in positive samples (dsDNA, RNA p = 0.021). For dsDNA, 16S rDNA copies were significantly greater than RPP30 in both groups (p < 0.0001), and RPP30 were significantly higher in positive samples (p < 0.0001). CONCLUSIONS: Archived NA quality after SARS-CoV-2 rRT-PCR was guaranteed for subsequent molecular research using human or bacterial DNA, especially for short targets.

A novel bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia: case report.

BACKGROUND: The germline mutations of DDX41, also known as DEAD box RNA helicase 41, have been found in about 1.5% of myeloid neoplasms (MNs). Development of MDS/AML is relatively common in germline DDX41 mutations. However, a variety of hematological malignancies (HMs) have been reported. CASE PRESENTATION: We report a novel case of bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia (B-ALL), with unusual location of DDX41 mutations. The gene expression profile (GEP) of Ph + B-ALL with bi-alleleic DDX41 mutations showed heterogeneously transitional GEP and altered gene expression levels of genes involved in the process essential for red blood cells and myeloid cell differentiation were noted. CONCLUSIONS: We report that DDX41 mutations are unusual but can be an underlying event in Ph + B-ALL and screening DDX41 mutations can be also informative for patients awaiting for haploidentical stem cell transplantation and choosing the therapy.

Structural Relationship between Korean Adolescent's Sports Participation, Optimism, Pessimism, Self-Regulation, and Coronavirus-Related Stress in the Pandemic Situation.

This study aimed to examine the relationships between sports participation, optimism/pessimism, self-regulation, and coronavirus-related stress in Korean adolescents during the pandemic situation. Specifically, we attempted to offer valuable information that could help to alleviate coronavirus-related stress in adolescents by promoting participation in sports and the development of optimism and self-regulation. To achieve this aim, we conducted an online survey of 836 Korean adolescents in the pilot and main studies. Confirmatory factor, frequency, path, reliability, descriptive statistical, and multimedia analyses were performed. Our findings indicated several differences for each variable according to demographic characteristics. Sports participation exerted a positive effect on optimism (p < 0.001) and self-regulation (p < 0.01) and negative effects on coronavirus-related stress (p < 0.05) and pessimism (p < 0.001). In addition, optimism exerted a positive effect on self-regulation (p < 0.001) and a negative effect on coronavirus-related stress (p < 0.001), while pessimism exerted a negative effect on self-regulation (p < 0.01) and a positive effect on coronavirus-related stress (p < 0.001). Further analysis indicated that self-regulation had a negative effect on coronavirus-related stress (p < 0.05). These findings highlight the need for youth educational institutions to encourage adolescents to participate in sports and for organizing bodies to suggest various policies and provide education that can assist them in properly coping with and overcoming coronavirus-related stress by strengthening their optimistic attitude and self-regulation ability.

Effects of School-Based Exercise Program on Obesity and Physical Fitness of Urban Youth: A Quasi-Experiment.

(1) Purpose: The purpose of this study was to evaluate if an after-school intervention program could prevent obesity and promote the physical fitness of urban sedentary school children. (2) Methods: A 16-week after-school physical fitness program was provided to 36 middle school students (7th, 8th, and 9th graders) recruited from a middle school for three days a week. They were high-risk youth showing poor health status (level 4 or 5) in the regular physical fitness evaluation conducted. Their body mass index (BMI), cardiovascular endurance, muscular strength and endurance, quickness, and flexibility were evaluated. (3) Results: A paired sample t-test was used (α = 0.05). There were statistical differences ((x ± s), p < 0.05) between the pre-Progressive Aerobic Cardiovascular Endurance Run (PACER) (13.36 ± 4.98 (# of laps)) and post-PACER (18.64 ± 6.31 (# of laps)) (p < 0.001), between the pre-sit-up (18.06 ± 7.22 (# of sit-ups)) and post-sit-up (24.89 ± 7.52 (# of sit-ups)) (p < 0.001), and between the pre-Trunk Flexion (2.64 ± 3.49 ((cm))) and post-Trunk Flexion (5.97 ± 2.78 ((cm)) (p < 0.001). There was no statistical difference between the pre-50m-Run (10.74 ± 1.30 ((sec))) and post-50m-Run results (10.69 ± 1.25 (sec)) (p = 0.063) or between the pre-BMI (24.84 ± 3.97 (kg/m2)) and post-BMI (24.76 ± 3.61 (kg/m2)) (p = 0.458). Overall, the physical fitness measures improved, whereas BMI did not change after 16 weeks. (4) Conclusion: Sixteen weeks of the school-based Health-related Physical Fitness (HrPF) program can be effective in improving overall physical fitness levels of adolescents, although additional treatments would be required to change BMI, which showed no improvement in the present study. It is concluded that in order for adolescents to maintain and promote physical fitness and health, participation in a school-based HrPF program is recommended for at least 30 min a day, three days a week. In addition, schools should provide high-risk youth with easy access to physical activities that are similar to the objectives of the physical education curriculum. In order to promote the health of school-age children, each school should establish and operate school-based systematic intervention programs.

A biodegradable poly(ester amine) based on polycaprolactone and polyethylenimine as a gene carrier.

The aim of research was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biodegradable polymers, in particular, poly(ester amine)s (PEAs), suitable for non-viral gene therapy. Poly(ester amine)s were successfully synthesized by Michael addition reaction between polycaprolactone (PCL) diacrylate and low molecular weight polyethylenimine (PEI). PEA/DNA complexes showed effective and stable DNA condensation with the particle sizes below 200nm, implicating its potential for intracellular delivery. PEAs showed controlled degradation and were essentially non-toxic in all three cells (293T: Human kidney carcinoma, HepG2: Human hepatoblastoma and HeLa: Human cervix epithelial carcinoma cell lines) at higher doses in contrast to PEI 25K. PEAs also revealed much higher transfection efficiencies in three cell lines as compared to PEI 25K. The highest reporter gene expression was observed for PCL/PEI-1.2 (MW 1200) complex having transfection efficiency 15-25 folds higher than PEI 25K in vitro. Also PEA/DNA complexes successfully transfected cells in vivo after aerosol administration than PEI 25K. These PEAs can be used as most efficient polymeric vectors which provide a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity and high transfection efficiency.

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus.

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine.

Heat killed spores of the Gram-positive bacterium Bacillus subtilis have been evaluated as a vaccine delivery system with mucosal adjuvant properties for influenza. Killed spores were able to bind H5N1 virions (NIBRG-14; clade 1) and, when intra-nasally administered to mice, resulting immune responses, both humoral and cell mediated, were enhanced compared to immunization with the virion alone. Levels of both systemic IgG and mucosal sIgA specific to the virion were elevated. Levels of IgG2a (a Th(1) antibody type) were strongly enhanced when the virion was co-administered with killed spores. Cytokine induction in stimulated splenocytes was also apparent indicating balanced T(h)1 and T(h)2 responses. Evidence of cross-neutralization of clade 2.2 viruses was shown. In a challenge experiment mice dosed two times with spores adsorbed with just 20 ng HA (hemagglutinin) of inactivated NIBRG-14 were fully protected against challenge with 20 LD(50) of H5N2 virus. Interestingly, partial protection (60%) was observed in animals dosed only with killed spores. Mice dosed only with killed spores were shown to be fully protected against H5N2 (5 LD(50)) infection indicating that innate immunity and its stimulation by spores may play an important role in protection. Supporting this killed spores were (i) shown to stimulate TLR-mediated expression of NF-κB, and (ii) able to recruit NK cells into lungs and induce maturation of DCs. This work demonstrates the potential and underlying mechanism for the use of bacterial spores as an adjuvant for H5N1 vaccination.