IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction.

Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor - alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations.

Genetic structure and population connection of two Bouyei populations in northern Vietnam based on short tandem repeat analysis.

OBJECTIVES: Genetic characteristics were investigated based on short tandem repeat (STR) data to assess the relationship between two Vietnamese Bouyei populations in Vietnam. METHODS: We collected hair and buccal swab samples from two separate Bouyei populations in the mountainous region of Northern Vietnam, which are the Bo Y in Ha Giang Province and the Tu Di in Lao Cai Province. The study included data of 23 autosomal and 27 Y-chromosome STRs loci of 96 unrelated participants from a total Vietnamese Bouyei population of under 3300 individuals. RESULTS: The results showed that these STR markers are valuable for differentiation of individuals and human genetic studies in Vietnamese Bouyei populations. Genetic analysis indicated that Tu Di and Bo Y people were from the same Bouyei population in China. CONCLUSIONS: The results supported the official historical records of the region and the classification of the Vietnamese government. Furthermore, the genetic data provided in this study will be helpful in investigating the genetic genealogy evolution and settlement or migration patterns of the Bouyei populations in Vietnam.

The mitochondrial DNA HVI and HVII sequences and haplogroup distribution in a population sample from Vietnam.

BACKGROUND: Mitochondrial DNA (mtDNA) analysis has been used in forensics and requires well-established population databases for statistical interpretations. However, high-quality mtDNA data from Vietnamese population samples have been limited. AIM: To examine the mtDNA sequences and haplogroup compositions of a Vietnamese population to provide an mtDNA dataset that can further be used to construct a Vietnamese-specific reference database. SUBJECTS AND METHODS: A total of 173 Vietnamese individuals were analysed for two hypervariable regions (HVI and HVII) of mtDNA. Forensic parameters were calculated and haplogroup assignment was performed based on the resulting mtDNA haplotypes. Genetic relationships between the Vietnamese and other Asian populations were investigated through principal component analysis (PCA) and pairwise Fst. RESULTS: The Vietnamese population sample consisted of 145 different haplotypes with a random match probability of 0.96%, a power of discrimination of 0.9904, and a haplotype diversity of 0.9962. The samples were assigned to 83 haplogroups that were commonly reported in Asia. PCA and pairwise Fst revealed close relationships of the Vietnamese population with other Asian populations, especially with populations in proximity. CONCLUSION: The results from this study can contribute to the current genetic information content as a supplementary mtDNA reference dataset for forensic investigations and phylogenetic research.

Massively parallel sequencing of human skeletal remains in Vietnam using the precision ID mtDNA control region panel on the Ion S5™ system.

Mitochondrial DNA (mtDNA) analysis using Sanger sequencing has been a routine practice for the identification of human skeletal remains. However, this process is usually challenging since DNA from the remains is highly degraded and at low concentration. Recently, the advent and implementation of massively parallel sequencing (MPS) have been offered the ability to improve mtDNA sequence data for forensic analysis. To assess the utility of the Ion S5™ system - an MPS platform for mtDNA analysis in challenging samples, we sequenced the mitochondrial control region of 52 age-old skeletal remains. Using the Precision ID mtDNA Control Region Panel, 50 full and two partial control region haplotypes at relatively high mean coverage of 2494 × were achieved for variant calling. Further variant analysis at 10% threshold for point heteroplasmy showed high degradation degree in terms of DNA damage in our bone samples. A higher point heteroplasmy threshold of 20% was required to diminish most of background noise caused by the damage. The results from this study indicated the potential application of the Ion S5™ system in sequencing degraded samples in Vietnam and provided valuable data sources for forensic analyses in the future.

Effect of an integral quality monitor on 4-, 6-, 10-MV, and 6-MV flattening filter-free photon beams.

PURPOSE: To investigate the effect of an integral quality monitor (IQM; iRT Systems GmbH, Koblenz, Germany) on 4, 6, 10, and 6-MV flattening filter-free (FFF) photon beams. METHODS: We assessed surface dose, PDD20,10 , TPR20,10 , PDD curves, inline and crossline profiles, transmission factor, and output factor with and without the IQM. PDD, transmission factor, and output factor were measured for square fields of 3, 5, 10, 15, 20, 25, and 30 cm and profiles were performed for square fields of 3, 5, 10, 20, and 30 cm at 5-, 10-, and 30-cm depth. RESULTS: The differences in surface dose of all energies for square fields of 3, 5, 10, 15, 20, and 25 cm were within 3.7% whereas for a square field of 30 cm, they were 4.6%, 6.8%, 6.7%, and 8.7% for 4-MV, 6-MV, 6-MV-FFF, and 10-MV, respectively. Differences in PDD20,10 , TPR20,10 , PDD, profiles, and output factors were within ±1%. Local and global gamma values (2%/2 mm) were below 1 for PDD beyond dmax and inline/crossline profiles in the central beam region, respectively. The gamma passing rates (10% threshold) for PDD curves and profiles were above 95% at 2%/2 mm. The transmission factors for 4-MV, 6-MV, 6-MV-FFF, and 10-MV for field sizes from 3 × 3 to 30 × 30 cm2 were 0.926-0.933, 0.937-0.941, 0.937-0.939, and 0.949-0.953, respectively. CONCLUSIONS: The influence of the IQM on the beam quality (in particular 4-MV X-ray has not verified before) was tested and introduced a slight beam perturbation at the surface and build-up region and the edge of the crossline/inline profiles. To use IQM in pre- and intra-treatment quality assurance, a tray factor should be put into treatment planning systems for the dose calculation for the 4-, 6-, 10-, and 6-MV flattening filter-free photon beams to compensate the beam attenuation of the IQM detector.

A novel p.A191D matrilin-3 variant in a Vietnamese family with multiple epiphyseal dysplasia: a case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia that is characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. Mutations in a gene encoding matrilin-3 (MATN3) have been reported as disease causing of autosomal dominant MED. The current study identified a novel c.572 C > A variant (p.A191D) in exon 2 of MATN3 in a Vietnamese family with MED. CASE PRESENTATION: A standard clinical tests and radiological examination were performed in an 8-year-old Vietnamese girl patient. The clinical examination showed that patient height was under average, with bent lower limbs, limited mobility and dislocation of the joints at both knees. Radiological documentation revealed abnormal cartilage development at the epiphysis of the femur and patella. The patient has a varus deformity of the lower limbs. The patient was diagnosed with autosomal dominant MED using molecular testing in the order of the coding sequences and flanking sequences of five genes: COMP (exons 8-19), MATN3 (exon 2), COL9A2 (exon 3), COL9A3 (exon 3), COL9A1 (exon 8) by Sanger sequencing. A novel heterozygous missense variant (c.572 C > A, p.A191D) in MATN3 was identified in this family, which were not inherited from parents. The p.A191D was predicted and classified as a pathogenic variant. When the two predicted structures of the wild type and mutant matrilin-3 were compared, the p.A191D substitution caused conformational changes near the substitution site, resulting in deformity of the ß-sheet of the single A domain of matrilin- 3. CONCLUSIONS: This is the first Vietnamese MED family attributed to p.A191D matrilin-3 variant, and our clinical, radiological and molecular data suggest that the novel de novo missense variant in MATN3 contributed to MED.

Genetic characteristics of 23 Y-chromosomal STRs in the Kinh population in Northern Vietnam.

We collected and analysed the genetic characteristics of 23 Y-STR data of 200 unrelated Kinh individuals living in the North of Vietnam. Haplotype frequencies and forensic parameters were calculated, showing high discrimination value. Population comparison analysis was performed to determine the genetic relationship with neighbouring ethnicities, in particular with Thai and Han populations.

Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

Boron nitride nanotubes reinforced gelatin hydrogel-based ink for bioprinting and tissue engineering applications.

The rapid evolution of 3D bioprinting technique, very few biomaterials have been studied and utilised as ink solutions to produce structures. In this work, a polymeric nanocomposite hydrogel-based ink solution was developed using boron nitride nanotubes (BNNTs) reinforced gelatin for 3D bioprinting of scaffolds. The ink solutions and printed scaffolds were characterised for their printability, mechanical, thermal, water uptake, and biological properties (cell viability and inflammation). The viscoelastic behaviour of the scaffolds indicated the increase in storage modulus with an increase in BNNTs composition. Additionally, the compressive strength of the scaffolds increased from 9.43 ± 1.3 kPa to 30.09 ± 1.5 kPa with the addition of BNNTs. Similarly, the thermal stability of the scaffolds enhanced with an increase in BNNTs composition. Furthermore, the scaffolds with a higher concentration of BNNTs displayed resilience in cell culture media at 37 °C for up to 14 days compared with pure gelatin scaffolds. The cell viability results showed a decreased viability rate with an increased concentration of BNNTs scaffolds. However, BNNTs incubated with cells did not display cytokine inflammation. Therefore, this work provides a potential hydrogel-based ink solution for 3D bioprinting of biomimetic tissue constructs with adequate structural stability for a wide range of tissue engineering and regenerative medicine applications.

Analysis of interleukin-1 receptor associated kinase-3 (IRAK3) function in modulating expression of inflammatory markers in cell culture models: A systematic review and meta-analysis.

BACKGROUND: IRAK3 is a critical modulator of inflammation in innate immunity. IRAK3 is associated with many inflammatory diseases, including sepsis, and is required in endotoxin tolerance to maintain homeostasis of inflammation. The impact of IRAK3 on inflammatory markers such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell culture models remains controversial. OBJECTIVE: To analyse temporal effects of IRAK3 on inflammatory markers after one- or two-challenge interventions in cell culture models. METHODS: A systematic search was performed to identify in vitro cell studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data were available. Comparisons of outcome measures were performed between different cell lines and human and mouse primary cells. RESULTS: The literature search identified 7766 studies for screening. After screening titles, abstracts and full-texts, a total of 89 studies were included in the systematic review. CONCLUSIONS: The review identifies significant effects of IRAK3 on decreasing NF-κB DNA binding activity in cell lines, TNF-α protein level at intermediate time intervals (4h-15h) in cell lines or at long term intervals (16h-48h) in mouse primary cells following one-challenge. The patterns of TNF-α protein expression in human cell lines and human primary cells in response to one-challenge are more similar than in mouse primary cells. Meta-analyses confirm a negative correlation between IRAK3 and inflammatory cytokine (IL-6 and TNF-α) expression after two-challenges.