Non-homologous End-joining Genotype, mRNA Expression, and DNA Repair Capacity in Childhood Acute Lymphocytic Leukemia.

BACKGROUND/AIM: The capacity for non-homologous end-joining (NHEJ) repair plays a pivotal role in maintaining genome stability and in carcinogenesis. However, there is little literature on the involvement of NHEJ-related genes in childhood acute lymphocytic leukemia (ALL). Our study aimed to elucidate the impact of polymorphisms of X-ray repair cross-complementing group 4 (XRCC4) (rs6869366, rs2075685, rs2075686, rs28360071, rs3734091, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and DNA ligase IV (LIG4) rs1805388, on the odds of childhood ALL. MATERIALS AND METHODS: Genotypes NHEJ-related genes of 266 cases and 266 controls were determined, and the genotype-phenotype correlation was investigated by examining mRNA transcript expression and the capacity for overall and precise NHEJ repair. RESULTS: The variant genotypes of XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 were significantly associated with increased odds of childhood ALL. Further analysis based on susceptibility genotypes showed no significant differences in mRNA transcript expression levels among childhood ALL cases with various putative high-risk genotypes, except XRCC6 rs5751129. Moreover, the overall NHEJ repair capacity was similar among carriers of different XRCC4, XRCC5, and XRCC6 genotypes. However, it is worth noting that individuals carrying the variant C allele at XRCC6 rs5751129 exhibited lower precise NHEJ repair capacity compared to those with the wild-type T allele. CONCLUSION: Our study identified significant associations between XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 genotypes and childhood ALL. Notably, lower transcriptional expression and reduced precise NHEJ repair capacity were observed in patients carrying the C allele of XRCC6 rs5751129. Further investigations are required to gain deeper insights into childhood ALL development.

Non-Homologous End-Joining Pathway Genotypes Significantly Associated with Nasopharyngeal Carcinoma Susceptibility.

Defects in the non-homologous end-joining (NHEJ) DNA repair pathway lead to genomic instability and carcinogenesis. However, the roles of individual NHEJ genes in nasopharyngeal carcinoma (NPC) etiology are not well-understood. The aim of this study was to assess the contribution of NHEJ genotypes, including XRCC4 (rs6869366, rs3734091, rs28360071, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and Ligase4 rs1805388, to NPC risk, with 208 NPC patients and 416 controls. Genotype-phenotype correlations were also investigated by measuring mRNA and protein expression in adjacent normal tissues and assessing the NHEJ repair capacity in blood lymphocytes from 43 NPC patients. The results showed significant differences in the distributions of variant genotypes at XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 between the cases and controls. The variant genotypes of these three polymorphisms were associated with significantly increased NPC risks. NPC patients with the risk genotypes at XRCC6 rs2267437 had significantly reduced expression levels of both mRNA and protein, as well as a lower NHEJ repair capacity, than those with the wild-type genotype. In conclusion, XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 in the NHEJ pathway were associated with NPC susceptibility. XRCC6 rs2267437 can modulate mRNA and protein expression and the NHEJ repair capacity.

The Contribution of Tissue Inhibitor of Metalloproteinase-2 Genotypes to Breast Cancer Risk in Taiwan.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinase-2 and is highly expressed in breast cancer (BC) cases at diagnosis. However, the genetic investigations for the association of TIMP-2 genotypes with BC risk are rather limited. In this study, contribution of TIMP-2 rs8179090, rs4789936, rs2009196 and rs7342880 genotypes to BC risk was examined among Taiwan's BC population. TIMP-2 genotypic profiles were revealed among 1232 BC cases and 1232 controls about their contribution to BC using a PCR-based RFLP methodology. The TIMP-2 rs8179090 homozygous variant CC genotype was significantly higher in BC cases than controls (odds ratio (OR) = 2.76, 95% confidence interval (95%CI) = 1.78-4.28, p = 0.0001). Allelic analysis showed that C allele carriers have increased risk for BC (OR = 1.39, 95%CI = 1.20-1.62, p = 0.0001). Genotypic together with allelic analysis showed that TIMP-2 rs4789936, rs2009196 or rs7342880 were not associated with BC risk. Stratification analysis showed that TIMP-2 rs8179090 genotypes were significantly associated with BC risk among younger (≤55) aged women, not among those of an elder (>55) age. Last, rs8179090 genotypes were also associated with triple negative BC. This study sheds light into the etiology of BC in Taiwanese women. Rs8179090 may be incorporated into polygenic risk scores and risk prediction models, which could aid in stratifying individuals for targeted breast cancer screening.

Association of EZH2 Genotypes With Oral Cancer Risk.

BACKGROUND/AIM: The over-expression of enhancer of zeste homolog 2 (EZH2) protein is found in oral cancer tissues. However, the genetic role of the enhancer of EZH2 in the etiology of oral cancer is unknown. The aim of this study was to evaluate the association of EZH2 genotypes with oral cancer risk among Taiwanese. MATERIALS AND METHODS: Three polymorphic variants of EZH2, rs887569 (C to T), rs41277434 (A to C), and rs3757441 (T to C), were analyzed regarding their association with oral cancer risk among 958 oral cancer patients and the same number of healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, the interaction of EZH2 rs887569, rs41277434, and rs3757441 genotypes with personal behaviors such as smoking, alcohol drinking, and betel quid chewing were also examined. RESULTS: The EZH2 genotypes rs887569, rs41277434, and rs3757441, were not significantly associated with oral cancer risk (p for trend=0.1735, 0.5658, and 0.4606, respectively). The analysis of allelic frequency distribution also supported the findings that the variant alleles at EZH2 rs887569, rs41277434, and rs3757441 may not serve as determinants of oral cancer risk (all p>0.05). There was no interaction between EZH2 rs887569, rs41277434, or rs3757441 genotypes with personal smoking, alcohol drinking or betel quid chewing behaviors. CONCLUSION: EZH2 genotypes cannot predict oral cancer risk in Taiwan.

The Contribution of Flap Endonuclease 1 Genotypes to Oral Cancer Risk.

BACKGROUND/AIM: Flap endonuclease 1 (FEN1) is a critical protein in DNA repair, genomic stability, and carcinogenesis. Functional polymorphisms in FEN1 promoter -69G>A (rs174538) and 3'UTR 4150G>T (rs4246215), have been associated with the susceptibility to several cancers, including lung, breast, esophageal, gastric, liver, colorectal, and gallbladder cancer, as well as glioma, endometriosis, and leukemia. However, the contribution of FEN1 variant genotypes to oral cancer has never been examined. Thus, we aimed to evaluate the contribution of FEN1 rs174538 and rs4246215 genotypes to oral cancer risk in Taiwan. MATERIALS AND METHODS: The contribution of FEN1 genotypes to oral cancer risk was examined in 958 oral cancer patients and 958 age- and sex-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The percentages of GG, AG, and AA genotypes at FEN1 rs174538 were 34.8%, 46.0%, and 19.2% among oral cancer patients and 37.8%, 45.2%, and 17.0% among healthy controls (p for trend=0.2788). The genotypic percentages of FEN1 rs4246215 were 35.9%, 45.9%, and 18.2% among oral cancer patients and 37.6%, 45.1%, and 17.3% among healthy controls (p for trend=0.7315). Overall, FEN1 rs174538 and rs4246215 were not differently distributed between the oral cancer patient and healthy control groups. The allele frequency analysis confirmed that FEN1 rs174538 and rs4246215 were non-differentially distributed among case and control groups (OR=1.11 and 1.05, 95%CI=0.98-1.27 and 0.93-1.20, p=0.1074 and 0.4491, respectively). CONCLUSION: FEN1 may contribute to oral cancer risk determination via protein expression and/or post-transcription modification, but may not be a practical genetic marker.

Protective Effects of Jujubosides on 6-OHDA-Induced Neurotoxicity in SH-SY5Y and SK-N-SH Cells.

6-hydroxydopamine (6-OHDA) is used to induce oxidative damage in neuronal cells, which can serve as an experimental model of Parkinson's disease (PD). Jujuboside A and B confer free radical scavenging effects but have never been examined for their neuroprotective effects, especially in PD; therefore, in this study, we aimed to investigate the feasibility of jujubosides as protectors of neurons against 6-OHDA and the underlying mechanisms. 6-OHDA-induced neurotoxicity in the human neuronal cell lines SH-SY5Y and SK-N-SH, was used to evaluate the protective effects of jujubosides. These findings indicated that jujuboside A and B were both capable of rescuing the 6-OHDA-induced loss of cell viability, activation of apoptosis, elevation of reactive oxygen species, and downregulation of the expression levels of superoxide dismutase, catalase, and glutathione peroxidase. In addition, jujuboside A and B can reverse a 6-OHDA-elevated Bax/Bcl-2 ratio, downregulate phosphorylated PI3K and AKT, and activate caspase-3, -7, and -9. These findings showed that jujubosides were capable of protecting both SH-SY5Y and SK-N-SH neuronal cells from 6-OHDA-induced toxicity via the rebalancing of the redox system, together with the resetting of the PI3K/AKT apoptotic signaling cascade. In conclusion, jujuboside may be a potential drug for PD prevention.

The Contribution of PDCD6 Polymorphisms to Oral Cancer Risk.

BACKGROUND/AIM: Programmed cell death 6 (PDCD6) is up-regulated and highly expressed in early apoptotic cells. In several types of cancer, such as cervical, breast and lung cancers, the association of PDCD6 genotypes have been investigated. However, the contribution of PDCD6 variant genotypes to oral cancer has never been examined. The current study aimed to evaluate the contribution of the PDCD6 rs4957014 and rs3756712 genotypes to the risk of oral cancer in Taiwan. PATIENTS AND METHODS: The contribution of PDCD6 genotypes to oral cancer risk was examined among 958 patients with lung cancer and 958 age- and sex-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP). RESULTS: The data showed that the hetero-variant GT and homo-variant GG genotypes of PDCD6 rs4957014 were associated with a decreased risk of oral cancer [odds ratio (OR)=0.81 and 0.39, 95% confidence interval (CI)=0.67-0.97 and 0.27-0.56, respectively]. The recessive and dominant models also showed that G carriers have protective effects (OR=0.43 and 0.72, 95% CI=0.30-0.61 and 0.61-0.87, respectively). The analysis of allelic frequency distributions showed that the G allele of PDCD6 rs4957014 was associated with reduced oral cancer risk (OR=0.71, 95% CI=0.62-0.82). There was no significant association between any PDCD6 rs3756712 genotype and oral cancer risk. In addition, the GG genotype at PDCD6 rs4957014 significantly decreased the risk of oral cancer among both males (adjusted OR=0.31, 95%CI=0.24-0.56) and females (adjusted OR=0.44, 95% CI=0.22-0.91). Furthermore, the GG genotype at PDCD6 rs4957014 significantly decreased the risk of oral cancer among smokers (adjusted OR=0.35, 95% CI=0.22-0.58), alcohol drinkers (adjusted OR=0.33, 95% CI=0.18-0.49), non-betel quid chewers (adjusted OR=0.33, 95% CI=0.17- 0.81), betel quid chewers (adjusted OR=0.34, 95% CI=0.21- 0.59), but not among never-smokers and non-alcohol drinkers. CONCLUSION: The G allele carriers of PDCD6 rs4957014 may have protective effects on oral cancer risk and serve as a practical marker for early detection of oral cancer in Taiwan.

Toxicity test of flavonoid compounds from the leaves of Dendrophthoe pentandra (L.) Miq. using in vitro culture cell models.

Background and Aim: The flavonoids from mistletoe are thought to have antimicrobial action. This encouraging finding supports the benefits of medicinal plants as a substitute for synthetic antimicrobials, thus promoting healthy lifestyles. In contrast, it is known that the use of topical drug formulations made from flavonoids of mistletoe (Dendrophthoe pentandra (L.) Miq. Loranthaceae) with Indonesian name, Benalu duku (BD) is required in skin cell irritation. This study aimed to assess the toxic effects of the flavonoid substances of BD, as an initial screening. Materials and Methods: A myeloma cell line was cultured in Roswell Park Memorial Institute medium, and the Baby Hamster Kidney clone 12 (BHK21) cell line was cultured in Dulbecco's Modified Eagle's Medium from stock (±9 × 107 cells/mL), and 1.2 mL of culture were distributed into each well of a microtiter plate. Subsequently, 0.2 mL of serially diluted flavonoid compounds (0.5-3 µg/mL) were added to 12 wells for each concentration, as trial groups (including control groups), followed by a 2-day incubation. Observations were performed based on the cytopathic effect (CPE) using an inverted microscope at a magnification of 100×. Results: Cytopathic effect was detected on the microtiter plate wells for the groups of myeloma and BHK21 cells at a flavonoid concentration of 0.5 µg/mL-3 µg/mL. Conclusion: Flavonoid compounds from BD were safely used for topical treatment of cancer cells at a concentration <2.491 µg/mL, whereas for non-cancerous cells, a concentration <2.582 µg/mL was sufficient (p < 0.05).

Protective Effects of Crocetin on Arsenic Trioxide-induced Oxidative Stress in Human Umbilical Vein Endothelial Cells.

BACKGROUND/AIM: The clinical use of arsenic trioxide (As2O3) is hampered due to its cardiotoxicity. Therefore, it is critical to prevent As2O3-induced loss of endothelial integrity. The purpose of this study was to examine As2O3-induced endothelial dysfunction and evaluate the efficacy of crocetin on reversing As2O3-induced cardiotoxicity. MATERIALS AND METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were used to examine As2O3-induced oxidative stress, apoptosis, production of reactive oxygen species (ROS) and DNA adducts. In addition, the impact of crocetin on As2O3-induced cardiotoxicity was evaluated. RESULTS: As2O3 decreased the viability of HUVEC cells and led to apoptosis. Additionally, As2O3 elevated NADPH oxidase activity, and the levels of intracellular ROS. Furthermore, the formamidopyrimidine DNA-glycosylase- and endonuclease III-digestible adducts were induced by As2O3 Crocetin treatment reversed the As2O3-induced reduction in cell viability, the induction of apoptosis, the activation of NADPH oxidase activity, ROS levels and DNA adducts. CONCLUSION: Crocetin protects from As2O3-induced cardio-toxicity.

Positive charge of Arg-201 on hemagglutinin is required for the binding of H6N1 avian influenza virus to its target through a two-step process.

In our previous study, we produced a monoclonal antibody EB2 that recognized an epitope in the HA1 domain on the hemagglutinin (HA) of H6N1 influenza virus (A/chicken/Taiwan/2838 V/00). The residue Arg-201 (R201) on this epitope was protected by the glycan at Asn-167 (N167) from tryptic digestion; therefore, the infectivity of the virus was retained. R201 was extremely conserved in various subtypes of the influenza virus. To explore the role of R201 and the protecting glycan, we developed a bi-cistronic baculovirus expression system for the production of H6HA1 and H6HA0 (nearly full-length HA), which were glycosylated in insect cells. The expressed H6HA1 was mostly found in the trimeric form, and the H6HA0 protein was only found in the monomeric form. The trimeric H6HA1 was resistant to tryptic digestion; however, it could not bind to fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans. By contrast, the monomeric H6HA0 could bind to fetuin but was sensitive to tryptic digestion. We found that the positive charge on R201 was critical for binding HA to the negatively charged surface of host cells because the mutant R201A of H6HA0 lost its binding capacity substantially. Moreover, this binding capacity was dependent on the pH value and inhibited by free electrically charged amino acids. We propose a two-step model for binding the influenza virus with a host cell. The first step involved the specific recognition of the receptor binding site on HA to the sialylated glycan on the host cell. After the virus is engulfed by the acidic endosome, R201 could bind to the cell surface with stronger interactions and trigger the fusion process.

A secretary bi-cistronic baculovirus expression system with improved production of the HA1 protein of H6 influenza virus in insect cells and Spodoptera litura larvae.

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.

Opto-Microfluidic Immunosensors: From Colorimetric to Plasmonic.

Optical detection has long been the most popular technique in immunosensing. Recent developments in the synthesis of luminescent probes and the fabrication of novel nanostructures enable more sensitive and efficient optical detection, which can be miniaturized and integrated with microfluidics to realize compact lab-on-a-chip immunosensors. These immunosensors are portable, economical and automated, but their sensitivity is not compromised. This review focuses on the incorporation and implementation of optical detection and microfluidics in immunosensors; it introduces the working principles of each optical detection technique and how it can be exploited in immunosensing. The recent progress in various opto-microfluidic immunosensor designs is described. Instead of being comprehensive to include all opto-microfluidic platforms, the report centers on the designs that are promising for point-of-care immunosensing diagnostics, in which ease of use, stability and cost-effective fabrication are emphasized.

Digital Microfluidics for Manipulation and Analysis of a Single Cell.

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

Glycosylation at hemagglutinin Asn-167 protects the H6N1 avian influenza virus from tryptic cleavage at Arg-201 and maintains the viral infectivity.

Cleavage of the hemagglutinin (HA) precursor (HA0) by trypsin, which produces the active HA1 and HA2 complex, is a critical step for activating the avian influenza virus (AIV). However, other tryptic cleavage sites on HA might also cause HA degradation and affect the virulence. Otherwise, HA is modified by glycosylation in the host cell. The conjugated glycans on HA may hinder the antigenic epitopes, and thus prevent the virus from being recognized and attacked by the antibodies. In this study, we observed that glycosylation at the Asn-167 (N167) site on the HA1 of the H6N1 AIV strain A/chicken/Taiwan/2838V/00 (2838V) protected Arg-201 (R201) from tryptic cleavage. The 2838V HA protein became sensitive to tryptic cleavage, whereas the glycans at N167 were removed by N-glycosidase F (PNGase-F). Furthermore, the infectivity of 2838V decreased when pretreated with PNGase-F and trypsin. Our observations suggest that the inaccessibility of the R201 residue of HA1 for tryptic cleavage, which is sterically hindered by glycosylation at N167, is a crucial factor for determining the infectivity of the AIV.

A monoclonal antibody recognizes a highly conserved neutralizing epitope on hemagglutinin of H6N1 avian influenza virus.

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

Preparation of monoclonal antibodies against poor immunogenic avian influenza virus proteins.

This study established a novel method of pre-screening peptides for monoclonal antibody (mAb) production. Whole virus particles were used as antigens to produce mAbs in the first stage. However, most mAbs obtained from this method were aimed toward hemagglutinin. For this reason, synthetic peptides were used as antigens for mAb production that aimed at the AIV proteins with low abundance or poor immunogenicity in the virus particle. The peptides that showed high immunogenicity were designed using bioinformatic tools for immunization. For high-throughput, a rabbit was used to screen the immunogenicities of the synthetic peptides. Those showed high immunity were used for mAb preparation in mice. Several new mAbs against PB2, PA, M1, M2, NS1 and NS2 proteins were successfully obtained in this study. Furthermore, the epitopes of M1 and NS1 mAbs were determined using competitive western blot assay and competitive ELISA. This study might simplify the mAb preparation and serves as the basis for developing mAb against poor immunogenic proteins.

Detection of H6 influenza antibody by blocking enzyme-linked immunosorbent assay.

Low pathogenic H6N1 avian influenza viruses (AIVs) have circulated in domestic chickens since 1972 in Taiwan. Detection of avian influenza (AI) antibody is a routine work for AI control in Taiwan. The currently available commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses between different subtypes. To this end, a panel of monoclonal antibodies (mAbs) to A/chicken/Taiwan/2838V/00 (H6N1) was developed and implemented a blocking ELISA (bELISA) to detect the H6 antibody. These mAbs were confirmed specific to H6 AIVs. One monoclonal antibody was purified and labeled with horseradish peroxidase to set up a bELISA. The cut-off value was calculated to be 30% inhibition percentage from 138 H6-negative sera. The sensitivity and specificity of the bELISA were 100% and 97%, respectively. The bELISA detected seroconversion in H6-infected farms earlier than hemagglutination inhibition test. The results show that this bELISA could be used to detect H6 infection in the field.