Framework Nucleic Acids-Based VEGF Signaling Activating System for Angiogenesis: A Dual Stimulation Strategy.

Angiogenesis is crucial for tissue engineering, wound healing, and regenerative medicine. Nanomaterials constructed based on specific goals can be employed to activate endogenous growth factor-related signaling. In this study, based on the conventional single-stranded DNA self-assembly into tetrahedral framework nucleic acids (tFNAs), the Apt02 nucleic acid aptamer and dimethyloxallyl glycine (DMOG) small molecule are integrated into a complex via a template-based click chemistry reaction and toehold-mediated strand displacement reaction. Thus, being able to simulate the VEGF (vascular endothelial growth factor) function and stabilize HIF (hypoxia-inducible factor), a functional whole is constructed and applied to angiogenesis. Cellular studies demonstrate that the tFNAs-Apt02 complex (TAC) has a conspicuous affinity to human umbilical vein endothelial cells (HUVECs). Further incubation with DMOG yields the tFNAs-Apt02-DMOG complex (TACD), which promotes VEGF secretion, in vitro blood vessel formation, sprouting, and migration of HUVECs. Additionally, TACD enhances angiogenesis by upregulating the VEGF/VEGFR and HIF signaling pathways. Moreover, in a diabetic mouse skin defect repair process, TACD increases blood vessel formation and collagen deposition, therefore accelerating wound healing. The novel strategy simulating VEGF and stabilizing HIF promotes blood-vessel formation in vivo and in vitro and has the potential for broad applications in the vascularization field.

Digital workflow in the design of individualized emergence profiles of implant restorations based on the contralateral tooth.

PURPOSE: To describe a novel digital design technique for creating an individualized emergence profile for implant restoration based on the contralateral tooth. METHODS: Cone beam computed tomography (CBCT) data were used to accurately obtain a three-dimensional (3D) model of the contralateral tooth, which was mirror-flipped to design the emergence profile. The emergence profile was further divided into critical and subcritical areas; the critical area precisely replicated the mirror-flipped 3D model, whereas the subcritical area featured a slight concavity on the buccal side, flatness on the lingual side, and slight convexity on the mesial and distal surfaces. Subsequently, a milling machine was used to fabricate healing abutments with individualized emergence profiles. The design of the definitive restoration completely duplicated the emergence profile of the individualized healing abutment and was fabricated using a milling machine. CONCLUSIONS: This technical procedure presents an alternative novel method for designing the emergence profiles of implant restorations, with the potential to improve esthetics and functions as well as to maintain the long-term stability of peri-implant soft and hard tissues.

Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation.

How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2 and Isl1, including those dependent on Atoh7, and uncover the sequential and combinatorial interactions of these factors with the epigenetic landscape to control gene expression along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).

Tetrahedral Framework Nucleic Acids Connected with MicroRNA-126 Mimics for Applications in Vascular Inflammation, Remodeling, and Homeostasis.

The repair of damaged endothelium is crucial for vascular homeostasis maintenance, which comprises the recovery of early stage impaired endothelial cells and migration of surrounding unimpaired endothelial cells. MicroRNAs (miRNAs) play an indispensable role in balancing gene expression in organisms. For vascular tissues, miR-126 is one of the most important regulators and might have substantial application potential in maintaining vascular homeostasis. In this study, a type of sticky-end-modified tetrahedral framework nucleic acids (tFNAs-SE) was employed to successfully link the miR-126 5p mimic duplex, which was termed tFNAs-miR-126 5p mimics (tFNAs-MMs). Existing vascular endothelial growth factors (VEGF), tFNAs-MMs can improve cell viability, resist apoptosis, and recover the state and functions of LPS-induced impaired human umbilical vein endothelial cells (HUVECs). The angiogenesis ability of impaired HUVECs was recovered by tFNAs-MMs in vitro and in vivo. The mechanisms underlying these phenomena were demonstrated to be related to the downregulation of caspase3 and negative regulators of VEGF (SPRED1 and PIK3R2). Moreover, tFNAs-MMs promoted the migration and proliferation of HUVECs. Briefly, the strategy of sticky-end-modified tFNAs connecting miRNA mimics is available for miRNA gain of function, while tFNAs-MMs might be a promising agent for repairing early stage vascular damage and maintaining vascular homeostasis.

What factors are associated with improvements in productivity in clinical laboratories in the Asia Pacific Region?

INTRODUCTION: Clinical laboratories usually have a quality management system such as ISO 15189, which provides a framework for quality and competence to perform medical testing and internal systems such as audit and nonconformance to ensure consistent processes. However, organizations need to have access to internal procedures and external competitors' performance to improve their operations. These are often seen as commercial or areas where it is difficult to agree on an acceptable goal. METHOD: In 2019, 1158 laboratories from 17 countries/regions in the Asia Pacific Region answered the survey, including 399 Chinese sites. The survey collected information on quality, turnaround time and productivity. RESULTS: Median productivity for laboratories in the Asia Pacific Region not including Chinese sites was 25 samples/FTE/day for small laboratories (workload: <250 samples/day), 100 for medium-sized laboratories (workload: 251-1000 samples/day) and 220 for large laboratories (workload: >1001 samples/day). The parameters associated with increased productivity in some laboratories were automation, middleware, Lean Six Sigma quality improvement activities and International Accreditation. CONCLUSION: This survey provides evidence of an association of quality improvement activities on laboratory productivity. There are differences in the effect of these activities in Chinese and non-Chinese laboratories in the Asia Pacific Region. The survey confirmed that the implementation of automation is associated with increased median productivity in all sites. Implementation of Lean Six Sigma and International Accreditation is associated with increased productivity in large laboratories.

Single cell transcriptomics reveals lineage trajectory of retinal ganglion cells in wild-type and Atoh7-null retinas.

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.

[Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure].

OBJECTIVE: To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA). METHODS: Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco's Modified Eagle's medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope. RESULTS: After incubating protamine sulfate, negative potential was transformed into positive one ( (-1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h ( P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less. CONCLUSION: Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins.

BACKGROUND: During development, all retinal cell types arise from retinal progenitor cells (RPCs) in a step-wise fashion. Atoh7 and Pou4f2 mark, and function in, two phases of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 participates in RGC fate specification and RGC differentiation. Despite extensive research on their roles, the properties of the two phases represented by these two factors have not been well studied, likely due to the retinal cellular heterogeneity. RESULTS: In this report, we describe two novel knock-in mouse alleles, Atoh7zsGreenCreERT2 and Pou4f2FlagtdTomato , which labeled retinal cells in the two phases of RGC development by fluorescent proteins. Also, the Atoh7zsGreenCreERT2 allele allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells in the different phases by fluorescence assisted cell sorting (FACS). Single cell RNA-seq analysis of purified cells from Atoh7zsGreenCreERT2 retinas further validated that this allele labeled both transitional/competent RPCs and their progenies including RGCs. CONCLUSIONS: Thus, these two alleles are very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.

Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment.

Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization and editable functionality, tetrahedral DNA nanostructures show promising potential as an alternative vehicle for drug delivery and biomedical treatment. Here, we describe the design, fabrication, purification, characterization and potential biomedical applications of a self-assembling tetrahedral DNA nanostructure (TDN)-based multifunctional delivery system. First, relying on Watson-Crick base pairing, four single DNA strands form a simple and typical pyramid structure via one hybridization step. Then, the protocol details four different modification approaches, including replacing a short sequence of a single DNA strand by an antisense peptide nucleic acid, appending an aptamer to the vertex, direct incubation with small-molecular-weight drugs such as paclitaxel and wogonin and coating with protective agents such as cationic polymers. These modified TDN-based complexes promote the intracellular uptake and biostability of the delivered molecules, and show promise in the fields of targeted therapy, antibacterial and anticancer treatment and tissue regeneration. The entire duration of assembly and characterization depends on the cargo type and modification method, which takes from 2 h to 3 d.

PEGylated Protamine-Based Adsorbing Improves the Biological Properties and Stability of Tetrahedral Framework Nucleic Acids.

Recently, many researchers have reported that DNA nanostructures, such as tetrahedral framework nucleic acids (tFNAs), have great potential to be useful tools in clinical and laboratory applications due to their programmable shapes, functional sites, and biological responses. However, finite endocytosis and stability in cells and body fluids compromise the functions of DNA nanostructures as a result of various adverse factors. In this study, we successfully synthesized PEGylated protamine, and tFNAs were adsorbed to it in a proper ratio of nitrogen in protamine to phosphorus in tFNAs (N/P ratio) as the functional complex. Furthermore, we demonstrated that PEGylated protamine-adsorbed tFNAs show a more prominent positive effect on cell viability and proliferation than naked tFNAs do. An increase in endocytosis can be observed in three different tissue-derived cells with the PEG-protamine-tFNA (PPT) complex. The increased endocytic ability is mediated by multiple pathways; moreover, the stimulatory effect of the PPT complex on the endocytic ability is dramatically blocked by the inhibition of the caveola-dependent pathway. Consistently, when tFNAs are stabilized by PEGylated protamine, they often tend to escape from lysosomes and survive for a longer period in biological fluids rather than being rapidly eliminated from the kidneys. The in vitro and in vivo results of our study demonstrate that the PPT complex method is a feasible, potent, and low-cost strategy that improves tFNA biocompatibility, stability, and internalization. This study provides evidence supporting the possibility of implementing PPTs for use in drug delivery, bioimaging, and gene transfection in the future.

GMDTC Chelating Agent Attenuates Cisplatin-Induced Systemic Toxicity without Affecting Antitumor Efficacy.

Cisplatin is a platinum-based chemotherapeutic drug widely used in the treatment of various cancers such as testicular, ovarian, lung, bladder, and cervical cancers. However, its use and the dosage range applied have been limited by severe side effects (e.g., nephrotoxicity and ototoxicity) and by the development of resistance to cisplatin in patients during treatment. Metal chelators have shown promising potential in overcoming these problems often associated with platinum drugs. Previously, a new chelating agent, sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-4(methylthio)butanoate (GMDTC), was developed. In this study, we examined the effect of GMDTC in modifying cisplatin-induced toxicities following in vitro and in vivo exposures. GMDTC treatment dramatically reduced cisplatin-induced apoptosis and cytotoxicity in HK2 cells by decreasing the amount of intracellular platinum. In the 4T1 breast cancer mouse model, GMDTC reduced cisplatin-induced nephrotoxicity by reducing cisplatin deposition in the kidney. GMDTC attenuated cisplatin-induced elevations in blood urea nitrogen and plasma creatinine, ameliorated renal tubular dilation and vacuolation, and prevented necrosis of glomeruli and renal tubular cells. GMDTC also inhibited cisplatin-induced ototoxicity as shown by improved hearing loss which was assessed using the auditory brainstem response test. Furthermore, GMDTC attenuated cisplatin-induced hematotoxicity and hepatotoxicity. Importantly, co-treatment of cisplatin with GMDTC did not affect cisplatin antitumor efficacy. Tumor growth, size, and metastasis were all comparable between the cisplatin only and cisplatin-GMDTC co-treatment groups. In conclusion, the current study suggests that GMDTC reduces cisplatin-induced systemic toxicity by preventing the accumulation and assisting in the removal of intracellular cisplatin, without compromising cisplatin therapeutic activity. These results support the development of GMDTC as a chemotherapy protector and rescue agent to overcome the toxicity of and resistance to platinum-based antineoplastic drugs.

Phytoextraction of Cd from a contaminated soil by tobacco and safe use of its metal-enriched biomass.

Successful phytoextraction produces a large quantity of contaminated biomass, which will cause secondary pollution unless properly treated. This study investigated the disposal of contaminated tobacco biomass after phytoextraction. We detected significantly high Cadmium concentrations in tobacco, especially in their stems and leaves. From the latter, nearly all the Cd and nicotine were removed by extractions with 0.5% HCl + 70% ethanol, and the nicotine completely recovered via steam distillation, whereas the protein content remained unaffected in the leaves, thus making them safe for use as animal feed. The highest biochar yield was 47%, obtained after slow pyrolysis at 300 °C. In this case, the biochar contained the highest amount of nutrients and metals. From stem biochar, 87% of Cd and a large amount K along with several other elements were extracted by deionized water at pH 1. After acid-extraction, metals were formed precipitation and then separated from the K-enriched solution when the pH was adjusted to 11 by using drops of 40% KOH. Therefore, with improved technology to remove metals and recover nutrients and nicotine from biomass, tobacco is an ideal candidate as profit yielding crop for use in phytoextraction while also providing renewable resources.

Mapping dynamic histone modification patterns during arsenic-induced malignant transformation of human bladder cells.

Arsenic is a known potent risk factor for bladder cancer. Increasing evidence suggests that epigenetic alterations, e.g., DNA methylation and histones posttranslational modifications (PTMs), contribute to arsenic carcinogenesis. Our previous studies have demonstrated that exposure of human urothelial cells (UROtsa cells) to monomethylarsonous acid (MMAIII), one of arsenic active metabolites, changes the histone acetylation marks across the genome that are correlated with MMAIII-induced UROtsa cell malignant transformation. In the current study, we employed a high-resolution and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitatively measure various PTM patterns during the MMAIII-induced malignant transformation. Our data showed that MMAIII exposure caused a time-dependent increase in histone H3 acetylation on lysine K4, K9, K14, K18, K23, and K27, but a decrease in acetylation on lysine K5, K8, K12, and K16 of histone H4. Consistent with this observation, H3K18ac was increased while H4K8ac was decreased in the leukocytes collected from people exposed to high concentrations of arsenic compared to those exposed to low concentrations. MMAIII was also able to alter histone methylation patterns: MMAIII transformed cells experienced a loss of H3K4me1, and an increase in H3K9me1 and H3K27me1. Collectively, our data shows that arsenic exposure causes dynamic changes in histone acetylation and methylation patterns during arsenic-induced cancer development. Exploring the genomic location of the altered histone marks and the resulting aberrant expression of genes will be of importance in deciphering the mechanism of arsenic-induced carcinogenesis.

Taxonomic and Functional Analyses of the Supragingival Microbiome from Caries-Affected and Caries-Free Hosts.

Caries is one of the most prevalent and costly infectious diseases affecting humans of all ages. It is initiated by cariogenic supragingival dental plaques forming on saliva-coated tooth surfaces, yet the etiology remains elusive. To determine which microbial populations may predispose a patient to caries, we report here an in-depth and comprehensive view of the microbial community associated with supragingival dental plaque collected from the healthy teeth of caries patients and healthy adults. We found that microbial communities from caries patients had a higher evenness and inter-individual variations but simpler ecological networks compared to healthy controls despite the overall taxonomic structure being similar. Genera including Selenomonas, Treponema, Atopobium, and Bergeriella were distributed differently between the caries and healthy groups with disturbed co-occurrence patterns. In addition, caries and healthy subjects carried different Treponema, Atopobium, and Prevotella species. Moreover, distinct populations of 13 function genes involved in organic acid synthesis, glycan biosynthesis, complex carbohydrate degradation, amino acid synthesis and metabolism, purine and pyrimidine metabolism, isoprenoid biosynthesis, lipid metabolism, and co-factor biosynthesis were present in each of the healthy and caries groups. Our results suggested that the fundamental differences in dental plaque ecology partially explained the patients' susceptibility to caries, and could be used for caries risk prediction in the future.

Phytoextraction of cadmium-contaminated soil and potential of regenerated tobacco biomass for recovery of cadmium.

The aim of this study was to estimate the influence of regenerated tobacco on the extraction of Cd from two acidic soils as well as to address the problem of how to deal with contaminated leaves following phytoextraction. Results showed that a coppicing tobacco led to a decline in Cd concentration in regenerated leaves and stalks when plants were grown in pots, but increased concentrations in regenerated lower and middle leaves when plants were grown under field conditions. The highest recorded bioconcentration factors in Chaling and Guanxi soil were 37.53 and 19.21 in lower leaves in the field, respectively. Total Cd extraction efficiency in practice (9.43% for Chaling soil and 6.24% for Guanxi soil) under field conditions confirmed our theoretical calculations (10.0% for Chaling soil and 6.73% for Guanxi soil). Use of a 0.5% hydrochloric acid(HCl) solution was sufficient to reduce Cd (98.4%) in tobacco leaves to permissible levels as required by the Hygienic Standard for Feeds in China (≤0.5 mg kg-1). Regenerated tobacco has the potential to allow cultivation of Cd contaminated farmland to produce animal feed, assist in lowering total Cd content of soil, and allow income generation for farmers.

Epstein-Barr virus-induced up-regulation of TCAB1 is involved in the DNA damage response in nasopharyngeal carcinoma.

Telomerase Cajal body protein 1 (TCAB1), which is involved in Cajal body maintenance, telomere elongation and ribonucleoprotein biogenesis, has been linked to cancer predisposition, including nasopharyngeal carcinoma (NPC), due to its oncogenic properties. However, there are no specific reports to date on the functional relevance of TCAB1 and Epstein-Barr virus (EBV), which is considered to be a risk factor for NPC. In this study, we first examined NPC clinical tissues and found a notable overexpression of TCAB1 in EBV-positive specimens. Secondly, on a cellular level, we also observed that TCAB1 expression rose gradually along with the increased duration of EBV exposure in NPC cell lines. Additionally, EBV infection promoted cell proliferation and telomerase activity, but the activation was significantly inhibited after TCAB1 knockdown. Moreover, depletion of TCAB1 caused both cell cycle arrest and apoptosis, and suppressed the activation of ataxia telangiectasia and Rad3 related protein (ATR) induced by EBV, resulting in accumulation of DNA damage. Taken together, we here demonstrate that up-regulated expression of TCAB1, induced by EBV in the development of NPC, is involved in stimulating telomerase activity and regulating the DNA damage response within the context of EBV infection.

Comparison of hematological alterations and markers of B-cell activation in workers exposed to benzene, formaldehyde and trichloroethylene.

Benzene, formaldehyde (FA) and trichloroethylene (TCE) are ubiquitous chemicals in workplaces and the general environment. Benzene is an established myeloid leukemogen and probable lymphomagen. FA is classified as a myeloid leukemogen but has not been associated with non-Hodgkin lymphoma (NHL), whereas TCE has been associated with NHL but not myeloid leukemia. Epidemiologic associations between FA and myeloid leukemia, and between benzene, TCE and NHL are, however, still debated. Previously, we showed that these chemicals are associated with hematotoxicity in cross-sectional studies of factory workers in China, which included extensive personal monitoring and biological sample collection. Here, we compare and contrast patterns of hematotoxicity, monosomy 7 in myeloid progenitor cells (MPCs), and B-cell activation biomarkers across these studies to further evaluate possible mechanisms of action and consistency of effects with observed hematologic cancer risks. Workers exposed to benzene or FA, but not TCE, showed declines in cell types derived from MPCs, including granulocytes and platelets. Alterations in lymphoid cell types, including B cells and CD4+ T cells, and B-cell activation markers were apparent in workers exposed to benzene or TCE. Given that alterations in myeloid and lymphoid cell types are associated with hematological malignancies, our data provide biologic insight into the epidemiological evidence linking benzene and FA exposure with myeloid leukemia risk, and TCE and benzene exposure with NHL risk.

Preliminary analysis of salivary microbiome and their potential roles in oral lichen planus.

Several studies have explored the origin and development mechanism of oral lichen planus (OLP) with limited attention to the role of bacteria in the progression of this common oral disease. Here we utilized MiSeq sequencing of 16S rRNA gene amplicons to identify complex oral microbiota associated with OLP from saliva samples of two subtypes (reticular and erosive) of OLP patients and healthy controls. Our analyses indicated that the overall structure of the salivary microbiome was not significantly affected by disease status. However, we did observe evident variations in abundance for several taxonomic groups in OLP. Porphyromonas and Solobacterium showed significantly higher relative abundances, whereas Haemophilus, Corynebacterium, Cellulosimicrobium and Campylobacter showed lower abundances in OLP patients, as compared with healthy controls. In addition, we explored specific microbial co-occurrence patterns in OLP, and revealed significantly fewer linkers of Streptococcus comprising species in erosive OLP. Furthermore, the disease severity and immune dysregulation were also genus-associated, including with Porphyromonas that correlated to disease scores and salivary levels of interleukin (IL)-17 and IL-23. Overall, this study provides a general description of oral microbiome in OLP, and it will be useful for further investigation of their potential roles in the initiation and immune modulation of OLP.

Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study.

INTRODUCTION: High-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. METHODS: KBM7-mu cells were exposed to 200 µM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h of treatment. After a 2-3 week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. RESULTS: We identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. DISCUSSION: The KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity.

Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation.

In humans, arsenic is primarily metabolized by arsenic (+3 oxidation state) methyltransferase (As3MT) to yield both trivalent and pentavalent methylated metabolites. We recently reported that the putative N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) can biotransform monomethylarsonous acid (MMA(III)) to dimethylarsinic acid, conferring resistance of human cells to arsenic exposure. To further decipher the role of N6AMT1 and its interaction with As3MT in arsenic biomethylation, we examined the relative contribution of N6AMT1 and As3MT in metabolizing arsenic using several newly modified UROtsa human urothelial cells, ie, UROtsa cells with either a constant level of N6AMT1 or As3MT in combination with an inducible level of As3MT or N6AMT1, respectively. Our analysis confirmed the involvement of N6AMT1 in MMA(III) biomethylation but not for inorganic arsenic. In a comparable level of N6AMT1 and As3MT, the effect of N6AMT1 mediated MMA(III) biomethylation was obscured by the action of As3MT. Furthermore, we showed that the levels of N6AMT1 and As3MT proteins varied among and within human normal and cancerous tissues. Overall, the data showed that N6AMT1 has a role in MMA(III) biomethylation, but its effect is relatively minor and limited compared with As3MT. In addition, the varied levels and distributions of N6AMT1 and As3MT among human tissues may potentially contribute to the tissue specificity and susceptibility to arsenic toxicity and carcinogenicity.