Association between polymorphism and haplotype of ATP2B1 gene and skeletal fluorosis in Han population.

To evaluate the association between ATP2B1 gene polymorphisms and skeletal fluorosis, a cross-sectional study was conducted. In China, 962 individuals were recruited, including 342 cases of skeletal fluorosis. Four TP2BA1 polymorphisms (rs2070759, rs12817819, rs17249754, and rs7136259) were analysed. The results suggested that rs17249754 and rs7136259 were associated with skeletal fluorosis. After controlling confounders, the protective effect of GG genotype in rs17249754 was apparent in individuals over 45 years old, female, with urine fluoride concentration below 1.6 mg/L, serum calcium above 2.25 mmol/L or serum phosphorus between 1.1 and 1.3. Heterozygote TC in rs7136259 increased the risk of skeletal fluorosis in subjects who are elderly, female, with urinary fluoride more than 1.6 mg/L, serum calcium more than 2.25 mmol/L and blood phosphorus between 1.1 and 1.3 mmol/L. Four loci were found to be tightly related by linkage disequilibrium analysis, and the frequency of distribution of haplotype GCGT was lower in the skeletal fluorosis group.

First Report of Neopestalotiopsis piceana Causing Gray Blight in Camellia sinensis L. in China.

Tea plants (Camellia sinensis L.) are an important cash crop and are cultivated worldwide for their commercial value (Palanisamy et al. 2014). Tea gray blight is an important tea plant disease as it can cause a decline in tea quality and reduce yields by 20-30% (Sanjay et al. 2008). In August 2018, a disease survey was conducted on 400 ha of organic tea plantations in the Pu'er area of Yunnan Province (22.48° N, 100.58° E). The survey found that widespread disease was causing damage to 40% of the tea plantations and that the most seriously affected tea variety was Yunkang No. 10, which had an average disease incidence of 30-35%. The affected leaves grew small yellow-green spots on their tips or margins in the early stage that expanded into round or irregular brown spots with distinct concentric whorls and black conidial disks arranged in whorls when the humidity was high (Fig. 1A-B), which is consistent with tea gray blight disease (Zheng et al. 2021). Twenty-four diseased leaf samples were collected from four different tea plantations and transported to the Pu-Erh Tea Research Laboratory. Leaves with disease spots were cut into 4 mm ×4 mm square pieces, surface-sterilized with 75% alcohol for 1 min, disinfected with 1% sodium hypochlorite for 3 min, and washed thrice with sterile water. The tissue pieces were placed on potato dextrose agar (PDA) plates containing 100 µg ml-1 of chloramphenicol (Wang et al. 2021). After 3 d of culturing in the dark at 28 C, twenty pure cultures with similar morphology were obtained, and two representative isolates were selected and transferred into new PDA media. After 7 d, circular fungal colonies with dense aerial mycelium produced black, wet spore masses that grew on the PDA media (Fig. 1C-D). The conidia were spindle-shaped with four septa, measuring 25.0 (21.0-26.0) × 6.0 (4.5-7.0) µm (n=15). The conidia had three median cells, two of which were dark brown in color with unclear separations, with a single basal hyaline appendage 3.8 (3.5-4.5) µm (n=30) in length and 2-3 apical hyaline appendages 31 (27-35) µm in length (n=30) (Fig. 1E), similar to the conidial characteristics of Neopestalotiopsis piceana (Maharachchikumbura et al. 2014). Two isolates were selected for DNA extraction. The internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (tef1-α) gene, and partial ß-tubulin (tub2) gene were amplified using the ITS1F-ITS4 primer set (White et al 1990), the EF-1α-F and EF-1α-R primer sets (Li et al. 2018), and the tub1 and tub2 primers, respectively (Chauhan et al. 2007). The ITS (OP535632 to OP535632), tef1-α (OP589285,OP589287), and tub2 (OP589286,OP589288) sequences were submitted to NCBI GenBank. Basic Local Alignment Search Tool analysis demonstrated that these sequences were 100% similar to those of N. piceana isolates available in GenBank. The sequences were compared using the Mafft software package, and sequences with the same ID were concatenated using scripts. A maximum likelihood phylogenetic tree was constructed using the MEGA (ver. 5.1) software package based on the concatenated sequences (ITS, tef1-α, and tub2). Phylogenetic analysis revealed that C-5 and B-3 showed 95% bootstrap support with N. piceana isolates in references (Fig. 2). According to the morphology and molecular characterization, C-5 and B-3 were identified as N. piceana. Pathogenicity tests on these two isolates were conducted using 36 healthy tea plants. The leaves were scratched slightly with sterile toothpick tips, after which pathogen cakes (6 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain a moist environment. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). Seven days later, the inoculated leaves exhibited similar symptoms observed under natural conditions, whereas the control leaves exhibited no symptoms. The same isolates as the introduced strains were isolated from the diseased tea leaves, completing Koch's postulates. To our knowledge, this is the first report of N. piceana causing gray blight on tea leaves in China. These results provide valuable information for the prevention and management of gray blight on tea leaves. References: Chauhan, J. B., et al. 2007. Indian J Biotechnol. 6: 404-406 Li, D. X., et al. 2018. J. Trop. Crops. 39:1827-1833. Maharachchikumbura, S. N., et al. 2014. Stud. Mycol. 79:121-186. Palanisamy, S., et al. 2014. Appl. Biochem. Biotechnol. 172:216-223. Sanjay, R., et al. 2008. Crop Protect. 27(3-5): 689-694. Wang, Q. M., et al. 2021. Front. Microbiol. 12:774438. White, T. J., et al. 1990. Academic, San Diego. 315-322 Zheng, S., et al. 2021. Plant Dis. 105:3723-3726.

MiR-96-5p Suppresses Progression of Arsenite-Induced Human Keratinocyte Proliferation and Malignant Transformation by Targeting Denticleless E3 Ubiquitin Protein Ligase Homolog.

Long-term exposure to arsenic has been linked to a variety of cancers, among which skin cancer is the most prevalent form. However, the mechanism underlying arsenic carcinogenesis is unclear, and there is still limited information on the role of miRNAs in arsenic-induced skin cancer. This study aims to explore the role of miR-96-5p in the arsenite-induced proliferation and malignant transformation of human HaCaT keratinocytes. The GEO database (accession numbers GSE97303, GSE97305, and GSE97306) was used to extract mRNA and miRNA expression profiles of HaCaT cells treated with or without 0.1 µmol/L sodium arsenite for 3 and 7 weeks. In this paper, according to the CCK8 assay result, HaCaT cells exposed to 0.1 µmol/L sodium arsenite for 48 h were finalized. CCK8, MTT, EdU incorporation, and colony formation assays were used to determine the viability and proliferation of HaCaT cells and transformed HaCaT (T-HaCaT) cells. The subcellular localization and relative expression levels of DTL, as well as miR-96-5p in HaCaT cells induced by arsenite, were determined via immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was performed to identify miR-96-5p bound directly to DTL. Transfection of miR-96-5p mimics or DTL siRNA was conducted to verify the arsenite-induced viability of HaCaT cells and T-HaCaT cells. T-HaCaT cells and nude mice were used to construct arsenite-induced malignant transformation and an in vivo xenograft model to demonstrate the over-expressed effect of miR-96-5p. The results showed that DTL was the target gene of miR-96-5p. Meanwhile, we also found that 0.1 µmol/L sodium arsenite upregulated DTL by decreasing the miR-96-5p level, leading to the proliferation and malignant transformation of HaCaT cells. MiR-96-5p agomir treatment slowed the growth of transplanted HaCaT cells transformed by arsenite in a manner associated with DTL downregulation in the nude mice xenograft model. Taken together, we confirmed that miR-96-5p, as a potent regulator of DTL, suppressed arsenite-induced HaCaT cell proliferation and malignant transformation, which might provide a novel therapeutic target for the treatment of arsenic-induced skin cancer.

miR-21-5p and canonical Wnt signaling pathway promote osteoblast function through a feed-forward loop induced by fluoride.

Long-term excessive exposure to fluoride from environmental sources can cause serious public health problems such as dental fluorosis and skeletal fluorosis. The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis and canonical Wnt signaling pathway participate in the progress. However, the specific mechanism that how canonical Wnt signaling pathway was mediated is not yet clear. In this study, we found that miR-21-5p induced the activation of canonical Wnt signaling pathway via targeting PTEN and DKK2 during fluoride induced osteoblasts activation and firstly demonstrated the forward loop between canonical Wnt signaling and miR-21-5p in the process. These findings suggested an important regulatory role of miR-21-5p on canonical Wnt signaling pathway during skeletal fluorosis and miR-21-5p might be a potential therapeutic target for skeletal fluorosis.

Mechanical nanosprings: induced coiling and uncoiling of ultrathin Au nanowires.

We report the controllable coiling of colloidal gold nanowires induced by the contraction of their polymer shells. The mechanical energy stored in this process can be released upon removal or swelling of the polymer shells.

sKlotho is associated with the severity of brick tea-type skeletal fluorosis in China.

The change of serum soluble Klotho (sKlotho) content is related to a variety of osteoarthropathy. However, its association with the severity of skeletal fluorosis (SF) is not clear. Here, the association of tea fluoride exposure with serum sKlotho levels and the severity of SF were investigated and further verified in a rat model of fluorosis. A cross sectional case control study was conducted in residents over 50 years old from brick-tea drinking areas in Qinghai and Xinjiang Provinces, China. Concentrations of fluoride in brick tea water and urine were determined by ion selective electrode method, and the levels of serum sKlotho were determined by ELISA method. Linear regression and ordered logistic regression models were constructed to examine the relationship among fluoride exposure, serum sKlotho levels and the severity of SF. The kidney and small intestine of Wistar rats were isolated for detection of Klotho by immunohistochemistry (IHC), and femoral artery blood was sampled to measure the serum levels of sKlotho. An increase of 1 mg/day in tea fluoride intake (TFI) was associated with a 12.070 pg/mL (95% CI: 0.452-23.689) increase in serum sKlotho levels and a 1.163-fold (95% CI: 1.007-1.342) increase in the severity of SF after adjusting for age, gender, and ethnicity. Serum sKlotho levels were also positively associated with the severity of SF (P < 0.05). The mediation analysis showed that serum sKlotho levels mediated 17.76% of the increase in the severity of SF caused by an increase of 1 mg/day of TFI. Moreover, a significant increase of serum sKlotho levels in fluoride-exposed groups was also seen in the rat model. The present study suggests that serum sKlotho may be a potential mediator of SF in brick tea-type fluorosis endemic areas.

Binding Process and Free Energy Characteristics of Cellulose Chain into the Catalytic Domain of Cellobiohydrolase TrCel7A.

It was observed in experiments that the catalytic domain (CD) of Trichoderma reesei Cel7A (TrCel7A) hydrolyzes crystalline cellulose in a processive manner, but the underlying binding mechanism is still unknown. Here, through replica-exchange molecular dynamics simulations, we find that the loading and sucking-in process of the cellulose chain into CD is entropy-driven and enthalpy-unfavorable, which firmly relate to the desolvation of the binding channel of CD. During the loading process, hydrophobic interactions play a dominant role because several aromatic residues have been identified to guide the cellulose chain processing. At the active site, a transition from enthalpy- to entropy-driven is detected for the driving force. Such a finding reveals the indispensability of the catalytic reaction of the glycosidic bond to provide the energy to drive the movements of the cellulose chain. Our study reveals the interaction pictures between the cellulose chain and TrCel7A at the atomic level, which helps better understand the catalytic mechanism of TrCel7A.

Paternal Low-Level Mosaicism-Caused SATB2-Associated Syndrome.

Mutations of SATB2 (OMIM#608148) gene at 2q33.1 have been associated with the autosomal dominant SATB2-associated syndrome (SAS), which is still short of comprehensive diagnosis technologies for small deletions and low-level mosaicism. In this Chinese Han family, single nucleotide polymorphism array identified a 4.9-kb deletion in the SATB2 gene in two consecutive siblings exhibiting obvious developmental delay and dental abnormalities but failed to find so in their parents. Prenatal diagnosis revealed that their third child carried the same deletion in SATB2 and the pregnancy was terminated. To determine the genetic causes behind the inheritance of SATB2 deletion, gap-PCR was performed on peripheral blood-derived genomic DNA of the family and semen-derived DNA from the father. Gap-PCR that revealed the deletions in the two affected siblings were inherited from the father, while the less intense mutant band indicated the mosaicism of this mutation in the father. The deletion was 3,013 bp in size, spanning from chr2: 200,191,313-200,194,324 (hg19), and covering the entire exon 9 and part of intron 8 and 9 sequences. Droplet digital PCR demonstrated mosaicism percentage of 13.2% and 16.7% in peripheral blood-derived genomic DNA and semen-derived DNA of the father, respectively. Hereby, we describe a family of special AT-rich sequence-binding protein 2-associated syndrome caused by paternal low-level mosaicism and provide effective diagnostic technologies for intragenic deletions.

Identification of miR-200c-3p as a major regulator of SaoS2 cells activation induced by fluoride.

The skeletal lesion of fluoride has become a major concern in many countries due to its damage to bone and joints and even leading to disability. Skeletal fluorosis is characterized by disturbance of bone metabolism, aberrant proliferation and activation of osteoblasts is critical for the pathogenesis. However, the mechanism underlying the osteotoxicity of fluoride has not been clearly illustrated and there is still limited information on the role of miRNAs in skeletal fluorosis. In this study, we found that NaF promoted SaoS2 proliferation and activation by activating BMP4/Smad pathway. NaF increased expression of miR-200c-3p and miR-200c-3p inhibitor reduced activation of SaoS2 induced by NaF via targeting Noggin to repress BMP4/Smad. These findings suggested an important regulatory role of miR-200c-3p on BMP4/Smad pathway during skeletal fluorosis. MiR-200c-3p might be a novel therapeutic target for skeletal fluorosis.

[Extraction and analysis of nuclear DNA from free margin of nail material].

To investigate the feasibility of DNA analysis from free margin of the nail, genomic DNA was extracted from the free margin of nail clipping of 10 volunteers using the proteinase K/SDS -based organic method, the Chelex-100 method, or a combined method. Target DNA was simultaneously amplified using a fluorescent multiplex AmpFlSTR Identifier kit. The PCR products were analyzed on the ABI PRISM 3130 Genetic Analyzer. The results showed that, compared with profiles achieved by genotyping of blood samples from each volunteer as reference, 100% concordance was achieved using the combined method. The STR genotype profiles obtained through the organic method were acceptable, despite preferential amplification at some loci. In contrast, no readable profiles could be determined when DNA was extracted by the Chelex-100 method, and there were a large number of alleles missing. Our data suggest that free margin of nail can be used for nuclear DNA analysis, but the type of DNA isolation method used is critical. The traditional organic extraction method works reasonably well for free margin nail DNA isolation, and combination of organic extraction and the Chelex-100 method works best.

ClC-7/Ostm1 contribute to the ability of tea polyphenols to maintain bone homeostasis in C57BL/6 mice, protecting against fluorosis.

Epidemiological investigations indicate that certain ingredients in tea bricks can antagonize the adverse effects of fluoride. Tea polyphenols (TPs), the most bioactive ingredient in tea bricks, have been demonstrated to be potent bone-supporting agents. ClC­7 is known to be crucial for osteoclast (OC) bone resorption. Thus, in this study, we investigated the potential protective effects of TPs against fluorosis using a mouse model and explored the underlying mechanisms with particular focus on ClC­7. A total of 40, healthy, 3­week­old male C57BL/6 mice were randomly divided into 4 groups (n=10/group) by weight as follows: distilled water (control group), 100 mg/l fluoridated water (F group), water containing 10 g/l TPs (TP group) and water containing 100 mg/l fluoride and 10 g/l TPs (F + TP group). After 15 weeks, and after the mice were sacrificed, the long bones were removed and bone marrow-derived macrophages were cultured ex vivo in order to perform several experiments. OCs were identified and counted by tartrate­resistant acid phosphatase (TRAP) staining. The consumption of fluoride resulted in severe fluorosis and in an impaired OC function [impaired bone resorption, and a low mRNA expression of nuclear factor of activated T-cells 1 (NFATc1), ATPase H+ transporting V0 subunit D2 (ATP6v0d2) and osteopetrosis­associated transmembrane protein 1 (Ostm1)]. In the F + TP group, fluorosis was attenuated and OC function was restored, but not the high bone fluoride content. Compared with the F group, mature OCs in the F + TP group expressed higher mRNA levels of ClC­7 and Ostm1; the transportation and retaining of Cl­ was improved, as shown by the fluorescence intensity experiment. On the whole, our findings indicate that TPs mitigate fluorosis in C57BL/6 mice by regulating OC bone resorption. Fluoride inhibits OC resorption by inhibiting ClC­7 and Ostm1, whereas TPs attenuate this inhibitory effect of fluoride.

Matrix Metallopeptidase-2 Gene rs2287074 Polymorphism is Associated with Brick Tea Skeletal Fluorosis in Tibetans and Kazaks, China.

Brick tea skeletal fluorosis is still a public health issue in the north-western area of China. However its pathogenesis remains unknown. Our previous study reveals that the severity of skeletal fluorosis in Tibetans is more serious than that in Kazaks, although they have similar fluoride exposure, suggesting the onset of brick tea type skeletal fluorosis might be genetically influenced. Here we show that MMP-2 rs2287074 SNP (G/A), but not rs243865, was associated with Brick tea type fluorosis in Tibetans and Kazaks, China. The trend test reveals a decline in probability for skeletal fluorosis with increasing number of A alleles in Tibetans. After controlling potential confounders, AA genotype had about 80 percent lower probability of developing skeletal fluorosis than GG genotype in Tibetans (odds ratio = 0.174, 95% CI: 0.053, 0.575), and approximately 53 percent lower probability in Kazaks (odds ratio = 0.462, 95% CI: 0.214, 0.996). A meta-analysis shows that the AA genotype had approximately 63 percent lower odds (odds ratio = 0.373, 95% CI: 0.202, 0.689) compared with GG genotype within the two ethnicities. A significant correlation was also found between the genotype of MMP2 rs2287074 and skeletal fluorosis severity. Therefore, the A allele of MMP2 rs2287074 could be a protective factor for brick tea skeletal fluorosis.

Modifying Role of GSTP1 Polymorphism on the Association between Tea Fluoride Exposure and the Brick-Tea Type Fluorosis.

BACKGROUND: Brick tea type fluorosis is a public health concern in the north-west area of China. The association between SNPs of genes influencing bone mass and fluorosis has attracted attention, but the association of SNPs with the risk of brick-tea type of fluorosis has not been reported. OBJECTIVE: To investigate the modifying roles of GSTP1 rs1695 polymorphisms on this association. METHODS: A cross-sectional study was conducted. Brick-tea water was tested by the standard of GB1996-2005 (China). Urinary fluoride was tested by the standard of WS/T 89-2006 (China). Skeletal fluorosis was diagnosed by X-ray, the part we scheduled was forearm, shank, and pelvic, then diagnosed the skeletal fluorosis by the standard of WS/192-2008 (China). Gene polymorphism was tested by Sequenom MassARRAY system. RESULT: The prevalence rate in different ethnical participants was different: Tibetan individuals had the highest prevalence rate of skeletal fluorosis. There were significant differences in genotype frequencies of GSTP1 Rs1695 among different ethnical participants (p<0.001): Tibetan, Mongolian and Han subjects with homozygous wild type (GSTP1-AA) genotype were numerically higher than Kazakh and Russian subjects (p<0.001). Compared to Tibetan participants who carried homozygous A allele of GSTP1 Rs1695, Tibetan participants who carried G allele had a significantly decreased risk of skeletal fluorosis (OR = 0.558 [95% CI, 0.326-0.955]). For Kazakh participants, a decreased risk of skeletal fluorosis among carriers of the G allele was limited to non high-loaded fluoride status (OR = 0. 166 [95% CI, 0.035-0.780] vs. OR = 1.478 [95% CI, 0.866-2.552] in participants with high-loaded fluoride status). Neither SNP-IF nor SNP-age for GSTP1 Rs1695 was observed. CONCLUSION: The prevalence rate of the brick tea type fluorosis might have ethnic difference. For Tibetan individuals, who had the highest prevalence rate, G allele of GSTP1 Rs1695 might be a protective factor for brick tea type skeletal fluorosis.

Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.