[Gene Mutation Types of Thalassemia in Chongzuo Childbearing-age Population of Guangxi Zhuang Autonomous Region of China].

OBJECTIVE: To investigate the gene mutation and genotype distribution of thalassemia in the population of childbearing age in Chongzuo area of Guangxi. METHODS: Six α-thalassemia and 17 ß-thalassemia gene mutations common in Chinese were detected by gap-polymerase chain reaction (gap-PCR) combined with agarose gel eletrophoresis and reserve dot bolt hybridization in 29 266 cases of child-bearing age suspected of thalassemia. RESULTS: A total of 19 128 (65.36%) cases were identified with thalassemia. The detection rate of α-thalassemia, ß-thalassemia and α-combining ß-thalassemia was 45.25% (13 242/29 266), 15.47% (4 526/29 266) and 4.65% (1 360/29 266), respectively. A total carrying rate of 8 kinds of α-thalassemia gene mutations was 26.74% (15 649/58 532), including 12.51% for --SEA, followed by 5.70% for -α3.7, and 0.24% for --Thai. Among 32 α-thalassemia genotypes, the most common five were --SEA/αα, -α3.7/αα, αCSα/αα, -α4.2/αα and αWSα/αα, accounting for 47.27%, 18.31%, 8.56%, 8.52% and 7.91%, respectively, as well as 0.97% for --Thai/αα. A total carrying rate of 13 kinds of ß-thalassemia gene mutations was 10.07% (5 897/58 532), including 3.63% for CD41-42, followed by 2.55% for CD17, and 0.003% for -50 (G>A). Among 17 ß-thalassemia genotypes, the most common six were CD41-42/N, CD17/N, CD71-72/N, CD26/N, 28/N and IVSI-1/N, accounting for 36.15%, 25.81%, 9.43%, 8.18%, 8.09% and 7.75%. The homozygous genotype CD26/CD26 [hemoglobin (Hb): 121 g/L] and -28/-28 (Hb: 56 g/L) were respectively detected in one case, and double heterozygous genotype were detected in 5 cases, including 3 cases of CD41-42/CD26 (Hb: 41 g/L, 51 g/L, 63 g/L, respectively), 1 case of -28/IVSI-1 (Hb: 53 g/L), and 1 case of CD71-72/CD26 (Hb: 89 g/L), in which patients with moderate or severe anemia had a history of blood transfusion. Among 104 α-combining ß-thalassemia genotypes, the most common were --SEA/αα, -α3.7/αα combining CD41-42/N and --SEA/αα combining CD17/N, accounting for 12.13%, 9.63% and 9.26%, respectively. In addition, 1 case of --SEA/-α3.7 combining -28/IVSI-1 (Hb: 83 g/L) and 1 case of -α3.7/αα combining CD41-42/ CD41-42 (Hb: 110 g/L) were detected without history of blood transfusion, while 1 case of αWSα/αα combining CD41-42/CD17 (Hb: 79 g/L) and 1 case of --SEA/αα combining CD17/-28 (Hb: 46 g/L) were detected with history. CONCLUSIONS: The detection rate of thalassemia genes is high and the mutations are diverse in the population of childbearing age in Chongzuo area of Guangxi. The common deletion genotype is --SEA/αα in α-thalassemia and CD41-42/N in ß-thalassemia, and deletion genotype --Thai is not rare. There is a certain incidence of intermediate and severe ß-thalassemia, and most patients require transfusion therapy. The results are beneficial for genetic consultation and intervention of thalassemia.

[Analysis of HKαα Genotype and Hematological Characteristics in South Guangxi].

OBJECTIVE: To investigate the detection rate and hematologic phenotype of HKαα thalassemia in south Guangxi, in order to provide reference for the prevention and control of thalassemia and prenatal and postnatal care consultation in this region. METHODS: Gene testing was performed on pre-marital medical examinations, pre-pregnancy eugenic health examinations, prenatal examinations and hospitalized thalassemia-positive persons in south of Guangxi, and the results were analyzed. RESULTS: A total of 183 190 thalassemia patients were included in this study, the age was mainly concentrated in 26-35 years old (101 709 cases, accounting for 55.521%), and 40 HKαα mutations were detected, detection rate was 0.022%, including 5 cases in Nanning, 22 cases in Qinzhou, 2 cases in Fangchenggang, 11 cases in Beihai. A total of 29 ethnic groups were included in the survey, but HKαα gene was observed only in Han nationality (0.0380%) and Zhuang nationality (0.0068%). A total of 8 genotypes carrying HKαα mutations were detected in this study ( HKαα/--SEA, ßN/ ßN, HKαα/αα, ß-28/ ßN, HKαα/αα, ß-50/ ßN, HKαα/αα, ßCD17/ ßN, HKαα/αα, ßCD27/28/ß N, HKαα/αα, ßCD41-42/ ßN, HKαα/αα, ßCD71-72/ ßN, and HKαα/αα, ßN/ ßN). Except for most cases with HKαα/αα, ßN/ ßN genotypes with no significant changes in the hematological indexes, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of other genotypes were decreased, showing microcytic hypochromic performance, mild anemia or no anemia. CONCLUSION: HKαα carrier is often misdiagnosed as -α3.7 carrier, which easily leads to missed diagnosis or misdiagnosis. Therefore, it is necessary to continuously improve the diagnostic level of laboratory testing personnels and genetic counselors to avoid unnecessary interventional puncture operations and birth of children with moderate and severe thalassemia.

Epidemiology of birth defects based on a birth defects surveillance system in southwestern China and the associated risk factors.

Background: Birth defects (BDs) are associated with many potential risk factors, and its causes are complex. Objectives: This study aimed to explore the epidemiological characteristics of BDs in Guangxi of China and the associated risk factors of BDs. Methods: BDs data of perinatal infants (PIs) were obtained from the Guangxi birth defects monitoring network between 2016 and 2020. Univariate Poisson regression was used to calculate the prevalence-rate ratios (PRR) to explore the changing trends of BDs prevalence by year and the correlation between the regarding of characteristics of BDs (including infant gender, maternal age, and quarter) and BDs. Clinical characteristics of PIs with BDs and general characteristics of their mothers were documented, and Spearman correlation analysis was used to explore the potential associated risk factors of BDs. Results: Between 2016 and 2020, 44,146 PIs with BDs were monitored, with an overall BDs prevalence of 121.71 (95% CI: 120.58-122.84) per 10,000 PIs, showing a significant increase trend (PRR = 1.116, 95% CI: 1.108-1.123), especially the prevalence of congenital heart defects (CHDs) that most significantly increased (PRR = 1.300, 95% CI: 1.283-1.318). The 10 most common BDs were CHDs, polydactyly, congenital talipes equinovarus, other malformation of external ear, syndactyly, hypospadias, cleft lip with cleft palate, cleft lip, hemoglobin Bart's hydrops fetalis syndrome (BHFS), and congenital atresia of the rectum and anus. BDs were positively correlated with pregnant women's age (R = 0.732, P < 0.01) and education level (R = 0.586, P < 0.05) and having pre-gestational diabetes mellitus (PGDM)/gestational diabetes mellitus (GDM) (R = 0.711, P < 0.01), while when the pregnant women had a family history of a dead fetus (R = -0.536, P < 0.05) and a birth of a fetus with BDs (R = -0.528, P < 0.05) were negatively correlated with BDs. Conclusion: A significant increase in the prevalence of BDs was detected between 2016 and 2020 in Guangxi, especially the prevalence of CHDs that most significantly increased. Older maternal age, higher maternal education level, and having PGDM before pregnancy or GDM in early pregnancy were the risk factors for BDs.

Genome Mining and Screening for Secondary Metabolite Production in the Endophytic Fungus Dactylonectria alcacerensis CT-6.

Endophytic fungi are a treasure trove of natural products with great chemical diversity that is largely unexploited. As an alternative to the traditional bioactivity-guided screening approach, the genome-mining-based approach provides a new methodology for obtaining novel natural products from endophytes. In our study, the whole genome of an endophyte, Dactylonectria alcacerensis CT-6, was obtained for the first time. Genomic analysis indicated that D. alcacerensis CT-6 has one 61.8 Mb genome with a G+C content of 49.86%. Gene annotation was extensively carried out using various BLAST databases. Genome collinearity analysis revealed that D. alcacerensis CT-6 has high homology with three other strains of the Dactylonectria genus. AntiSMASH analysis displayed 45 secondary metabolite biosynthetic gene clusters (BGCs) in D. alcacerensis CT-6, and most of them were unknown and yet to be unveiled. Furthermore, only six known substances had been isolated from the fermented products of D. alcacerensis CT-6, suggesting that a great number of cryptic BGCs in D. alcacerensis CT-6 are silent and/or expressed at low levels under conventional conditions. Therefore, our study provides an important basis for further chemical study of D. alcacerensis CT-6 using the gene-mining strategy to awaken these cryptic BGCs for the production of bioactive secondary metabolites.

Diversity of endophytic fungi in Coptis chinensis Franch. and their activity against methicillin-resistant Staphylococcus aureus.

This study aimed at investigating the diversity of endophytic fungi from Coptis chinensis and their activity against methicillin-resistant Staphylococcus aureus (MRSA). Seventy-nine fungal isolates obtained from C. chinensis were identified to belong to 27 species based on morphological features and internal transcript spacer (ITS) gene sequencing analysis. Comparing relative frequency values, the most frequent genera were Colletotrichum and Fusarium, while most frequent species were C. gloeosporioides and F. avenaceum. Analysis of diversity indices indicated that C. chinensis harbored abundant fungal resources. Methanol extracts of fungal endophyte cultures were evaluated for antibacterial activity against S. aureus ATCC 25923 and two other MRSA clinical strains. Nine of 27 endophytic fungi exhibited inhibitory activities against S. aureus ATCC 25923. Among them, Paraboeremia litseae HL-17, Fusarium sp. HL-23, and Fusarium sp. HL-27 exhibited obvious inhibition against the three S. aureus strains. Our findings suggest that the endophytic fungi in C. chinensis have a high diversity and an obvious tissue specificity, and could be of potential interest in screening anti-MRSA agents. To the best of our knowledge, this is the first report on the diversity and anti-MRSA activity of fungal endophytes from C. chinensis.

Xanthones and benzophenones isolated from the endophytic fungus Penicillium sp. ct-28 of Corydlis tomentella and their cytotoxic activity.

One new xanthone, griseophenexanthone A (1), one new benzophenone, digriseophene A (2), and 14 previously reported compounds were isolated from the culture of Penicillium sp. ct-28, an endophytic fungus of Corydlis tomentella. The structures of the isolated compounds were identified by an extensive analysis of HRESIMS, 1D and 2D NMR. MTT assay showed that six xanthones (1 and 3-7) significantly inhibited cell proliferation in four cancer cell lines, with IC50 values ranging from 18.12 ± 2.42 to 85.55 ± 7.66 µM. Our results showed that slight structural changes led to obvious activity differences among these compounds. We also investigated the effects of the six xanthones on cell cycle and apoptosis in human hepatoma HepG2 cells. Compound 7 caused cell cycle arrest at G1 phase, compounds 5 and 6 caused cell cycle arrest at S phase, whereas compounds 1, 3 and 4 had no effects on cell cycle distribution. All six xanthones induced apoptosis in dose-dependent manners in HepG2 cells accompanied by degradation of PARP and activation of caspase 3. The structure-activity relationship analysis revealed that the effects of these xanthones on cell cycle and apoptosis in HepG2 cells were closely related to the substituent groups on their skeleton. Our studies provide novel insights for the structural optimization of xanthones in the development of new anticancer drugs.

Molecular Characterization of α- and ß-Thalassaemia Among Children From 1 to 10 Years of Age in Guangxi, A Multi-Ethnic Region in Southern China.

Background: Thalassemia is one of the most common genetic diseases in southern China. Howerver, population in different regions or different population has their own spectrums of thalassemia. To investigate the prevalence and spectrum features of thalassemia among children in Guangxi. Hematology and genetic analysis were performed on 71,459 children aged 1-10 years in various regions of Guangxi. Results: A total of 11,821 children were diagnoses with thalassemia including 7,615 (10.66%) subjects of α-thalassemia, 3,507 (4.90%) subjects of ß-thalassemia, and 699 (0.98%) cases with both α- and ß-thalassemia. Nine α-thalassemia mutations and 30 genotypes were identified among the α-thalassemia children. The - -SEA and - -SEA/αα were the most frequent mutation and genotype, respectively. One α-thalassemia fusion gene and a rare 2.4 kb deletion both causing α+-thalassemia were identified, respectively. Thirteen ß-thalassemia mutations and 31 genotypes were characterized among the ß-thalassemia children, with the most common mutation CD41-42 (-CTTT) accounting for 46.05% of the ß-mutations. Two rare mutations IVS-II-5 (G>C), and IVS-I-2 (T>C) were firstly identified. Furthermore, 92 genotypes were identified among 699 children with both α- and ß-thalassemia. Conclusions: Our findings highlight the great heterogeneity and the extensive spectrum of thalassemia among children in Guangxi, which provide an available reference for prevention of thalassemia in this area.

DNA Damage Response Genes in Osteosarcoma.

BACKGROUND: Improving the osteosarcoma (OS) patients' survival has long been a challenge, even though the disease's treatment is on the verge of progress. DNA damage response (DDR) has traditionally been associated with carcinogenesis, tumor growth, and genomic instability. No study has used DDR genes as a signature to identify the prognosis of OS. The goal of this work was to find an effective possible DDR gene biomarker for predicting OS prognosis, which may be useful in clinical diagnosis and therapy. METHODS: To assess gene methylation, univariate and multivariate cox regression analyses were performed on data from OS patients. The data were retrieved from public databases, including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and the Gene Expression Omnibus (GEO). RESULTS: The DDR gene signature was chosen, which included seven genes (NHEJ1, RMI2, SWI5, ERCC2, CLK2, POLG, and MLH1). In the TARGET dataset, patients were categorized into two groups: high-risk and low-risk. Patients with a high-risk score revealed a shorter OS rate (hazard ratio (HR): 3.15, 95% confidence interval (CI): 1.38-4.34, P < 0.001) in comparison with the patients with a low-risk score in the TARGET as a training group. The validation of the prognostic signature accuracy was carried out in relapse and validation cohorts (TARGET, n = 75; GSE21257, n = 53). The signature was found to be an independent predictive factor for OS in multivariate cox regression analysis, and a nomogram model was developed to predict an individual's risk of OS. DDR gene signature involved in Fanconi anemia pathway, nonhomologous end-joining pathway, mismatch repair, and nucleotide excision repair pathway. CONCLUSIONS: Our study suggests that the identified novel DDR genes could be a powerful prognostic tool for prognosis evaluation and a valuable tool in predicting the risk factors in OS patients.

Depletion of Ars2 inhibits cell proliferation and leukemogenesis in acute myeloid leukemia by modulating the miR-6734-3p/p27 axis.

Ars2 is a component of the nuclear cap-binding complex (CBC) that contributes to microRNA biogenesis and is required for cellular proliferation. Little is known regarding the functional role of Ars2 in cell proliferation and leukemogenesis of acute myeloid leukemia. Here, we show that the elevated expression of Ars2 was observed in acute myeloid leukemia (AML) cell lines and bone marrow samples from AML patients and was correlated with poorer overall survival. Overexpression of Ars2 promoted cell proliferation and colony formation in AML cells, whereas depletion of Ars2 inhibited cell proliferation and colony formation. Mechanistic studies reveal that depletion of Ars2 suppressed the interaction of Ars2 with CBC and led to alterations in miRNA processing. Furthermore, Ars2 depletion reduced the levels of miR-6734-3p, resulting in upregulation of p27 and culminating in cell cycle arrest at the G1 phase. In vivo studies indicate that depletion of Ars2 significantly reduced leukemic cell burden and prolonged the survival time of the leukemia-bearing mice. These findings indicate that Ars2 may not only play a crucial role in the regulation of cell proliferation and leukemogenesis, but could also be identified as a critical therapeutic target for treatment of AML.

Investigation on the association of occupational stress with risk of polycystic ovary syndrome and mediating effects of HOMA-IR.

We aimed to evaluate the association between occupational stress and PCOS risk in a Chinese population and whether insulin resistance mediates the association. A total of 366 patients with PCOS and 325 controls were included in this study. Three logistic regression analyzes were applied in statistical analysis. In the first logistic regression analysis, the occupational stress significantly influenced development of PCOS (cumulative R2 = 0.737). In model 2, the environmental factors cumulatively accounted for 4.2% of the variance in PCOS risk. In model 3, which contained HOMA-IR, the R2 of HOMA-IR to PCOS risk was as high as 0.41, but the R2 of occupational stress reduced to 0.22. HOMA-IR became the main risk factor for PCOS. SEM model showed that ORQ, PSQ and PRQ had a direct and indirect effect on PCOS, and the indirect effect was through HOMA-IR. Occupational stress has a direct and indirect relationship with PCOS, which is mediated by HOMA-IR.

NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer.

A large nuclear protein of 2089 amino acids, NFBD1/MDC1 has recently been implicated in tumorigenesis and tumor growth. In this study, we investigated its expression in cervical cancers and explored its function using gene knockdown approaches. We report here that NFBD1 expression is substantial increased in 24 of 39 cases (61.5%) of cervical cancer tissues at the mRNA level and in 35 of 60 cases (58.3%) at the protein level compared with the case matched normal tissues. Tumors with higher grade of malignancy tend to have higher levels of NFBD1 expression. By infecting cells with retroviruses expressing NFBD1 shRNA, we successfully knocked down NFBD1 expression in cervical cancer cell lines HeLa, SiHa, and CaSki. NFBD1 knockdown cells display significant growth inhibition, cell cycle arrest, higher apoptotic rate, and enhanced sensitivity to adriamycin. Furthermore, NFBD1 knockdown also inhibits the growth of HeLa cells in nude mice. Western blot analyses further revealed that NFBD1 knockdown induced Bax, Puma, and Noxa while down-regulating Bcl-2; it also up-regulated cytochrome C and activated caspases 3 and 9. Therefore, the function of NFBD1 may be involved in the CDC25C-CyclinB1/CDC2 pathway at the G2/M checkpoint, and the cytochrome C/caspase 3 apoptotic pathway. Since expression of NFBD1 seems to be related to the oncogenic potential of cervical cancer, and suppression of its expression can inhibit cancer cell growth both in vitro and in vivo, NFBD1 may be a potential therapeutic target in human cervical cancer.

Characterization and identification of iridoid glucosides, flavonoids and anthraquinones in Hedyotis diffusa by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS(n)). The optimized separation condition was obtained using an Agilent ZorBax SB-C18 column (4.6×150 mm, 5 µm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC-ESI-MS(n) method.

2ME and 2OHE2 exhibit growth inhibitory effects and cell cycle arrest at G2/M in RL95-2 human endometrial cancer cells through activation of p53 and Chk1.

Evidence is accumulating that estradiol (E2) may play a dual role in carcinogenic and anticarcinogenic effects by different metabolic pathways. It has been shown that some metabolites of E2 exert proliferative and others anti-proliferative properties on human cancer cells. In the present study, the effects of E2 and its four primary metabolites including 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME), and 4-methoxyestradiol (4ME) on proliferation and cell cycle in RL95-2 human endometrial cells were investigated. Our results indicate that 2ME and 2OHE2, but not E2, 4ME, and 4OHE2, exhibit the inhibitory effect through cell cycle arrest at G2/M. 2ME and 2OHE2-induced G2/M cell cycle arrest associated with activation of p53 (Ser15), upregulation of p21(WAF1/Cip1) (p21) and GADD45, inactivation of Cdc2 (Tyr15), as well as downregulation of Cyclin B1. 2ME and 2OHE2-mediated cell cycle arrest at G2/M was also related to activation of protein kinase Chk1 which is associated with p53 (Ser20) activation and downstream responses.

Antioxidative and immunoprotective effects of Pyracantha fortuneana (Maxim.) Li polysaccharides in mice.

This study was undertaken to determine the effects of Pyracantha fortuneana (Maxim.) Li polysaccharides (PFP) on antioxidant and immune functions in mice. Results from this study showed that PFP administration significantly increased thymus and spleen indices, promoted splenocyte proliferation and natural killer (NK) cell activity, and elevated CD4 T cell numbers as well as CD4(+)/CD8(+) ratios. PFP also increased interleukin-2 (IL-2) levels, and decreased the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in splenocytes. In addition, PFP treatment led to remarkable increases in glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities, and dramatic decreases in malondialdehyde (MDA) levels in splenocytes. Moreover, PFP increased mRNA and protein expression of nuclear factor erythroid 2-related factor (Nrf2) in splenocytes. Taken together, these results suggest that PFP treatment enhances the immune function and decreases the oxidative stress in mice.

Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular characteristics.

The whole length of MCHR2 gene cDNA fragment was amplified by PCR using human fetal brain cDNA library as template. The pcDNA3.1 (+)/MCHR2 eukaryotic expression vector was constructed successfully. The recombinant pcDNA3.1 (+)/MCHR2 plasmid was transfected into Chinese hamster ovary (CHO) cell by lipofectamine 2000, after G418 selection and then the CHO cell line expressing MCHR2 gene was established. The MCHR2 gene expression was tested by RT-PCR, western blotting and immunofluorescence. The maximum binding (B(max)) of CHO cell line was 309.97 +/-1.14 fM x mg(-1) protein and the dissociation constant (K(d) value) was 0.170 +/- 0.0006 nM. MCH could stimulate Ca2+ release, its 50% effective concentration (EC50) was 2.32 +/- 0.01 nM. The construction of the CHO cell line and the research of MCHR2 molecular characteristics have established a good experimental basis for the further research about the function of MCHR2 gene.