Construction of SNP-STR Multiplex Amplification System with Genetic Markers and Its Forensic Application.

ABSTRACT: Objective To select and develop a SNP-STR multiplex amplification system with genetic markers compatible with current STR databases. To understand its genetic polymorphisms in Sichuan Han population and its application value in DNA mixture analysis. Methods Based on the STR genetic markers in commercial kits, SNPs adjacent to these STR markers were selected to be SNP-STR genetic markers. A SNP-STR multiplex amplification system with genetic markers based on allele-specific amplification was constructed using allele-specific amplification primers. The genetic polymorphism of the system in the Sichuan Han population was investigated and the efficiency of systems with different numbers of loci to detect the two individual DNA mixture samples was evaluated. Results An allele-specific multiplex amplification system constituted of 13 SNP-STR genetic markers was selected and constructed. In Sichuan Han population, the heterozygosity of each locus ranged from 0.76 to 0.88, and the combined discrimination power reached 0.999 999 999 999 999 968. In the analysis of the two individual DNA mixture samples： for single-locus amplification, the genotype of the minor components can still be detected when the mixture ratio reaches 1 000∶1; for multiple loci multiplex amplification, the maximum mixture ratio can reach 500∶1. As the number of loci in the system increased, the detection efficiency of the minor components in the DNA mixture decreased. Conclusion SNP-STR genetic markers have a higher polymorphism than STR. The multiplex amplification system made of SNP-STR genetic markers has a better analysis efficiency for mixed samples than traditional STR multiplex amplification system.

[Investigation and analysis of reproductive health status of female street cleaners in a district].

Objective: To explore the reproductive health status of the female street cleaners in Chaoyang district of Beijing and its influencing factors. Methods: In July 2018, a total of 647 questionnaires were sent out to female road cleaning and sanitation workers in the whole district, 613 of which were valid, with an effective rate of 94.7%. Frequency or percentage (%) is used for statistical description of counting data, and mean standard deviation is used for measurement data. The influencing factors were analyzed by 2 test. Results: The average age of 613 female road sweepers in Chaoyang District of Beijing was 42.01 (SD=6.69) years old, including 535 married female workers (87.28%) , 356 middle school and below educated (58.08%) , 292 non Beijing registered (47.63%) , accounting for (/613) , (/613) female workers working hours >8 hours per day 110 (17.94%) , weekly rest <2 days 341 (55.63%) . 144 (23.49%) women workers did not have regular gynecological examination, 119 (19.41%) had reproductive tract infections, 177 (28.87%) had abnormal menstruation in recent 6 months. Drinking, sexual behavior outside marriage, night shift and job satisfaction were all the influencing factors (P<0.05) . The increasing age, working years, drinking, household registration in other places and the decreasing satisfaction of women workers＇ rights and interests protection were all the influencing factors (P<0.05) . Conclusion: The reproductive health status of female road sweepers in Chaoyang District of Beijing is not optimistic.

Effects of APELIN-13 on the expression of IL-6, TNF-α, and IFN-γ in rats with experimental autoimmune neuritis.

The objective of this paper was to study the effects of PYR-ARG-PRO-ARG-LEU-SER-HIS-YSGLY-PRO-MET-PRO-PHE-OH (APELIN-13) on the expression of inflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in rats with experimental autoimmune neuritis (EAN). A total of 30 rats were divided into a control group, an EAN group, and an APELIN-13 group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-6, TNF-α, and IFN-γ in rat plasma. Real-time quantitative Polymerase Chain Reaction (PCR) and Western blot were used to detect the protein and mRNA expression of IL-6, TNF-α, and IFN-γ in rat lymph nodes. In the EAN group, the infiltration of various types of inflammatory cells and focal demyelination were observed near the nerve fascicles of sciatic nerves. Compared with the EAN group, the infiltration of inflammatory cells and demyelination in the APELIN-13 group decreased significantly. The levels of plasma IL-6, TNF-α, and IFN-γ in the EAN group were significantly higher than those in the control group (P < 0.05) but significantly lower than those in the APELIN-13 group (P < 0.05). Compared with the control group, the mRNA and protein expression of IL-6, TNF-α, and IFN-γ increased significantly (P < 0.05) in the EAN group but decreased significantly in the APELIN-13 group (P < 0.05). In conclusion, APELIN-13 exerted a protective effect against EAN in rats.

[Effect of long-term deep slow-wave sleep deprivation on the reproductive system in male rats].

Objective: To investigate the effect of long-term deep slow-wave sleep deprivation on the gonad axis, sperm abnormality rate, and structure of the testis in male rats and possible mechanisms. Methods: A total of 30 specific pathogen-free male Wistar rats aged 5 weeks were randomly divided into slow-wave sleep deprivation group 1 (SD1 group) , slow-wave sleep and sleep time deprivation group 2 (SD2 group) , and control group, with 10 rats in each group. The flower pot method was used to establish a model of sleep deprivation. In addition to 12-hour sleep deprivation at night, the rats in the SD1 group were given interference once every 24 minutes, and those in the SD2 group were deprived of sleep for 8 minutes every 24 minutes; the rats in the control group were given 12-hour light illumination and then placed in dark environment for 12 hours. All rats were sacrificed by exsanguination from the femoral artery, and the testis, the epididymis, and blood were collected for analysis. Sperm abnormality rate and sperm motility rate were measured, and cauda epididymal sperm counting was performed. ELISA was used to measure the serum levels of testosterone (T) , follicle-stimulating hormone (FSH) , and luteinizing hormone (LH) . Results: Compared with the control group, the SD2 group had a significant increase in organ coefficient of the epididymis (P<0.05) and a significant reduction in sperm motility rate (P<0.05) . There were significant differences between the SD1 group and the SD2 group in the increase in sperm abnormality rate (P<0.05) and the reduction in cauda epididymal sperm count (P<0.05) . The levels of FSH and T tended to increase, and the level of LH tended to decrease. Pathological examination showed degeneration and vacuolization of a small amount of spermatogenic cells in the SD1 group; in the SD2 group, there were significant degeneration, edema, and vacuolization of most spermatogenic cells, some spermatogenic cells were observed in the lumen, and there were no sperms in the lumen. Conclusion: Long-term deep slow-wave sleep deprivation impairs the structure of the testis, affects sperm motility rate and sex hormones, and increases the risk of sperm abnormality.

Immunomagnetic separation combined with colony immunoblotting for selective enrichment and detection of piliated Lactobacillus rhamnosus strains.

AIMS: Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). METHODS AND RESULTS: Magnetic nanobeads (diameter: 180 nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimized experimental conditions, IMS-CIB selectively recovered pLR strains from 107  CFU ml-1 of faecal microbiota samples spiked with 2·9 × 101 to 2·4 × 106  CFU ml-1 of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29 CFU pLR ml-1 of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2·0 × 104  CFU ml-1 ). CONCLUSIONS: IMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings established an effective method for selective enrichment and detection of pLR strains.

The timing of intravenous antibiotics and clinical outcomes of Mycobacterium abscessus complex lung diseases.

BACKGROUND: The importance of early intravenous (IV) antibiotic use for Mycobacterium abscessus complex lung diseases (MABC-LD) treatment remains unknown. METHODS: A retrospective multi-centre observational study was conducted in Taiwan. Patients who were diagnosed with and received treatment for MABC-LD from January 2007 to April 2021 were included. Treatment outcome was defined as modified microbiological cure of MABC-LD.RESULTS: Of the 89 enrolled patients, 34 (38.2%) received IV antibiotics as part of the treatment regimen. The median time to IV initiation was 1 day (IQR 1???49); 24 (70.6%) of these patients received IV agents within 4 weeks, defined as early-use. Forty-two (47.2%) patients achieved modified microbiological cure. In the multivariable logistic analysis, early IV antibiotic use was an independent factor associated with modified microbiological cure (aOR 5.32, 95% CI 1.66???17.00), whereas high radiological score (aOR 0.86, 95% CI 0.73???1.00) demonstrated negative association.CONCLUSIONS: In the present study, early use of effective IV antibiotic was prescribed in a low percentage (27%) for MABC-LD. By contrast, early IV antibiotic use was correlated with higher microbiological cure than were late or non-use. Future larger and prospective studies are needed to validate the association.

Imaging patterns and prognosis of patients with gefitinib-related interstitial lung disease.

PURPOSE: We aimed to summarize the imaging findings of 25 patients with gefitinib-related interstitial lung disease (ILD), and identify the factors related to prognosis of gefitinib-related ILD in patients with non-small-cell-lung cancer. MATERIALS AND METHODS: Diagnosis of gefitinib-induced ILD by at least two chest radiologists was based on a review and analysis of the chest radiography and CT findings plus clinical data in the medical records. All patients were diagnosed with Stage III - IV non-small-cell carcinoma (adenocarcinoma (n = 24), bronchioalveolar cell carcinoma (n = 1)) and essential clinical data such as gefitinib as first-line use and survival status were recorded and analyzed to determine whether these were prognosis predictors. The imaging findings were classified into four patterns according to the previous largest study in Japan. RESULTS: The 25 chest radiographs were classified as Pattern A (n = 8), Pattern B (n = 3), Pattern C (n = 6), and pattern D (n = 8). Likewise the 23 CT images were classified as pattern A (n = 8; 34.8%), B (n = 3; 13%), C (n = 5; 21.7%), and D (n = 7; 30.4%). The mortality rate was significantly higher in patients with pattern D than in patients with the other patterns. Pattern D imaging findings were also significantly correlated with non first-line use of gefitinib (p = 0.007). CONCLUSIONS: We found an increase in mortality rate in patients with gefitinib associated ILD/pattern D compared to other radiological patterns. Familiarity with these imaging patterns can facilitate early and accurate diagnosis and help physicians gauge clinical prognosis of gefitinib-related ILD.

Problems with and a system to eliminate single-primer PCR product contamination in simple sequence repeat molecular marker-assisted selection in soybean.

Polymerase chain reaction (PCR) provides a foundation for simple sequence repeat molecular marker-assisted selection (SSR MAS) in soybean. This PCR system and its various conditions have been optimized by many researchers. However, current research on the optimization of the PCR system focuses on double-primer PCR products. We compared single- and double-SSR primer PCR products from 50 soybean samples and found that the use of single-PCR primers in the reaction system can lead to amplified fragments of portions of the SSR primers in the PCR process, resulting in both false-positives and fragment impurity of double-primer PCR amplification, inconvenient for subsequent analysis. We used "single-primer PCR correction" to eliminate interference caused by single-primer nonspecific PCR amplification and improve PCR quality. Using this method, the precision and success rates of SSR MAS in soybean can be increased.

Abnormal ventral attention network homogeneity in patients with right temporal lobe epilepsy.

OBJECTIVE: The attention network is the structural basis of cognitive function. As one of the two known attention networks, the ventral attention network (VAN) has a significant impact on the cognitive impairment of patients with epilepsy. Nevertheless, changes in network homogeneity (NH) are rarely reported in the VAN of right temporal lobe epilepsy (rTLE) patients. Therefore, we explored the NH of the VAN in rTLE patients in this study. PATIENTS AND METHODS: Seventy rTLE patients and 69 healthy controls were recruited. All participants underwent resting-state functional magnetic resonance imaging (fMRI), which was the primary method of evaluation. The executive control reaction time (ECRT) was examined via the attentional network test. The Data Processing Assistant for Resting-State fMRI (DPARSF) was used to analyze NH. The independent component analysis (ICA) and correlation analysis were used in data analysis. RESULTS: Compared to the control group, patients with right temporal lobe epilepsy showed a lower NH in the right superior temporal gyrus, and a longer ECRT. However, abnormal NH values had no significant association with the clinical measurements. CONCLUSIONS: Patients with right temporal lobe epilepsy have abnormal NH values in the VAN, and the executive functions in rTLE patients are also altered. The altered NH values in VAN may help provide new insights into the pathophysiology of cognitive impairment in rTLE.

[Interpretation for the group standards in technical specification for health risk investigation of central air conditioning ventilation system during coronavirus disease 2019 epidemic].

The central air conditioning ventilation system plays an important role in the air circulation of buildings such as centralized isolation medical observation points and general public buildings. In order to meet the requirements of COVID-19 epidemic prevention and control, Beijing Preventive Medicine Association organized Beijing CDC and other professional institutes to write up the group standard entitled "Technical specification for health risk investigation of central air conditioning ventilation system during the COVID-19 epidemic (T/BPMA 0006-2020)" . According to the particularity of central air conditioning ventilation system risk control during the outbreak of similar respiratory infectious diseases, based on current laws and regulations and the principle of scientific, practical, consistency and normative, 8 key points of risk investigations were summarized, which were the location of fresh air outlet, air conditioning mode, air return mode, air system, air distribution, fresh air volume, exhaust and air conditioner components. The contents, process, method, data analysis and conclusion of the investigation implementation were also defined and unified. It could standardize and guide institutions such as disease control and health supervision to carry out relevant risk managements, and provided solutions and technical supports for such major public health emergencies in city operations.

An antagonist decoy receptor and a death domain-containing receptor for TRAIL.

TRAIL, also called Apo2L, is a cytotoxic protein that induces apoptosis of many transformed cell lines but not of normal tissues, even though its death domain-containing receptor, DR4, is expressed on both cell types. An antagonist decoy receptor (designated as TRID for TRAIL receptor without an intracellular domain) that may explain the resistant phenotype of normal tissues was identified. TRID is a distinct gene product with an extracellular TRAIL-binding domain and a transmembrane domain but no intracellular signaling domain. TRID transcripts were detected in many normal human tissues but not in most cancer cell lines examined. Ectopic expression of TRID protected mammalian cells from TRAIL-induced apoptosis, which is consistent with a protective role. Another death domain-containing receptor for TRAIL (designated as death receptor-5), which preferentially engaged a FLICE (caspase-8)-related death protease, was also identified.

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Spirometra erinaceieuropaei severely infect frogs and snakes from food markets in Guangdong, China: implications a highly risk for zoonotic sparganosis.

Sparganosis is a parasitic disease caused by plerocercoid larvae of the genus Spirometra. In China, the main source of sparganosis is from Guangdong, 16.1% of the country's human sparganosis cases occur in this province. Frequent international trade of amphibians and reptiles in Guangdong may introduce new species of Spirometra into the local market. In this study, a large-scale, high-intensity sampling survey was conducted to find out the causative species and epidemic situation of Sparganosis in Guangdong. The prevalence of sparganum infection in five species of frogs (Boulengerana guentheri, Fejervarya multistriata, Hoplobatrachus chinensis, Pelophylax nigromaculatus and Quasipaa spinosa) and nine species of snakes (Elaphe carinata, Lycodon rufozonatum, Hypsiscopus plumbea, Ptyas dhumnades, P. korros, P. mucosa, Naja atra, Sinonatrix annularis and Xenochrophis piscator) was investigated in Guangdong, Southern China from May 2014 to August 2015. The results showed that 9.8% (50/511) of the frogs and 40.8% (141/ 346) of snakes were found to be infected by plerocercoids (spargana). To identify the species of the collected spargana, a partial sequence of the mitochondrial cytochrome c oxidase subunit1 gene (cox1) was amplified and sequenced. Phylogenetic analysis identified all the spargana specimens as Spirometra erinaceieuropaei. Our study indicated that S. erinaceieuropaei, a highly pathogenic parasite, is the only causative agent of sparganosis in Guangdong, China. This study suggests that the large numbers of frogs and snakes in food markets in Guangdong may impact public health in China by transmitting S. erinaceieuropaei sparganum. Additional steps should be considered by the governments and public health agencies to prevent the risk of food-associated Spirometra infections in humans in China.

Developmental changes in the rat atriopeptin hormonal system.

We undertook a study of fetal synthesis, storage, and release of atriopeptin (AP). Plasma levels of both atriopeptin immunoreactivity (APir) and the NH2-terminal fragment of the prohormone immunoreactivity (NTFir) were very high in the fetus (4 and 20 times the maternal plasma, respectively). However, the atrial content of the AP was low, but surprisingly, ventricular content of AP was quite high (relative to the adult) in the fetus and fell postnatally. Atrial AP messenger RNA (mRNA) increased with postnatal age, whereas ventricular mRNA was extremely high in the fetus and fell rapidly after birth. High fetal plasma peptide levels may derive from the mother since infusion of exogenous atriopeptin 24 into the mother resulted in parallel increases in fetal and maternal peptide levels. Fetal plasma APir and NTFir levels partially reflect the markedly reduced total renal metabolic capacity compared with that of the adult. Plasma levels fell progressively after birth; whereas neonatal atrial content rose substantially. Plasma AP and NTF were simultaneously elevated in both the maternal and fetal circulation after vasopressin injection of the mother. The fetus can also respond to exogenous stimuli (vasopressin or indomethacin--presumably via ductal closure) and promptly release substantial amounts of peptide into its circulation. Thus, it appears that the AP hormonal system is functional during fetal life and responds avidly to increases in intracardiac pressure as does the mature animal.

Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation.

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.