[Twelve-week of sofosbuvir/velpatasvir therapeutic regimen for chronic hepatitis C patients in northwest region of China: a real-world multicenter clinical study].

Objective: To study the real-world outcome of China FDA-approved Sofosbuvir (SOF)/Velpatasvir (VEL) in Northwest China. Methods: In this multicenter, prospective, real-world cohort study, we recruited patients from 10 sites from Northwest China, who were chronically infected with HCV GTs 1-6 from 06/2018 to 09/2019. Patients received SOF (400mg)/VEL (100mg) for 12 weeks, and with ribavirin 900-1200 mg for GT3 cirrhosis and for any genotype decompensated cirrhosis. The primary endpoint was sustained virological response at 12-weeks post-treatment (SVR12) and safety. The secondary endpoint was the change of liver function after the achievement of SVR12. Results: Totally, 143 patients were enrolled in the study, four patients were lost to follow-up and one died during the follow-up, 138 patients were included in per-protocol analysis. Of the 138 patients, the mean age 53 years, 53.6% male, 94.2% Han nationality, 53.6% liver cirrhosis, 10.1% HBsAg(+), 6.5% renal dysfunction, 5.1% treatment-experienced, and 16.7% patients received ribavirin treatment. The genotype distribution was as follows: 35.5% GT1, 42.8% GT2, 15.9% GT3, and 5.8% un-typed. The SVR12 rate was 96.5% (138/143, 95%CI: 93.5%-99.6%) for intention-to-treat analysis, and in per-protocol analysis, all 138 patients obtained SVR12 (100%). Compared with baseline, the serum total bilirubin, ALT and AFP levels decreased (all P < 0.05), as well as increased ALB and platelet count (all P < 0.001) at post-treatment 12-weeks. Overall adverse events (AEs) rate is 29.0%, and the most common AEs were anemia (14.5%) and fatigue (8.0%). Severe side effects (edema and fatigue) occurred in 2 patients, one of whom needed a short-term interruption of treatment due to fatigue. Conclusion: In this real-world cohort study, 12-week SOF/VEL regimen with or without ribavirin achieved high SVR12 rates (96.5%-100% overall) with excellent safety profile among patients with HCV GT1/2/3 infection including patients with GT3 and cirrhosis, and led to improvement of liver function.

[The associations between comorbidity and exacerbation risk in patients with chronic obstructive pulmonary disease].

Objective: To study the association between comorbidity and acute exacerbation risk in patients with chronic obstructive pulmonary disease (COPD). Methods: This was a prospective cohort study with 64 stable COPD patients included. There were 64 males and 18 females with an average age of (68±9) years. Clinical characteristics, the number and type of comorbidities were recorded, and Charlson comorbidity index (CCI) was calculated. The patients were interviewed by phone calls every 3 months since baseline in which the number of acute exacerbations was recorded until 12 months. The impact of CCI, the number of comorbidities and certain comorbidities in the prediction of COPD exacerbation risk were analyzed. Results: Compared to patients with a lower CCI score, patients with a higher CCI score were older (75±6 vs 62±8), and had more severe lung function impairment [FEV(1)%pred: (40±18)% vs (52±18)% ], higher number of comorbidities [4(3, 7) vs 1(1, 3)] and higher frequency of hospital admission due to acute exacerbation [1(0, 2) vs 0(0, 0.25)]. In comparison with patients with lower number of comorbidities, patients with higher number of comorbidities were older (72±7 vs 64±10), and had higher mMRC score [2(1, 3) vs 2(1, 2)] and more severe lung function impairment [FEV(1)%pred: (42±15)% vs (53±19)% ], higher age adjusted CCI score [5(3, 5) vs 3(2, 3) ] and more courses of systemic corticosteroids [2(0, 3) vs 0(0, 0.75)] and/or antibiotics use [3(2, 4) vs 1.5(1, 2.75)]. The number of hospitalizations and total number of exacerbations were higher in COPD patients with bronchiectasis than those without (P<0.005). Conclusion: The inclusion of clinically meaningful comorbidities into the combined assessment of COPD for the prediction of disease prognosis deserves further study.

Development and significance of SCAR marker QG12-5 for Canarium album (Lour.) Raeusch by molecular cloning from improved RAPD amplification.

Sequence-characterized amplified region (SCAR) is a valuable molecular marker for the genetic identification of any species. This marker is mainly derived from molecular cloning of random amplified polymorphic DNA (RAPD). We have previously reported the use of an improved RAPD technique for the genetic characterization of different samples of Canarium album (Lour.) Raeusch (C. album). In this study, DNA fragments were amplified using improved RAPD amplified from different samples of C. album. The amplified DNA fragment was excised, purified from an agarose gel and cloned into a pGM-T vector; subsequently, a positive clone, called QG12-5 was identified by PCR amplification and enzymatic digestion and sequenced by Sanger di-deoxy sequencing method. This clone was revealed consisting of 510 nucleotides of C. album. The SCAR marker QG12-5 was developed using specifically designed PCR primers and optimized PCR conditions. This SCAR marker expressed seven continuous "TATG" [(TATG)n] tandem repeats, which was found to characterize C. album. Subsequently, this novel SCAR marker was deposited in GenBank with accession No. KT359568. Therefore, we successfully developed a C. album-specific SCAR marker for the identification and authentication of different C. album species in this study.

An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers.

Random amplified polymorphic DNA (RAPD) is a widely used molecular marker technique. As traditional RAPD has poor reproducibility and productivity, we previously developed an improved RAPD method (termed RAMP-PCR), which increased the reproducibility, number of bands, and efficiency of studies on polymorphism. To further develop the efficiency of this method, we used high-GC content primers for improved RAMP-PCR with DNA samples from Lonicera japonica. Comparison of amplification profiles obtained by standard RAPD primers with those obtained by regular PCR and RAMP-PCR, and high-GC primers with regular PCR and RAMP-PCR showed that the average number of bands and polymorphisms per primer gradually and significantly increased (from 6.4 to 15.0 and from 4.6 to 10.2, respectively). Cluster dendrograms showed similar results, indicating that this new method is consistent and reproducible. A total of 22 samples from different species, including plants, animals, and humans, were used for RAMP-PCR with high-GC primers. Multiple bands were successfully amplified from all samples, demonstrating that this method is a reliable technique with consistent results and may be of general interest in studies on different genera and species. We developed highly effective DNA markers, which can provide a more effective and potentially valuable approach than traditional RAPD for the genetic identification of various organisms, particularly of medicinal plants.

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst.

Development of sequence-characterized amplified region (SCAR) markers from random-amplified polymorphic DNA (RAPD) fragments is a valuable molecular approach for the genetic identification of different species. By using SCAR markers, molecular analysis is reduced to a simple polymerase chain reaction (PCR) analysis using primers designed from the amplicon sequence of RAPD. In this study, the DNA fragments from an improved RAPD amplification of Ganoderma species were cloned into a pGM-T vector; positive clones were identified by PCR amplification and enzymatic digestion, and finally, DNA fragments were sequenced using the Sanger sequencing method for developing the SCAR markers. Two SCAR markers, named LZ4-1 with 534 nucleotides, and LZ5-2 with 337 nucleotides were identified, which are specific to Ganoderma lucidium (Leysser: Fr) Karst species. BLAST of these two nucleotide sequences in the GenBank database showed no identity to other species. We deposited these sequences into the GenBank database (LZ4-1 accession No. KM391933, LZ5-2 accession No. KM391934). PCR assays confirmed them as novel molecular markers for G. lucidium (Leysser: Fr) Karst, which might be used for genetic authentication of adulterant samples. Thus, our study developed two specific SCAR markers for identifying and distinguishing the medicinal mushroom G. lucidium (Leysser: Fr) Karst from other Ganoderma species.

The guiding significance of NT-proBNP and PCT levels in mechanical ventilator of patients with chronic respiratory failure.

OBJECTIVE: To analyze the guiding significance of N-terminal pro-brain natriuretic peptide (NT-proBNP) and procalcitonin (PCT) level in mechanical ventilator used for patients with chronic respiratory failure. PATIENTS AND METHODS: Eighty-two patients with simple chronic respiratory failure who were treated by mechanical ventilator were selected for this study. They were treated offline after they reached the standards of spontaneous breathing trial, and were divided into two groups: 1- the successful offline group with 59 patients and 2- the failure group with 23 patients. Differences of NT-proBNP and PCT levels, oxygenation index, average heart rate and mean arterial pressure between two groups were compared. RESULTS: The NT-proBNP and PCT levels in the failure group were significantly higher than those in the success group, while oxygenation index was significantly lower in the success group. All differences were statistically significant (p<0.05). Comparison of average heart rate and mean arterial pressure between two groups showed no statistically significant difference (p>0.05). CONCLUSIONS: Through multi-factor regression analyses we observed that NT-proBNP and PCT levels were independent risk factors for guiding the success rate of offline (p<0.05). Spontaneous breathing trial in combination with NT-proBNP and PCT levels improved the success rate of offline.

Development of novel SCAR markers for genetic characterization of Lonicera japonica from high GC-RAMP-PCR and DNA cloning.

Sequence-characterized amplified region (SCAR) markers were further developed from high-GC primer RAMP-PCR-amplified fragments from Lonicera japonica DNA by molecular cloning. The four DNA fragments from three high-GC primers (FY-27, FY-28, and FY-29) were successfully cloned into a pGM-T vector. The positive clones were sequenced; their names, sizes, and GenBank numbers were JYHGC1-1, 345 bp, KJ620024; YJHGC2-1, 388 bp, KJ620025; JYHGC7-2, 1036 bp, KJ620026; and JYHGC6-2, 715 bp, KJ620027, respectively. Four novel SCAR markers were developed by designing specific primers, optimizing conditions, and PCR validation. The developed SCAR markers were used for the genetic authentication of L. japonica from its substitutes. This technique provides another means of developing DNA markers for the characterization and authentication of various organisms including medicinal plants and their substitutes.

A 425 T>C polymorphism in complement C7 association with resistance to Aeromonas hydrophila in grass carp.

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes hemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortalities and tremendous economic loss in cultured fish. The complement component C7 is a terminal component of complement that interacts in a sequence of polymerization reactions with other terminal complement components to form a membrane attack complex. The formation of the membrane attack complex creates a pore in the membranes of certain pathogen that can lead to their death. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C7 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. We sequenced a total of 7826 bp of the C7 gene and identified 6 SNPs that were genotyped in the resource population. The SNP -1575 A>C was positioned in the promoter region of the gene. The SNP 425 C>T identified in the coding exon was a synonymous substitution in the fourth exon. Statistical analysis showed that SNP 425 C>T was associated with the incidence of hemorrhagic septicemia. The SNPs -1575 A>C, -688 T>C, and -266 A>C were highly linked together (r(2) > 0.85). No haplotypes generated with these 3 SNPs were associated with resistance to A. hydrophila in grass carp. These findings suggest that the 425 C>T polymorphism in C7 gene may be a significant molecular marker for resistance to A. hydrophila in grass carp.

Effects of hemodialysis and hemoperfusion on inflammatory factors and nuclear transcription factors in peripheral blood cell of multiple organ dysfunction syndrome.

OBJECTIVE: To investigate the effects of hemodialysis (HD) and hemoperfusion (HP) on inflammatory factors and nuclear transcription factors in peripheral blood cell of multiple organ dysfunction syndrome (MODS) patients. PATIENTS AND METHODS: 92 cases of MODS patients undergoing maintained hemodialysis in our hospital were randomly divided into the control group and observation group. The control group was treated with conventional hemodialysis (HD), the observation group was treated with hemoperfusion combined therapy (HD+HP) based on the control group. The levels of serum creatinine (SCR), serum total cholesterol (TC), blood urea nitrogen (BUN) and serum albumin (Alb) were compared and analyzed between two groups before and after treatment. The levels of NK-κB p65 in the white cell of peripheral blood were compared between two groups before and after treatment by Western blot. The levels of inflammatory factors TNF-α and IL-6 in peripheral blood were detected between two groups before and after treatment using ELISA method. RESULTS: Compared with the control group, the levels of Scr, BUN and TC were significantly decreased, while the level of Alb was significantly increased (p < 0.05). The level of cytoplasm NK-κB p65 protein in peripheral blood was significantly increased in the observation group, while the level of nuclear NK-κB p65 in peripheral blood was significantly decreased (p < 0.05). CONCLUSIONS: Hemodialysis combined with hemoperfusion in treating MODS patients could significantly improve the biochemical indicators, effectively remove the inflammatory mediums, and significantly inhibit the activation of NK-κB.

[Effect of thermo-sensitive Matrigel on minimization of thermal injury to the nearby structures in radiofrequency ablation of subcapsular hepatic tumors in a rat model].

OBJECTIVE: To evaluate the effect and safety of using thermo-sensitive Matrigel as artificial ascites on minimizing thermal injury to the nearby structures in a rat model of radiofrequency (RF) ablation of the liver. METHODS: In this research, the morphological characterization of Matrigel was observed at different temperatures in vitro. In vivo study was conducted by using 42 SD rats in Matrigel, Saline and control groups of 14 rats each. Artificial ascites with 10 ml of Matrigel or saline was produced under ultrasound guidance before RF in the experimental groups. Using a 0.7 cm exposed 17 G RF electrode for 5 minutes (90 ± 2) ℃, 41 hepatic ablations was performed abutting the diaphragm, abdominal wall and stomach. The 41 rats were sacrificed at 24 h, and necropsy was performed.Gross and histopathologic examinations were performed to compare the frequency and extent of thermal injury to the nearby organs.The animal status was followed up to achieve long term safety evaluation of Matrigel. RESULTS: Matrigel was in liquid formation at 0-4 ℃, but was not moving in vials at temperature 37 ℃ in 1 min, presenting gel formation.In vivo experiment, the artificial fluid thickness of Matrigel in site was significant larger than that of saline group, especially at 5 min of ablation ((8.1 ± 1.1) mm vs (2.4 ± 0.8)mm, P<0.01). Thermal injury to the nearby structures was found in 12 of 13 cases in control group, 8 of 14 cases (P=0.037) in saline group, and 1 of 14 cases (P<0.01) in Matrigel group. The sizes of the ablation zone of the liver did not differ among the three groups. There was no difference in the incidence of operation related complications among the three groups. There was no significant difference in body weight and health related parameters between Matrigel and saline group during 60 days of follow up. CONCLUSIONS: Using thermo-sensitive Matrigel as artificial ascites may be a safe and useful technique and help to further reduce the frequency and severity of collateral thermal injury to the nearby structures compare to conventional saline, during RF ablation of subcapsular hepatic tumors.

Identification of the origin of marker chromosomes by two-color fluorescence in situ hybridization and polymerase chain reaction in azoospermic patients.

Y chromosomal microdeletions at the azoospermia factor locus and chromosome abnormalities have been implicated as the major causes of idiopathic male infertility. A marker chromosome is a structurally abnormal chromosome in which no part can be identified by cytogenetics. In this study, to identify the origin of the marker chromosomes and to perform a genetic diagnosis of patients with azoospermia, two-color fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) techniques were carried out. The marker chromosomes for the two patients with azoospermia originated in the Y chromosome; it was ascertained that the karyotype of both patients was 46,X, ish del(Y)(q11)(DYZ3+, DXZ1-). The combination of two-color FISH and PCR techniques is an important method for the identification of the origin of marker chromosomes. Thus, genetic counseling and a clear genetic diagnosis of patients with azoospermia before intracytoplasmic sperm injection or other clinical managements are important.

Relationship between SPOP mutation and breast cancer in Chinese population.

SPOP protein has been found to have ubiquitin ligase activity. Mutations in SPOP gene have been recently reported in some cancers such as prostate, gastric, colorectal cancer. We investigated SPOP DNA mutation in tumor tissues collected from 70 Chinese female breast cancer patients in Southwestern China by DNA sequencing. The results did not show mutation in our tissue samples, indicating that a mutation in the SPOP gene may not be associated with breast cancer, particularly in Chinese women. This DNA mutation analysis or DNA genotyping may provide useful and important information for genetic counseling and personalized medical treatment for different types of cancers.

Establishment and rapid detection of a heterozygous missense mutation in the CACNA1F gene by ARMS technique with double-base mismatched primers.

Retinitis pigmentosa (RP) is a retinal degenerative disorder that often causes complete blindness. Mutations of more than 50 genes have been identified as associated with RP, including the CACNA1F gene. In a recent study, by employing next-generation sequencing, we identified a novel mutation in the CACNA1F gene. In this study, we used the amplification refractory mutation system (ARMS) and identified a single nucleotide change c.1555C>T in exon 13 of the CACNA1F gene, leading to the substitution of arginine by tryptophan (p.R519W) in a Chinese individual affected by RP. This study actually confirms this novel mutation, and establishes the ARMS technique for the detection of mutations in RP.

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/µL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

Field evidence for earlier leaf-out dates in alpine grassland on the eastern Tibetan Plateau from 1990 to 2006.

Worldwide, many plant species are experiencing an earlier onset of spring phenophases due to climate warming. Rapid recent temperature increases on the Tibetan Plateau (TP) have triggered changes in the spring phenology of the local vegetation. However, remote sensing studies of the land surface phenology have reached conflicting interpretations about green-up patterns observed on the TP since the mid-1990s. We investigated this issue using field phenological observations from 1990 to 2006, for 11 dominant plants on the TP at the levels of species, families (Gramineae-grasses and Cyperaceae-sedges) and vegetation communities (alpine meadow and alpine steppe). We found a significant trend of earlier leaf-out dates for one species (Koeleria cristata). The leaf-out dates of both Gramineae and Cyperaceae had advanced (the latter significantly, starting an average of 9 days later per year than the former), but the correlation between them was significant. The leaf-out dates of both vegetation communities also advanced, but the pattern was only significant in the alpine meadow. This study provides the first field evidence of advancement in spring leaf phenology on the TP and suggests that the phenology of the alpine steppe can differ from that of the alpine meadow. These findings will be useful for understanding ecosystem responses to climate change and for grassland management on the TP.

Genetic characterization and authentication of Dimocarpus longan Lour. using an improved RAPD technique.

Dimocarpus longan Lour. is an edible and traditional herb in China, commonly referred to as longon. An improved randomly amplified polymorphic DNA (RAPD) protocol was here developed in order to determine the geographical origins of D. longan samples collected from 5 provinces in the southern and southwestern areas of China, including Sichuan, Hainan, Fujian, Guangdong, and Guangxi. Generally, the improved RAPD method generated good fingerprinting of the 5 samples using the selected 17 primers. In particular, primers SBS-A5, SBS-A13, SBS-I9, SBS-I20, SBS-M1, and SBS-Q12 produced distinguishable bands that clearly separated all 5 cultivars, suggesting that there are variations in RAPD genetic sites among the samples. The similarity index ranged from 0.69 to 0.76. The Sichuan and Hainan clades clustered together with a 0.73 similarity index. The Guangxi and Fujian clades clustered together with a 0.76 similarity index, and they formed the sister clade to the Sichuan/Hainan clade with a 0.71 similarity index. The Guangdong clade was in a basal polytomy with a 0.70 similarity index. Based on the abundant DNA polymorphisms, these longan accessions are distinguishable using our improved RAPD technique. Therefore, RAPD analysis is an effective technique in distinguishing the geographical origins of D. longan. Moreover, the improved method could also be employed for a variety of applications including genetic diversity and fingerprinting analyses.

HBcAg-specific IL-21-producing CD4+ T cells are associated with relative viral control in patients with chronic hepatitis B.

Function exhaustion of specific cytotoxic CD8+ T cell in chronic virus infection partly results from the low levels of CD4 help, but the mechanisms by which CD4 help T cell required to control hepatitis B virus infection are not well understood. In this study, we investigated the role of interleukin-21-producing CD4+ T cell response in viral control of hepatitis B virus infection. HBcAg-specific interleukin-21-producing CD4+ T cells in blood were detected in patients with hepatitis B virus infection. Patients with acute hepatitis B had greater HBcAg-specific interleukin-21-producing CD4+ T cells in blood compared with chronic hepatitis B patients, and there was no statistical significance between immune active chronic hepatitis B patients and inactive healthy carrier patients for these cells, whereas frequencies of these cells negatively correlated with HBV DNA levels but positively correlated with HBc18-27-specific IFN-γ-producing CD8+ T cells. Moreover, interleukin-21 sustained HBc18-27-specific CD8+ T cells in vitro, and interleukin-21 production by HBcAg-specific IL-21-producing CD4+ T cells of acute hepatitis B patients enhanced IFN-γ and perforin expression by CD8+ T cells from chronic hepatitis B patients. Our results demonstrate that HBcAg-specific interleukin-21-producing CD4+ T cell responses might contribute to viral control by sustaining CD8+ T cell antiviral function.