AIM　To explore the biotransformation of compound 7-(4-chlorbenzyl)-7,8,13,13a-tetrahydroberberine in the rabbit. METHODS　Analyze the rabbit bile sample with HPLC, LC/MS and LC/NMR. RESULTS　A metabolite and unchanged 7-(4-chlorbenzyl)-7,8,13,13a-tetrahydroberberine were found in the rabit bile, the metabolite was characterized and its structure was elucidated. CONCLUSION　Compound 7-(4-chlorbenzyl)-7,8,13,13a-tetrahydroberberine is metabolized by demethylation at 10-OCH3 position.

Objective To explore the relationship between operation scores and the expression of caveolin-1 ( Cav-1 ) protein in the cortex of mice.Methods Male Kunming mice were used and divided into two groups (excellent group and bad group) according to the training scores after 4 days' Y-maze learning training.The expression of cav-1 protein in cortex and hippocampus of the mice with different scores were measured through Western blot technology.Results Cav-1 protein expression of excellent group( Cav-1/β-actin was 5.71 + 1.11 ) in the cortex was significantly higher than that of the bad group ( Cav-1/β-actin was 1.69 + 0.20) (P ＜ 0.01 ).While there was no significant difference in the two groups in the expression of Cav-1 protein in hippocampus(P ＞ 0.05 ).Conclusion The operation scores have distinct correlation with the expression of Cav-1 protein in the cortex of mice.

OBJECTIVETo establish a high-frequency regeneration system of Astragalus and an aFGF transformation system.

METHODCotyledon node of the Astragalus explants was used for organogenesis to establish a high-frequency regeneration system. GV3101 was used to transform cotyledon node, and aFGF gene was introduced into Astragalus, renewable strain was detected by PCR.

RESULTAll cotyledon node was explants, adventitious buds were induced in the medium of MS +2.0 mg x L(-1) BA +0.5 mg x L(-1) IBA, the root was taken in the medium of 1/2MS +5.0 mg x L(-1) NAA to give a high frequency regeneration system. All cotyledon node was precultured in medium for 3 days and infected with Agrobacterium (A600 0.3) for 10 min and then cocultured for 2 days. The aFGF gene was confirmed to transform into genome of Astragalus.

CONCLUSIONA high-frequency regeneration system of Astragalus and an aFGF transformation system were established.

Astragalus Plant ; genetics ; metabolism ; Fibroblast Growth Factor 1 ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Engineering ; methods ; Humans ; Plants, Genetically Modified ; genetics ; metabolism ; Transformation, Genetic

Acid fibroblast growth factor (aFGF) has great potential in clinical application, but it is very expensive. In order to reduce the cost of production and to make full use of the merits integrated with plant bioreator, we have explored the aFGF in transgenic Tobacco expression. AFGF gene was inserted into plant expression vector pBI121; the acquired plants contained aFGF gene expression vector pBI121-TOAB-aF. Using Agrobacterium-mediated gene transformation of Tobacco and using transgenic Tobacco containing kanamycin and cephalosporin culture medium, we obtained kanamycin resistant transgenic Tobacco plants. PCR detection, RT-PCR detection and Western blot detection confirmed that foreign genes were successfully expressed in Tobacco. These data could serve as a theoretical foundation on which to use the plant bioreactor for production of aFGF.
Agrobacterium ; genetics ; Fibroblast Growth Factor 1 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Tobacco ; genetics ; metabolism

OBJECTIVETo assess the correlation of cytogenetic changes with serum vascular endothelial growth factor (VEGF) and serum tartrate resistant acid phosphatase (TRacp-5b) levels among elderly patients with multiple myeloma (MM).

METHODSChromosomal changes were analyzed with a modified culturing method in the presence of IL-6. Serum levels of VEGF and TRacp-5b were determined with enzyme-linked immunosorbent assays (ELISA).

RESULTSAmong the 60 MM patients, chromosomal abnormalities were found in 27 cases, including 22 with numerical abnormalities and 15 with structural abnormalities. Many patients had both numerical and structural abnormalities. For 33 patients with a normal karyotype, the levels of VEGF and TRacp-5b were 117.35 ± 55.26 pg/mL and 4.15 ± 2.15 U/L, respectively, while for 27 patients with an abnormal karyotype, the levels of VEGF and TRacp-5b were 190.26 ± 85.74 pg/ml and 5.96 ± 2.24 U/L, respectively. The difference between the two groups was significant (P<0.05).

CONCLUSIONCompared with MM patients with a normal karyotype, the levels of VEGF and TRacp-5b are higher in those with cytogenetic abnormalities.

Aged ; Aged, 80 and over ; Chromosome Aberrations ; Cytogenetic Analysis ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotype ; Male ; Multiple Myeloma ; blood ; diagnosis ; genetics ; Tartrate-Resistant Acid Phosphatase ; blood ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo explore the mechanism of NK cell dysfunction in patients with multiple myeloma (MM).

METHODSThe expression of inhibitory receptors (CD158a and CD158b) and activating receptors NKG2D and NCRs (NKp30, NKp44 and NKp46) on CD3-CD56+NK cell of 13 MM patients and 30 healthy controls were analyzed by flow cytometry. The concentration of soluble NKG2D ligands (MICA, MICB, ULBP1, ULBP2 and ULBP3) in serum was detected by enzyme- linked immunosorbent assay (ELISA), and the cytotoxicity of NK cell against MM cell line by flow cytometry.

RESULTSThere are no significant differences of percentage and absolute number of NK cells, and the expression level of CD158a and CD158b between MM patients and healthy individuals (P>0.05). No NKp44 expression was detected on fresh isolated NK cells from both groups. There is no difference in inhibitor receptors expression between MM patients and healthy individuals but the expression of NKG2D, NKp30 and NKp46 on NK cells were higher in MM patients as compared with that in healthy individuals. The concentration of soluble NKG2D ligands in serum was higher in MM patients as compared with that in healthy individuals (P<0.05). Cultured healthy individual's NK cells with MM patient's serum could significantly decrease its cytotoxicity against MM cell line U266 cells [(38.5 ± 6.5) % vs (25.4 ± 5.9)%, P=0.044］.

CONCLUSIONThe higher level of soluble NKG2D ligands in serum may be the mechanism of NK cell dysfunction in MM patient.

Cells, Cultured ; Flow Cytometry ; Humans ; Killer Cells, Natural ; metabolism ; pathology ; Multiple Myeloma ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; metabolism ; Natural Cytotoxicity Triggering Receptor 1 ; metabolism ; Natural Cytotoxicity Triggering Receptor 2 ; metabolism ; Natural Cytotoxicity Triggering Receptor 3 ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.
Bacteria/genetics* ; Gastrointestinal Microbiome/genetics* ; High-Throughput Nucleotide Sequencing ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics*

Objective: To understand the prevalence, diagnosis and treatment of chronic critical illness (CCI) in China. Methods: The clinical data of 472 adult patients admitted to ICU in 53 hospitals, including basic information, disease-related data, nutrition program, etc., were collected on May 10, 2019, by means of multi-center cross-sectional study. If surgical intervention was needed or the occurrence of the disease was directly related to the surgery, ICU patients were regarded as surgical ICU cases (n=211). In this study, the diagnostic criteria for CCI were: (1) admission to ICU >14 days;(2) combined with persistent organ dysfunction. The prevalence,distribution and treatment of CCI and surgery-related CCI were recorded and analyzed. The Mann-Whitney U test, chi-square test or Fisher exact test were used for comparative analysis. Results: Among the 472 ICU patients from 53 hospitals, 326 were male (69.1%) and 146 were female (30.9%). The prevalence of CCI was 30.7% (145/472). Among 211 surgery-related ICU patients, 57 developed CCI with a prevalence of 27.0%. As compared to non-CCI patients, higher APACHE II score [median (IQR) 13.5 (10.0, 18.3) vs. 11.0 (7.0, 16.0), U=2970.000, P=0.007], higher Charlson comorbidity index [median (IQR) 4.0 (2.0, 7.0) vs. 3.0 (1.0, 5.0), U= 3570.000, P=0.036] and higher ratio of breath dysfunction [68.4% (39/57) vs. 48.1% (74/154), χ²=6.939, P=0.008] and renal dysfunction [42.1% (24/57) vs. 18.2% (28/154), χ²=12.821, P<0.001] were found in surgery-related CCI patients. While SOFA score, Glasgow coma score and other visceral function were not significantly different between surgery-related CCI and non-CCI patients (all P>0.05). NUTRIC score showed that surgery-related CCI patients had higher nutritional risk [43.9% (25/57) vs. 26.6%(41/154), U=5.750, P=0.016] and higher ratio of mechanical ventilation [66.7% (38/57) vs. 52.3% (79/154), χ²=3.977, P=0.046] than non-CCI patients. On the survey day, the daily caloric requirements of 50.2% (106/211) of surgery-related ICU patients were calculated according to the standard adult caloric intake index (104.6 to 125.5 kJ·kg^-1·d^-1, 1 kJ=0.239 kcal), and the daily caloric requirements of 46.4% (98/211) of patients were calculated by physicians according to the severity of the patient′s condition. 60.2% (127/211) of nutritional support therapy was enteral nutrition (including a combination of enteral and parenteral nutrition), while the remaining patients received parenteral nutrition (24.6%, 52/211), simple glucose infusion (9.0%, 19/211), or oral diet (6.2%, 13/211). The target calorie of CCI group was 104.6 (87.9, 125.5) kJ·kg^-1·d^-1, and the actual calorie intake accounted for 0.98 (0.80, 1.00) of the target calory. In the non-CCI group, the target calorie was 104.6 (87.9, 125.5) kJ·kg^-1·d^-1, and the actual calorie consumed accounted for 0.91 (0.66, 1.00) of the target calorie. There was no statistically significant difference between two groups (P=0.248, P=0.150). Conclusion The prevalence of CCI and surgery-related CCI in ICU is high, along with severe complications, respiratory and renal dysfunction and mechanical ventilation. Surgical patients admitted to ICU are at high nutritional risk, and active and correct nutritional support is essential for such patients.

Objective: To investigate the diagnosis and treatment for enterocutaneous fistula (ECF) in China, and to explore the prognostic factors of ECF. Methods: A multi-center cross-sectional study was conducted based on the Registration System of Chinese Gastrointestinal Fistula and Intra-Abdominal Infections to collect the clinical data of ECF patients from 54 medical centers in 22 provinces/municipalities from January 1, 2018 to December 31, 2018. The clinical data included patient gender, age, length of hospital stay, intensive care unit (ICU) admission, underlying diseases, primary diseases, direct causes of ECF, location and type of ECF, complications, treatment and outcomes. All medical records were carefully filled in by the attending physicians, and then re-examined by more than two specialists. The diagnosis of ECF was based on the clinical manifestations, laboratory/imaging findings and intraoperative exploration. Results: A total of 1521 patients with ECF were enrolled, including 1099 males and 422 females, with a median age of 55 years. The top three primary diseases of ECF were malignant tumors in 626 cases (41.2%, including 540 gastrointestinal tumors, accounting for 86.3% of malignant tumors), gastrointestinal ulcers and perforations in 202 cases (13.3%), and trauma in 157 cases (10.3%). The direct causes of ECF were mainly surgical operation in 1194 cases (78.5%), followed by trauma in 156 (10.3%), spontaneous fistula due to Crohn′s disease in 92 (6.0%), radiation intestinal injury in 41 (2.7%), severe pancreatitis in 20 (1.3%), endoscopic treatment in 13 (0.9%) and 5 cases (0.3%) of unknown reasons. All the patients were divided into three groups: 1350 cases (88.7%) with simple ECF, 150 (9.9%) with multiple ECF, and 21 (1.4%) with combined internal fistula. Among the patients with simple ECF, 438 cases (28.8%) were jejuno-ileal fistula, 313 (20.6%) colon fistula, 170 (11.2%) rectal fistula, 111 (7.3%) duodenal fistula, 76 (5.0%) ileocecal fistula, 65 (4.3%) ileocolic anastomotic fistula, 55 (3.6%) duodenal stump fistula, 36 (2.4%) gastrointestinal anastomotic fistula, 36 (2.4%) esophagogastric/esophagojejunal anastomotic fistula, 29 (1.9%) gastric fistula and 21 (1.4%) cholangiopancreatiointestinal. Among all the simple ECF patients, 991 were tubular fistula and 359 were labial fistula. A total of 1146 patients finished the treatment, of whom 1061 (92.6%) were healed (586 by surgery and 475 self-healing) and 85 (7.4%) died. A total of 1043 patients (91.0%) received nutritional support therapy, and 77 (6.7%) received fistuloclysis. Infectious source control procedures were applied to 1042 patients, including 711 (62.0%) with active lavage and drainage and 331 (28.9%) with passive drainage. Among them, 841 patients (73.4%) underwent minimally invasive procedures of infectious source control (replacement of drainage tube through sinus tract, puncture drainage, etc.), 201 (17.5%) underwent laparotomy drainage, while 104 (9.1%) did not undergo any drainage measures. A total of 610 patients (53.2%) received definitive operation, 24 patients died within postoperative 30-day with mortality of 3.9% (24/610), 69 (11.3%) developed surgical site infection (SSI), and 24 (3.9%) had a relapse of fistula. The highest cure rate was achieved in ileocecal fistula (100%), followed by rectal fistula (96.2%, 128/133) and duodenal stump fistula (95.7%,44/46). The highest mortality was found in combined internal fistula (3/12) and no death in ileocecal fistula. Univariate prognostic analysis showed that primary diseases as Crohn′s disease (χ²=6.570, P=0.010) and appendicitis/appendiceal abscess (P=0.012), intestinal fistula combining with internal fistula (χ²=5.460, P=0.019), multiple ECF (χ²=7.135, P=0.008), esophagogastric / esophagojejunal anastomotic fistula (χ²=9.501, P=0.002), ECF at ileocecal junction (P=0.012), non-drainage/passive drainage before the diagnosis of intestinal fistula (χ²=9.688, P=0.008), non-drainage/passive drainage after the diagnosis of intestinal fistula (χ²=9.711, P=0.008), complicating with multiple organ dysfunction syndrome (MODS) (χ²=179.699, P<0.001), sepsis (χ²=211.851, P<0.001), hemorrhage (χ²=85.300, P<0.001), pulmonary infection (χ²=60.096, P<0.001), catheter-associated infection (χ²=10.617, P=0.001) and malnutrition (χ²=21.199, P<0.001) were associated with mortality. Multivariate prognostic analysis cofirmed that sepsis (OR=7.103, 95%CI:3.694-13.657, P<0.001), complicating with MODS (OR=5.018, 95%CI:2.170-11.604, P<0.001), and hemorrhage (OR=4.703, 95%CI: 2.300-9.618, P<0.001) were independent risk factors of the death for ECF patients. Meanwhile, active lavage and drainage after the definite ECF diagnosis was the protective factor (OR=0.223, 95%CI: 0.067-0.745, P=0.015). Conclusions The overall mortality of ECF is still high. Surgical operation is the most common cause of ECF. Complications e.g. sepsis, MODS, hemorrhage, and catheter-associated infection, are the main causes of death. Active lavage and drainage is important to improve the prognosis of ECF.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.
Humans ; Antibodies, Viral ; CD8-Positive T-Lymphocytes ; COVID-19/complications* ; COVID-19 Vaccines/immunology* ; HIV Infections/complications* ; Receptors, Immunologic ; SARS-CoV-2