AIM: To construct a recombinant lentivirus RNAi vector carrying cytochrome C oxidase gene to obtain the titer of the lentiviral stock for investigation of the expression in the eukaryotic cell and the affection of the COX gene silencing in the eukaryotic cells. METHODS: According to the DNA of the cytochrome C oxidase gene, we designed and synthesized complementary single-strand DNA oligos, annealed the single-stranded oligos to generate a ds oligo, cloned the ds oligo into pENTR/U6 to obtain an entry clone; An LR recombination reaction was performed between the pENTR/U6 entry construct and pLenti6/BLOCK-iT-Dest to generate expression construct, the 293FT cell line was cotransfected with pLenti6/BLOCK-iT expression construct, and the viral packaging mix, viral supernatant was harvested to determine the titer. RESULTS: The DNA sequence of interest clone to the vector was constructd to generate an entry clone and an expression clone successfully, which were proved by sequence determination. A vector producing cell line 293FT was established, and the titer for transfection was obtained. Western blotting analysis demonstrated that COX shRNA expression construction could suppress the expression of MTCOX-I. CONCLUSION: A lentivirus RNAi vector containing cytochrome C oxidase gene was successfully constructed.

Purpose To analyze EGFR exon 18 ~21 gene mutations in lung cancer, and compare xTAG liquidchip technology and Sanger sequencing technology in clinical practice. Methods 1 139 tumor tissue samples from phaseⅠtoⅣlung cancer patients were randomly collected. DNA was extracted from the samples. EGFR gene mutation status in exon 18~21 was detected by xTAG liquidchip technology and Sanger sequencing technology respectively. Results The mutation status of EGFR was obtained by xTAG liquidchip technology in 1 134 patients, and 1 105 by Sanger sequencing technology, detection success rate was 99. 56% and 97. 01% respective-ly. The sensitivity and specificity of xTAG liquidchip technology Comparing with Sanger sequencing was 99. 59% and 94. 54%. Sever-al cases of multiple mutations were detected by both methods. All mutation types detected by two methods are fully consistent. Conclu-sions Comparing with Sanger sequencing technology, xTAG liquidchip technology, which is able to detect mutations of exon 18~21 simultaneously, is more convenient and efficient for EGFR gene mutation detection in lung cancer.

Objective: To analyze the characteristics of individuals positive for SARS-CoV-2 nucleic acid in a centralized isolation site for people entering China in Huzhou City of Zhejiang Province from December 18, 2021 to January 12, 2022, so as to provide insights into the prevention and control of overseas imported COVID-19. Methods: The basic characteristics, nucleic acid detection and epidemiological investigations were collected from individuals positive for SARS-CoV-2 nucleic acid in a centralized isolation site for people entering China from December 18, 2021 to January 12, 2022, and the temporal distribution, population distribution, source of importation, and virus typing were descriptively analyzed. Results : From December 18, 2021 to January 12, 2022, a total of 2 974 individuals in 19 flights were recorded in this centralized isolation site, and 33 cases were tested positive for SARS-CoV-2 nucleic acid, including 21 confirmed cases with common type, 9 confirmed cases with mild type, and 3 cases with asymptomatic infections. There were 11 cases with Omicron infections ( 33.33% ), 5 cases with Delta infections ( 15.15% ), and 17 cases with infection of unidentified types ( 51.52% ). The median interval ( interquartile range ) from the time of entry to the time of a positive test was 4.0 ( 7.0 ) days among all positive cases, 0 ( 4.0 ) day among cases with Omicron infections and 4.5 ( 8.5 ) days among cases with infections of Delta and unidentified types. The positive cases had a mean age of ( 36.97±8.58 ) years, and included 27 men (81.82%). There were 30 cases ( 90.91% ) receiving two and more doses of COVID-19 vaccines, and 7 cases ( 21.21% ) with a previous history of SARS-CoV-2 infections. There were 19 cases ( 57.58% ) from African countries, and 7 of 11 cases with Omicron infections were imported from African countries. Conclusion Omicron infection was predominant among individuals positive for SARS-CoV-2 nucleic acid in this centralized isolation site for people entering China from December 18, 2021 to January 12, 2022, with no severe cases detected, and most positive cases were imported from African countries.

OBJECTIVETo study the chemical constituents of Hemsleya chensnsis.

METHODVarious chromatographic techniques were used to separate and purify the constituents. The spectral data (MS, NMR) were obtained for structure elucidation.

RESULTEight known triterpenoid saponins were isolated from the root of H. chensnsis. Their structures were elucidated as dihydrocucurbitacin B (1), 25-O-acetyl-dihydrocucurbitacin F (2), cucurbitacin F (3), 3-O-(6'-ethyl ester) -beta-D-glucuropyranosyl oleanolic acid-28-O-alpha-L-arabinopyranoside (4), oleanolic acid-3-(6'-methyl ester) -beta-D-glucuropyranosyl (1-3) -alpha-L-arabinopyranoside (5), oleanolic acid-28-O-beta-D-glucopyranosyl-(1-6) -beta-D-glucopyranoside (6), 3-O-(6'-methyl ester) -beta-D-glucuropyranosyl oleanolic acid-28-O-beta-D-glucopyranosyl-(1-6) -beta-D-glucopyranoside (7), 3-O-(6'-methyl ester) -beta-D-glucuropyranosyl (1-2) -beta-D-glucopyranoside-oleanolic acid-28-O-beta-D-glucopyranoside (8).

CONCLUSIONAll compounds except for 2 were isolated from H. chensnsis for the first time.

Cucurbitaceae ; chemistry ; Magnetic Resonance Spectroscopy ; Medicine, Chinese Traditional ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Saponins ; chemistry ; isolation & purification ; Triterpenes ; chemistry ; isolation & purification

OBJECTIVETo evaluate the diagnostic value of serum miR-103 for breast cancer and its correlation to the clinicopathological features of the patients.

METHODSWe collected the serum samples and corresponding formalin-fixed paraffin-embedded (FFPE) surgical specimens from 50 breast cancer patients, using the serum samples from 50 healthy women as the control. The total RNA was extracted from the samples for quantitative analysis of miR-103 using real-time RT-PCR.

RESULTSThe serum levels of miR-103 expression were significantly higher in the cancer patients than in the healthy control group (P<0.01). In the cancer patients, high miR-103 expression was significantly correlated to advanced clinical stage (P<0.05) and lymph node metastasis (P<0.05). miR-103 showed a receiver operating characteristic (ROC) curve area of 84.3%, and a sensitivity of 84% and a specificity of 70% in discriminating breast cancer patients from the control subjects.

CONCLUSIONSerum miR-103 can serve as a potential diagnostic marker for breast cancer and provides useful information of the clinicopathological features of the patients.

Adult ; Aged ; Biomarkers, Tumor ; blood ; Breast Neoplasms ; diagnosis ; Case-Control Studies ; Female ; Humans ; MicroRNAs ; blood ; Middle Aged ; Sensitivity and Specificity

OBJECTIVETo investigate the loss of heterozygosity (LOH) on chromosomal arms 13q and 14q in nasopharyngeal carcinoma (NPC) using 21 microsatellite polymorphic markers and to study whether there is a correlation between LOH and clinicopathologic parameters and/or Epstein-Barr virus (EBV) infection in NPC.

METHODSSixty cases of NPC were studied using polymerase chain reaction based microsatellite analysis with genescan and genotyping techniques.

RESULTSLOH was detected on 13q in 78% of NPC tumors, high frequency LOH loci (more than 30%) clustered to 13q12.3-q14.3 and 13q32. On chromosome 14q, LOH was detected in 80% of NPC tumors; high frequency LOH loci clustered to 14q11-q13, 14q21-q24 and 14q32. High frequency LOH at 13q31-q32 correlated with a lower level of EBV infection; LOH on chromosome 14q was closely associated with poor differentiation of NPC tumor cells.

CONCLUSIONOur results suggest that in NPC, LOH on chromosome 13q and 14q are common genetic events, and putative tumor suppressor genes (TSG) residing in these regions may be involved in tumorigenesis.

Adult ; Aged ; Chromosomes, Human, Pair 13 ; genetics ; Chromosomes, Human, Pair 14 ; genetics ; DNA, Neoplasm ; genetics ; Female ; Gene Frequency ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Statistics as Topic

Objective: To learn the epidemiological characteristics of coronavirus disease 2019（COVID-19）in Huzhou,so as to provide reference for prevention and control of COVID-19. Methods: All the confirmed cases of COVID-19 in Huzhou,diagnosed according to the COVID-19 Diagnosis and Treatment Plan（Sixth Version Trial）and reported from January 25 to February 7,2020,were recruited. The process of diagnosis and treatment,clinical manifestation,exposure history and close contacts were collected to analyze the epidemiological characteristics. Results: On January 25,the first confirmed cases of COVID-19 in Huzhou was reported. By February 7,totally 10 confirmed cases were reported and no asymptomatic infection was found. They were all imported,including three Wuhan residents,two with a trip to Wuhan,three with a trip to Suizhou,one with a trip to Hangzhou and one with a trip to Thailand（two Wuhan passengers on the same flight）. The ratio of male to female cases was 1∶1. The median age was 32 years old. Seven cases were found when they went to a doctor by themselves,and three cases were found during the quarantine. The main clinical manifestations were fever,dry cough and fatigue. The median time from onset to diagnosis was 3 days. By March 3,all the cases were discharged,with median course of 24 days. There were 312 close contacts,and all of them were released after 14 days of quarantine. Conclusions To prevent imported cases from outside and stop spread inside taken by Huzhou government was proved to be effective. All the COVID-19 cases in Huzhou were imported,mostly from Wuhan. No local cases were reported.

Objective:To explore the effect of NOD-like receptor thermal protein 3 ( NLRP3) knockout in γ-aminobutyric acid (GABA)-ergic neurons in the hippocampal CA1 area on improving cognitive dysfunction in mice after traumatic brain injury (TBI). Methods:Forty-eight healthy male NLRP3 flox/flox mice weighing 25-28 g were randomly divided into 4 groups ( n=12): sham-operated+control virus group (SV group), sham-operated+ NLRP3 specific knockout group (SG group), TBI+control virus group (TV group), TBI+ NLRP3 specific knockout group (TG group). TBI in the TV and TG groups was established by free-fall method, while surgical procedures such as scalp incision and cranial window opening without impact were given to the SV and SG groups. Adenovirus was injected into the hippocampal CA1 area of SG and TG groups 21 d before TBI to induce NLRP3 specific knockout in GABA-ergic neurons in the hippocampal CA1 area; empty virus was injected into the CA1 area of SV and TV groups. Cognitive function was evaluated using novel object recognition test 30 and 31 d after TBI, and learning and memory functions were assessed using Morris water maze test 32-36 d after TBI. Field potentials in the hippocampal CA1 area were recorded during novel object recognition 31 d after TBI. After behavioral tests, these mice were sacrificed. Immunofluorescent staining was used to detect the fluorescent intensity of microtubule-associated protein2 (MAP2), glutamic acid decarboxylase 67 (GAD67), and postsynaptic density protein 95 (PSD95) in the hippocampal CA1 area, as well as percentage of pyroptosis-associated inflammatory factor interleukin-18 (IL-18)/GAD67 double-positive neurons in total GAD67 positive neurons. Results:Compared with the SV and SG groups, the TV and TG groups had decreased novel object recognition index, decreased number of platform crossings during the experimental period, increased escape latency on day 3 and day 4 of the training period in Morris water maze test, decreased θ and γ oscillation power in the hippocampal CA1 area during novel object recognition, decreased fluorescent intensity of MAP2, GAD67, and PSD95 in the hippocampal CA1 area, increased percentage of IL-18/GAD67 double-positive neurons, with significant differences ( P<0.05). Compared with the TV group, the TG group had increased novel object recognition index, increased number of platform crossings in Morris water maze test, decreased escape latency during the training period, increased θ and γ oscillation power in the hippocampal CA1 area during novel object recognition, increased fluorescence intensity of MAP2, GAD67, and PSD95 in the hippocampal CA1 area, decreased percentage of IL-18/GAD67 double-positive neurons, with significant differences ( P<0.05). Conclusion:Specific inhibition of NLRP3 expression in GABA-ergic neurons in the hippocampal CA1 area can improve cognitive dysfunction in mice after TBI, whose mechanism may be related to inhibited GABA-ergic neuronal pyroptosis in the hippocampal CA1 area.