Performance of probe polymerization-conjunction-agarose gel electrophoresis in the rapid detection of KRAS gene mutation.

This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. The genotype of mutation sites was determined based on the occurrence of target bands in the corresponding lanes of the reaction tubes through polymerization-conjunction of the probes, probe purification and amplification, and agarose gel electrophoresis. Circulating DNA samples were obtained from the plasma of 72 patients with lung cancer, which were identified based on six mutation sites (G12S, G12R, G12C, G12D, G12A, and G12V) of codon 12 of the KRAS gene. The detection results were compared with direct sequencing data. The proposed detection method is characterized by simple operation, high specificity, and high sensitivity (2%). This method can detect the mutations of three samples at G12S, G12R, and G12A. In the direct sequencing spectra of these samples, the genotype could not be determined due to the lack of evident sequencing peaks that correspond to the basic group of mutations. In conclusion, a simple and rapid method was established based on probe polymerization-conjunction-agarose gel electrophoresis for detecting KRAS gene mutations. This method can be applied to the conventional mutation detection of inhomogeneous samples.

Molecular epidemiology and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolates in Xiangyang, China.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide. This study aimed to investigate the antimicrobial susceptibility and molecular epidemiological characteristics of MRSA strains in Xiangyang, China, during 2012-2014. METHODS: Eighty non-duplicate S. aureus isolates from clinical specimens were collected from four tertiary hospitals. MRSA strains were identified and were tested for antibacterial susceptibility. Staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing were performed to explore molecular characteristics. RESULTS: Among the 80 S. aureus isolates, 43 MRSA (53.8%) were detected. MRSA strains exhibited resistance against non-ß-lactam antibiotics to varying degrees. SCCmec type III was the predominant type (39/43; 90.7%), and the remainder were SCCmec type IVa (4/43; 9.3%). Thirteen MLST types were found, mainly ST239 (12/43; 27.9%) and ST59 (7/43; 16.3%). Fifteen spa types were found, mainly t437 (13/43; 30.2%) and t030 (6/43; 14.0%). PFGE grouped the 43 MRSA isolates into five types. SCCmecIII-ST239-t030/t632 and SCCmecIII-ST59/ST338-t437 were the dominant epidemic clones in this region. ST239-t030/t632/t037 was the epidemic clone with the most serious drug resistance. CONCLUSIONS: This region presented a high MRSA rate and the MRSA isolates demonstrated strong antimicrobial resistance. The existence of four strains of community-acquired MRSA (SCCmec type IVa) indicated the dissemination of MRSA strains from the community to hospitals. The epidemic situation and drug resistance of MRSA should be regularly monitored. Effective measures should be adopted to prevent and control the occurrence of infection in hospitals.

Detection of mutations by fill-in ligation reaction with enzyme-linked immunosorbent assay for rapid medical diagnosis.

Several approaches for parallel genotyping have been developed with increasingly available information on DNA variation. However, these methods require either complex laboratory procedures or expensive instrumentation. None of these procedures is readily performed in local clinical laboratories. In this study, we developed a flexible genotyping method involving fill-in ligation reaction with enzyme-linked immunosorbent assay successfully applied to detect important single-nucleotide polymorphisms (SNPs) for EGFR c.2573T > G (L858R), EGFR c.2582T > A (L861Q), and EGFR c.2155G > T (G719C). This assay exhibited excellent specificity, with a sensitivity as low as 0.5%. Eight out of 62 clinical samples were identified as heterozygotes for the SNP site of L858R, whereas only two samples were identified as heterozygotes by direct sequencing. The developed method enabled accurate identification of SNP in a simple and cost-effective manner adapted to routine analysis.

Application of video-assisted thoracic surgery in the standard operation for thoracic tumors.

OBJECTIVE: To evaluate the short-term outcomes of video-assisted thoracic surgery (VATS) for thoracic tumors. METHODS: The data of 1,790 consecutive patients were retrospectively reviewed. These patients underwent VATS pulmonary resections, VATS esophagectomies, and VATS resections of mediastinal tumors or biopsies at the Cancer Institute & Hospital, Chinese Academy of Medical Sciences between January 2009 and January 2012. RESULTS: There were 33 patients converted to open thoracotomy (OT, 1.84%). The overall morbidity and mortality rate was 2.79% (50/1790) and 0.28% (5/1790), respectively. The overall hospitalization and chest tube duration were shorter in the VATS lobectomy group (n=949) than in the open thoracotomy (OT) lobectomy group (n=753). There were no significant differences in morbidity rate, mortality rate and operation time between the two groups. In the esophageal cancer patients, no significant difference was found in the number of nodal dissection, chest tube duration, morbidity rate, mortality rate, and hospital length of stay between the VATS esophagectomy group (n=81) and open esophagectomy group (n=81). However, the operation time was longer in the VATS esophagectomy group. In the thymoma patients, there was no significant difference in the chest tube duration, morbidity rate, mortality rate, and hospital length of stay between the VATS thymectomy group (n=41) and open thymectomy group (n=41). However, the operation time was longer in the VATS group. The median tumor size in the VATS thymectomy group was comparable with that in the OT group. CONCLUSIONS: In early-stage (I/II) non-small cell lung cancer patients who underwent lobectomies, VATS is comparable with the OT approach with similar short-term outcomes. In patients with resectable esophageal cancer, VATS esophagectomy is comparable with OT esophagectomy with similar morbidity and mortality. VATS thymectomy for Masaoka stage I and II thymoma is feasible and safe, and tumor size is not contraindicated. Longer follow-ups are needed to determine the oncologic equivalency of VATS lobectomy, esophagectomy, and thymectomy for thymoma vs. OT.

[Comparison of the short-term outcomes of surgical treatment for non-small cell lung cancer via video assisted thoracoscopic surgery and open thoracotomy].

OBJECTIVE: To compare the short-term outcomes of surgical treatment for non-small cell lung cancer (NSCLC) by video-assisted thoracoscopic surgery (VATS) and open thoracotomy (OT). METHODS: Data of 737 consecutive NSCLC patients who underwent surgical treatment for non-small cell lung cancer by video-assisted thoracoscopic surgery and 630 patients who underwent pulmonary resection via open thoracotomy (as controls) in Cancer Institute & Hospital, Chinese Academy of Medical Sciences between January 2009 and August 2011 were retrospectively reviewed. The risk factors after lobectomy were also analyzed. RESULTS: In the 506 NSCLC patients who received VATS lobectomy, postoperative complications occurred in 13 patients (2.6%) and one patient died of acute respiratory distress syndrome (0.2%). In the 521 patients who received open thoracotomy (OT) lobectomy, postoperative complications occurred in 21 patients (4.0%) and one patient died of pulmonary infection (0.2%). There was no significant difference in the morbidity rate (P > 0.05) and mortality rate (P > 0.05) between the VATS group and OT group. In the 190 patients who received VATS wedge resections, postoperative complications occurred in 3 patients (1.6%). One hundred and nine patients received OT wedge resections. Postoperative complications occurred in 4 patients (3.7%). There were no significant differences for morbidity rate (P = 0.262) between these two groups, and there was no perioperative death in these two groups. Univariate and multivariate analyses demonstrated that age (OR = 1.047, 95%CI: 1.004 - 1.091), history of smoking (OR = 6.374, 95%CI: 2.588 - 15.695) and operation time (OR = 1.418, 95%CI: 1.075 - 1.871) were independent risk factors of postoperative complications. CONCLUSIONS: To compare with the NSCLC patients who should undergo lobectomy or wedge resection via open thoracotomy, a similar short-term outcome can be achieved via VATS approach.

[Application of video assisted thoracic surgery for chest tumors: experience of 144 cases in a single institution].

OBJECTIVE: To evaluate the indication and safety of video assisted thoracic surgery (VATS) for chest tumors. METHODS: Data of 144 consecutive patients receiving VATS between January and November 2009 in Cancer hospital Chinese Academy of Medical Sciences were retrospectively reviewed. RESULTS: There was no conversion to open thoracotomy. Overall morbidity rate was 2.08% (3/144) and mortality rate was 0.69% (1/144). There were no significant differences for operative time, number of nodal dissection, morbidity rate, mortality rate, overall hospitalization and postoperative length of stay between VATS lobectomy group and open thoracotomy (OT) lobectomy group. Chest tube duration was shorter in the VATS lobectomy group than OT lobectomy group and more early-stage lung cancer patients were found in VATS group. There were no significant differences for number of nodal dissection, chest tube duration, morbidity rate, mortality rate, and postoperative length of stay between VATS lung wedge resection group and OT lung wedge resection group. Operative time and overall hospitalization were shorter in the VATS wedge resection group than OT wedge resection group. CONCLUSION: Morbidity and mortality rate of VATS were acceptable. VATS lobectomy can be used as an alternative surgical technique for early-stage lung cancer. For lung wedge resection, VATS was superior than OT.

[Paragonimus skrjabini infection in animal reservoir hosts and questionnairing in residents at a village of Hubei Province].

Freshwater crabs (Sinopotamon denticulatum) were examined for metacercariae. Cats and dogs were also examined for Paragonimus infection. Questionnairing was carried out on health knowledge and behaviors among local residents in a village of Baokang County, Hubei Province. Results showed that the infection rate of Paragonimus skrjabini metacercariae in Sinopotamon denticulatum was 20.5% (46/214), with 15.6% (20/128) in a mining area and 30.2% (26/86) for the non-mining area respectively (chi2 = 6.5, P < 0.05). The prevalence in cats and dogs was 25.0% (6/24) and 17.6% (6/34) respectively (chi2 = 0.46, P > 0.05). Questionnairing showed that dogs and cats were with the habit of foraging and defecating at streams and children had the habits of eating raw or under-cooked crabs. The natural and ecological environments are in favor of the life cycle of P. skrjabini.

[In vitro effect of medicinal scorpion on the larvae of Ancylostoma caninum].

Five gram stool sample from dog infected with Ancylostoma caninum was smeared on filter paper for routine cultivation under 35 degrees C with moist condition for 24 hours. Hookworm larvae were separated and divided into 3 bottles through precipitation-washing method. Traditional Chinese medicine scorpion (2 ml), albendazole (10 mg) and normal saline (2 ml) (control) were added respectively into the 3 bottles, which were cultured for another 24 hours. The growth and development of the larvae were observed under microscope. In scorpion group, the larva body shrank, development stopped, and with an unclear internal structure. Treated with albendazole, the larvae became rigid and shriveled with rough body surface and vague internal structure. The results indicate that both medicinal scorpion and albendazole have considerable effect in inhibiting the development of hookworm larvae and the effect of albendazole is stronger.

[Solid medium-filter paper cultivation of Ancylostoma caninum larvae].

Consisted of beef extract (3 grams), peptone (10 grams), sodium chloride (5 grams), agar (20 grams) and distilled water, the solid medium was used with filter paper for cultivation of the larvae of Ancylostoma caninum. The method shows higher detection rate and simpler operation than the traditional test tube-filter paper culture technique.

Physiological modulation of intestinal motility by enteric dopaminergic neurons and the D2 receptor: analysis of dopamine receptor expression, location, development, and function in wild-type and knock-out mice.

Dopaminergic neurons are present in both plexuses of the murine bowel and are upregulated after extrinsic denervation but play unknown roles in enteric nervous system (ENS) physiology. Transcripts encoding dopamine (DA) receptors D1-D5 were analyzed by reverse transcription-PCR in stomach approximately duodenum approximately ileum approximately proximal > > distal colon. Dissected muscle and myenteric plexus contained transcripts encoding D1-D3 and D5, whereas mucosa contained D1 and D3-D5. D1-D5 expression began in fetal gut [embryonic day 10 (E10)], before the appearance of neurons (E12), and was sustained without developmental regulation through postnatal day 1. In situ hybridization revealed that subsets of submucosal and myenteric neurons contained mRNA encoding D2 or D3. Immunoblots confirmed that D1, D2, and D5 receptor proteins were present from stomach through distal colon. Subsets of submucosal and myenteric neurons were also D1, D2, or D3 immunoreactive. When double labeled by in situ hybridization, these neurons contained mRNA encoding the respective receptors. Total gastrointestinal transit time (TGTT) and colonic transit time (CTT) were measured in mice lacking D2, D3, or D2 plus D3. Both TGTT and CTT were decreased significantly (motility increased) in D2 and D2 plus D3, but not D3, knock-out animals. Mice lacking D2 and D2 plus D3 but not D3 were smaller than wild-type littermates, yet ate significantly more and had greater stool frequency, water content, and mass. Because motility is abnormal when D2 is absent, the net inhibitory DA effect on motility is physiologically significant. The early expression of DA receptors is also consistent with the possibility that DA affects ENS development.

Latent and lytic infection of isolated guinea pig enteric ganglia by varicella zoster virus.

Varicella zoster virus (VZV) has been demonstrated to infect guinea pig enteric neurons in vitro. Latent infection of isolated enteric neurons is established when the cultures predominantly consist of neurons and they are exposed to cell-free VZV. Neurons harboring latent infection survive for weeks in vitro and express mRNA encoding ORFs 4, 21, 29, 40, 62, and 63, but not 14(gC) or 68 (gE) (although DNA encoding the glycoproteins is present). The expressed proteins are the same as those that are also expressed in human sensory neurons harboring latent VZV. In addition to mRNA, the immunoreactivities of ORFs 4, 21, 29, 62, and 63 can be detected. ORF 62 and 29 proteins are cytoplasmic and not intranuclear. VZV does not preferentially infect and/or become latent in intrinsic enteric primary afferent neurons indicating that the virus is latent in these neurons. Lytic infection occurs when mixed cultures of neurons and non-neuronal cells of the bowel wall are exposed to cell-free VZV or when isolated enteric neurons are exposed to cell-associated VZV. When lytic infection occurs, enteric neurons die within 48 hr. Prior to their death, neurons express VZV glycoproteins, including gE and gB, and ORF 62 and 29 proteins are intranuclear. This new animal model should facilitate studies of VZV latency and the efficacy of therapies designed to prevent VZV infection, latency, and reactivation.