[Detection of drug resistance genes of mycobacterium tuberculosis by rolling circle amplification technique with multicolor fluorescent probes].

The aim of this study was to construct a simple, rapid and ultra-sensitive optical biosensing technique based on rolling circle amplification (RCA), and to apply it to multiple detection of drug-resistant genes of mycobacterium tuberculosis. The common mutation sites of isoniazid, rifampicin and streptomycin resistance genes are katG315 (AGC➝ACC), rpoB531 (CAC➝TAC) and rpsL43 (AAG➝AGG). For these three gene sites, from February 2020 to May 2021, in the Department of Laboratory Medicine of the First Affiliated Hospital of Army Military Medical University, the padlock probe (PLP), primers and capture probes were designed. And a solid-phase RCA constant temperature amplification reaction system based on magnetic beads was constructed and the experimental parameters were optimized. The RCA products were accurately captured by the multicolor fluorescent probes (Cy3/Cy5/ROX), and the single-tube multiple detection of three mutation genes was realized. The sensitivity, specificity and linear range of this method were further verified. The results showed that the response range of katG315 in the same reaction system ranged from 1.0 pmol/L to 0.1 nmol/L. The response range of rpoB531 and rpsL43 ranged from 1.0 pmol/L to 50.0 pmol/L and 1.0 pmol/L to 20.0 pmol/L, and the method had good specificity and sensitivity, and could accurately identify single base mutations in mixed targets, with the minimum detection limit as low as 1.0 pmol/L. The recoveries of simulated serum samples were 95.0%-105.2%. In conclusion, the constant temperature amplification multiple detection method constructed in this study can quickly realize the single-tube multiple detection of three drug resistance mutation sites. This technology is low-cost, simple and rapid, and does not rely on large equipment, providing a new analysis method for pathogen drug resistance gene detection.

[Characteristic analysis of patients with type 2 diabetes mellitus combined with advanced stage chronic liver disease: a community population-based cross-sectional study in Heping District, Shenyang City (CHESS-LN 2101)].

Objective: To investigate the prevalence and independent risk factors of non-alcoholic fatty liver disease (NAFLD) and advanced chronic liver disease among the type 2 diabetes mellitus (T2DM) population in the Shenyang community, so as to provide evidence for the prevention and control of T2DM combined with NAFLD. Methods: This cross-sectional study was conducted in July 2021. 644 T2DM cases from 13 communities in Heping District, Shenyang City were selected. All the surveyed subjects underwent physical examination (measurements of height, body mass index, neck circumference, waist circumference, abdominal circumference, hip circumference, and blood pressure), infection screening (excluding hepatitis B and C, AIDS, and syphilis), random fingertip blood glucose, controlled attenuation parameter (CAP), and liver stiffness measurement (LSM). The study subjects were divided into the non-advanced chronic liver disease group and the advanced chronic liver disease group according to whether the LSM value was greater than 10 kPa. Cirrhotic portal hypertension development was indicated in patients with LSM ≥ 15 kPa. The comparison of multiple mean values among the sample groups was performed by analysis of variance when the normal distribution was met. Results: In the T2DM community population, there were 401 cases (62.27%) combined with NAFLD, 63 cases (9.78%) combined with advanced chronic liver disease, and 14 cases (2.17%) combined with portal hypertension. There were 581 cases in the non-advanced chronic liver disease group and 63 cases (9.78%) in the advanced chronic liver disease group (LSM ≥10 kPa), including 49 cases (7.61%) with 10 kPa≤LSM<15 kPa, 11 cases (1.71%) with 15 kPa ≤LSM<25 kPa, and 3 cases (0.47%) with LSM ≥ 25 kPa. Age, body mass, body mass index, neck circumference, waist circumference, hip circumference, waist-to-height ratio, systolic blood pressure, and CAP were all statistically different between the non-advanced chronic liver disease group and the advanced chronic liver disease group (F=-1.983,-2.598,-4.091,-2.062,-3.909, -4.581,-4.295,-2.474, and -5.191, respectively; P<0.05). There was a statistically significant difference in terms of whether or not there was combined cerebrovascular disease (2=4.632, P=0.031); however, there were no statistically significant differences in terms of lifestyle, diabetes complications, and other complications (P>0.05). Conclusion: Patients with T2DM have a higher prevalence of NAFLD (62.27%) than those with advanced chronic liver disease (9.78%). 2.17% of T2DM cases in the community may not have had early diagnosis and early intervention, and they might have been combined with cirrhotic portal hypertension. So, the management of these patients should be strengthened.

[Construction of microRNA-21 terahertz metamaterial sensing method based on duplex-specific nuclease triggered rolling circle amplification].

Objective: To construct of a terahertz metamaterial sensing method for detection of microRNA-21 (miRNA-21) based on duplex-specific nuclease triggered rolling circle amplification. Methods: First, a THz metamaterial sensing method was constructed, and verified by polyacrylamide gel electrophoresis and zeta potential; after optimizing the detection conditions of the sensor, different concentrations of miRNA-21 and other different miRNAs were detected. And this biosensor was compared with other miRNA detection methods; finally, the recovery rate of the biosensor was evaluated. Results: Under the optimal experimental conditions, through the dual signal amplification strategy of duplex-specific nuclease (DSN) cycle recognition and rolling circle amplification (RCA), the THz metamaterial sensor has a response range of 10 fmol/L to 10 nmol/L to the target miRNA-21, with a detection limit of 8.49 fmol/L. And the biosensor has good specificity with the ability to recognize the target miRNA-21 from a variety of microRNAs. And the experiment with recovery rate from 94.33% to 115.33% has been further verified in commercial human serum samples. Conclusion: The terahertz biosensor can achieve highly sensitive and specific detection of the target miRNA-21, which proves the potential for label-free diagnosis and early warning of miRNA-related diseases.

Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis.

OBJECTIVE: To downregulate the expression of leptin receptor functional isoform (Ob-Rb) on chondrocytes using lentiviral vector-mediated short-hairpin RNA (LV-shRNA) and to determine its effects on cartilage degeneration. METHOD: In vitro, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to select an optimal Ob-Rb LV-shRNA (LV-shRNA3) and to determine its effects on nine OA-related mediators in cultured rat chondrocytes. In vivo, an OA model was surgically induced in the right knees of rats, and LV-shRNA3, lentiviral vector-mediated non-targeting control sequence (LV-NTC) or phosphate buffered saline was injected into the joints. Osteoarthritis Research Society International (OARSI) scoring was performed to assess cartilage degeneration, and immunohistochemistry was performed to evaluate OA-related mediator expression in the above groups. RESULTS: Ob-Rb expression was significantly downregulated by LV-shRNA3 in cultured chondrocytes. In conjunction with Ob-Rb downregulation, the expression levels of pro-inflammatory mediators (TNF-α, IL-1ß and IL-6) and catabolic mediators (ADAMTS-5, MMP-9, NOS-2 and COX-2) were also significantly decreased, and the expression levels of anabolic type II collagen were significantly increased. The in vivo study results showed that OARSI scores were significantly decreased by LV-shRNA3. Immunohistochemistry analysis demonstrated that Ob-Rb expression levels on chondrocytes were significantly downregulated by LV-shRNA3. In conjunction with Ob-Rb downregulation, ADAMTS-5 and MMP-9 expression levels were also significantly decreased, and type II collagen expression levels were increased. CONCLUSION: These results indicate that LV-shRNA3-mediated Ob-Rb downregulation on chondrocytes inhibits cartilage degeneration in a rat model of OA, suggesting that Ob-Rb may be a novel target in the treatment of OA.

Comparison of cryobiopsy and forceps biopsy for the diagnosis of mediastinal lesions: A randomised clinical trial.

INTRODUCTION: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is the standard approach for lung cancer staging. However, its diagnostic utility for other mediastinal diseases might be hampered by the limited tissue retrieved. Recent evidence suggests the novel sampling strategies of forceps biopsy and cryobiopsy as auxiliary techniques to EBUS-TBNA, considering their capacity for larger diagnostic samples. METHODS: This study determined the added value of forceps biopsy and cryobiopsy for the diagnosis of mediastinal diseases. Consecutive patients with mediastinal lesions of 1 cm or more in the short axis were enrolled. Following completion of needle aspiration, three forceps biopsies and one cryobiopsy were performed in a randomised pattern. Primary endpoints included diagnostic yield defined as the percentage of patients for whom mediastinal biopsy led to a definite diagnosis, and procedure-related complications. RESULTS: In total, 155 patients were recruited and randomly assigned. Supplementing EBUS-TBNA with either forceps biopsy or cryobiopsy increased diagnostic yield, with no significant difference between EBUS-TBNA plus forceps biopsy and EBUS-TBNA plus cryobiopsy (85.7 % versus 91.6 %, P = 0.106). Yet, samples obtained by additional cryobiopsies were more qualified for lung cancer molecular testing than those from forceps biopsies (100.0 % versus 89.5 %, P = 0.036). When compared directly, the overall diagnostic yield of cryobiopsy was superior to forceps biopsy (85.7 % versus 70.8 %, P = 0.001). Cryobiopsies produced greater samples in shorter procedural time than forceps biopsies. Two (1.3 %) cases of postprocedural pneumothorax were detected. CONCLUSIONS: Transbronchial mediastinal cryobiopsy might be a promising complementary tool to supplement traditional needle biopsy for increased diagnostic yield and tissue harvesting. TRIAL REGISTRATION: ChiCTR2000030373.

Ingenious floral structure drives explosive pollination in Hydrilla verticillata (Hydrocharitaceae).

In explosive pollination, many structures and mechanisms have evolved to achieve high-speed stamen movement. The male flower of the submerged plant Hydrilla verticillata is reported to be able to release pollen explosively some time after leaving the mother plant time, but the mechanism of stamen movement and the related functional structure in this species are unclear. In this study, we observed the male flower structure and pollen dispersal process of H. verticillata. We analysed the stamen movements during the pollen dispersal process and conducted several controlled experiments to study the process of storage and release of elastic potential energy in explosive pollination. When the male flower of H. verticillata is bound to the united bracts, the sepals accumulate elastic potential energy through the expansion of basal extensor cells. After the male flower is liberated from the mother plant, the stamens unfold rapidly with the sepals under adhesion and transfer the elastic potential energy to the filament in seconds. Once stamens unfold to a critical angle, at which the elasticity of the filament just exceeds the adhesion between sepals and anthers, the stamens automatically rebound and release pollen in milliseconds. These results reveal that Catapult-like stamens, spoon-shaped sepals and enclosed united bracts in the spathe together constitute the functional structure in rapid stamen movement of H. verticillata. They ensure that the pollen can be released on the water surface, and thus adapt successfully to the pollen-epihydrophilous pollination.

Chondrosarcoma in a paediatric population: a study of 247 cases.

PURPOSE: The aims of present study are to clarify the follow questions: 1) what constitutes paediatric chondrosarcoma?; 2) what are the effects of the demographic and tumour characteristics on survival in patients with paediatric chondrosarcoma?; 3) which prognostic factors of paediatric chondrosarcoma differ from those of the adult population, which have been reported previously? METHODS: Paediatric patients who were diagnosed with chondrosarcoma were searched for using the case listing session protocol of the National Cancer Institute's Surveillance, Epidemiology, and End Results 18 databases (1973 to 2014). The extracted demographic information includes: age, race, gender, year of diagnosis, tumour sites, tumour histological subtype, grade, stage and treatment. RESULTS: A total of 247 paediatric chondrosarcoma patients were extracted and included in our present study. We find that the paediatric patients have significantly better survival rates than the adult patients. The year of diagnosis, tumour sites, tumour histological subtype, grade, stage and surgery received are independent prognostic factors for the survival rate of paediatric chondrosarcoma patients, but race, gender and age are not. CONCLUSION: The paediatric chondrosarcoma patients have better survival rates than the adults. Paediatric patients with a diagnosis at an early age, tumour site at the vertebral column and pelvis/sacrococcyx, myxoid variants, high grade, distant stage and who did not have surgery have a poorer prognosis than patients with a diagnosis at a later age, tumour site at limbs, head and base, chondrosarcoma not otherwise specified, lower grade, localized stage and who received surgery. LEVEL OF EVIDENCE: II -Prognostic Study.

Monitoring and control of infection in patients after renal transplantation.

Infection is a leading cause of death of renal transplantation and how to control it is still an urgent problem to be solved. Bacteria culture, computerized modified McGuckin's monitoring methods, OKT monoclonal antibody technique and radioimmunoassay (RIA) were used on studying the cause, monitoring and prevention of the infection. Results showed that 22 out of 24 cases (91.7%) were colonized by Gram-negative bacilli in pharynx and groin region. Of them, 15 were infected with the same bacteria in wound, urine, blood and lung. Six out of 7 cases with serious infection were colonized by Gram-negative bacilli on the surface of the whole body. The computerized modified McGuckin's method predicted and analyzed the infection outbreak much more fast and accurate, with a rate of over 80%. Helper/suppressor lymphocyte ratio of 1.08 was taken as a threshold value for the diagnosis of infection. The results suggested that the change of skin flora is closely related with the infection. Monitoring the alteration of skin flora is an effective method for the prevention and control of severe infection. The pharynx and groin regions of the patients are stable reservoirs of Gram-negative bacilli which may cause infection easily. Computerized quantitative analysis may increase the efficiency and accuracy of infection surveillance. In order to preserve the immune system as much as possible and prevent rejection of transplant graft, proper use of immunosuppressive drugs and regulation of the immune system of the patients are the key for preventing infection.

[Cytological features of small cell lung cancer--relation between nuclear chromatin change and chemotherapeutic effect].

This paper describes the relation between the cytologic features, change of chromatin and effect of chemotherapy in 75 cases of small cell lung cancer. Basing on the microscopic changes of the chromatin in the cancer cells, 4 types were observed: (1) chromatin evenly distributed with fine granules (2) chromatin evenly distributed with rough granules (3) chromatin sparsely distributed with rough granules (4) ink spot-like nuclei. Type 1 was found in 75.7% of the responders to chemotherapy while Type 3 was frequently observed in 69.2% of the non-responders. The results suggest that the changes of nuclear chromatin be used as an index to predict the response to chemotherapy, conforming to Takeshi's observation. Yet, in this series, the exfoliative cells were obtained from the sputum rather than Takeshi's brush during bronchoscopy. The authors believe that the changes of chromatin may be observed clearly if the sputum is fresh, smeared promptly and stained in the standard way.

[Cataract and implantation in the vitrectomized eyes]. / Cataracte et implantation sur oeil vitrectomisé.

Cataract is a frequent complication of vitreous surgery after some months or years. In these cases cataract extraction is somewhat difficult. We performed cataract surgery with implantation in 38 eyes from 37 patients between 1986 and 1991. Twenty patients underwent vitreous or vitreoretinal surgery with or without internal tamponade by gas, 17 underwent silicone oil tamponade and silicone oil removal, prior to cataract surgery. Timing of surgery, number of surgical procedures and complications previous to cataract surgery are detailed in the paper. Cataract extraction was extracapsular in 7 cases, intracapsular in 6 cases; phako was performed in 24 cases and phacophagy in one case. A posterior IOL was inserted in 31 cases, an anterior chamber IOL in 7 cases. Anatomical and visual results were satisfying. Visual acuity improved postoperatively in 29 cases (up to 20/20 in 4 cases), was unchanged in 6 cases, decreased in 3 cases. Among these, loss of vision occurred in 5 cases due to retinal detachment or redetachment. For anatomical reasons, lack of vitreous support and the frequent zonular weakness of these eyes, account for surgical difficulties. Thus, phakoemulsification appears the best way to manage cataract in vitrectomized eyes.

[Relationship between gut origin bacteria and wound infection after thermal injury].

The pUC19 plasmid vector trace with restriction map analysis and fluorescence labelling bacteria method were applied to study the relationship between the gut origin bacteria and wound infection. According to the characteristic of pUC19 plasmid, a special animal model was designed. 110 Wistar rats received 30% TBSA full thickness burns. On hours 6, 12, 24, 48 and day 12 postburn, injured animal were killed. Subeschar tissue homogenates were examined under fluorescence microscope, and bacterial culture, isolation of plasmids and restriction map analysis were also carried out. The results show that during early stage of burns, 32.5% of fluorescence labelling bacteria and 10.81% of pUC19 plasmid vectors could be detected from the subeschar specimens. 12 day postburn, the detectable rage of pUC19 plasmid vector increased to 62.5%. Beside the factor of early colonization, the contaminative route form gut perineum and then wounds should be considered.

Age-dependence of the chronotropic response to noradrenaline, acetylcholine and transmural stimulation in isolated rabbit atria.

The chonotropic response to noradrenaline, tyramine, acetylcholine and transmural electrical stimulation was compared in atria isolated from rabbits at different stages of developmental after birth (day 2 to day 210). Pacemaker rates understeady state conditions were related inversely to days after birth; the rate in atria from rabbits at day 2 was significantly greater than that at days 10-210. The rate of neonatal rabbit atria was not significantly reduced by propranolol and the positive chontropic response to noradrenaline was not significantly reduced by propranolol and the positive chronotropic response to noradrenaline was not significantly different in atria from ages of rabbits as far as threshold concentrations for inducing tachycardia and ED50's were concerned. The maximum rate induced by noradrenaline was higher in neonatal rabbit atria than in adult rabbit atria. The effect of tyramine was approx. the same regardless of age. Increase in the pacemaker rate induced by transmural neural stimulation varied directly with age. The negative chronotropic effect of acetylcholine was greater in neonatal than in adult rabbit atria; the ED50 in the former was significantly less than in the latter. Bradycardia induced by transmural stimulation of intracardiac cholinergic nerves was related directly to age. Tachycardia in the neonatal rabbit atria may be due to electrogenic characteristics of pacemaker cells which differ from those in adult rabbit atria. Our evidence strongly suggests that the adrenergic and cholinergic nerves innervating the S-A node develop at an early postnatal stage in the rabbit.

Serotonin antagonism in isolated canine cerebral arteries.

In helically-cut strips of canine cerebral arteries, the dose-response curve of serotonin was not influenced by 10(-7) M phentolamine but was slightly moved to the right and downward at 10(-6) M. The contractile response to serotonin was unaffected by cocaine (3 X 10(-6)M), atropine (10(-6) M) and propranolol (10(-6) M). The addition of lysergic acid diethylamide (LSD), ergotamine and methysergide caused a dose-dependent contraction. Treatment with LSD (10(-9) and 10(-8) M), ergotamine (10(-10) to 10(-8) M) and methysergide (10(-8) to 10(-6) M) shifted the dose-response curve of serotonin to the right and downward in a dose-dependent manner. The inhibitory effect of methysergide was reversed by washing, while that of ergotamine was not reversed. Apparent pA2 values of LSD, ergotamine and methysergide were 9.17, 9.63 and 7.92, respectively. Contractile responses to 20 mM K+ were not significantly influenced by these blocking agents even in the highest concentrations used. It may be concluded that an alpha-adrenergic mechanism is not involved in the genesis of serotonin-induced contractions and that serotonin acts directly on serotonergic receptors in canine cerebral arteries.