[Expert consensus on the application of critical care ultrasonography in invasive procedures].

The evolution of critical care medicine is inextricably linked to the development of critical care procedures. These procedures not only facilitate diagnosis and treatment of critically ill patients, but also provide valuable insights into disease pathophysiology. While critical care interventions offer undeniable benefits, the potential for iatrogenic complications necessitates careful consideration. The recent surge in critical care ultrasound (US) utilization is a testament to its unique advantages: non-invasiveness, real-time bedside availability, direct visualization of internal structures, elimination of ionizing radiation exposure, repeatability, and relative ease of learning. Recognizing the need to optimize procedures and minimize complications, critical care utrasound study group of Beijing critical care ultrasound research assocition convened a panel of critical care experts to generate this consensus statement. This document serves as a guide for healthcare providers, aiming to ensure patient safety and best practices in critical care.

[Bioinformatics-based analysis of the effect of general Transcription Factor IIH on prognosis of Prostate cancer].

Objective: To investigate the genetic and expression characteristics of transcription factor IIH (TFIIH) in pre-initiationcomplex in prostate cancer (PCa) and its relationship with prostate cancer progression. Methods: Analyzing the expression characteristics and clinical signification of TFIIH subunits about 495 cases of PCa and 52 cases of adjacent cancer in The Cancer Genome Atlas-Prostate adenocarcinoma (TCGA-PRAD) database. PCa microarray chip was used to verify the correlation between the key factor General Transcription Factor IIH Subunit 4 (GTF2H4) in TFIIH and clinical features. Results: The 495 patients with PCa were (61.01±6.82) years old.The mRNA expression of ERCC3、GTF2H4 and MNAT1 were high in PCa tissues with GS≥8(P<0.05). The expression of GTF2H4 and MNAT1 were relevant to the pathological stages(P<0.05). High expression of GTF2H4 has higher biochemical recurrence (BCR) rate in PCa patients(HR=2.47, 95%CI:1.62-3.77, P<0.001), which has better predictive effect of BCR in PCa patients(The 3rd, 5th, and 7th year AUC all>0.7) than other subunits, and it has been verified in four additional databases. Single-factor Cox regression analysis showed that GTF2H4 were risk factors for BCR (HR=2.470, 95%CI:1.620-3.767, P<0.001) and GTF2H5 were protective factors(HR=0.506,95%CI: 0.336-0.762, P=0.001). The results of immunohistochemical staining showed that the protein expression of GTF2H4 was correlated with the clinical features of PCa patients.The differences of the above results were statistically significant. Conclusion: GTF2H4, the key factor of TFIIH, is highly expressed in PCa and indicates a poor prognosis.

[Recommendations for the treatment of severe coronavirus disease 2019 based on critical care ultrasound].

Severe patients with coronaviras disease 2019 (COVID-19) are characterized by persistent lung damage, causing respiratory failure, secondary circulatory changes and multiple organ dysfunction after virus invasion. Because of its dynamic, real-time, non-invasive, repeatable and other advantages, critical ultrasonography can be widely used in the diagnosis, assessment and guidance of treatment for severe patients. Based on the recommendations of critical care experts from all over the country who fight against the epidemic in Wuhan, this article summarizes the guidelines for the treatment of COVID-19 based on critical ultrasonography, hoping to provide help for the treatment of severe patients. The recommendations mainly cover the following aspects: (1) lung ultrasound in patients with COVID-19 is mainly manifested by thickened and irregular pleural lines, different types of B-lines, shred signs, and other consolidation like dynamic air bronchogram; (2) Echocardiography may show right heart dysfunction, diffuse cardiac function enhancement, stress cardiomyopathy, diffuse cardiac depression and other multiple abnormalities; (3) Critical ultrasonography helps with initiating early treatment in the suspect patient, screening confirmed patients after intensive care unit admission, early assessment of sudden critical events, rapid grading assessment and treatment based on it; (4) Critical ultrasonography helps to quickly screen for the etiology of respiratory failure in patients with COVID-19, make oxygen therapeutic strategy, guide the implementation of lung protective ventilation, graded management and precise off-ventilator; (5) Critical ultrasonography is helpful for assessing the circulatory status of patients with COVID-19, finding chronic cardiopulmonary diseases and guiding extracorporeal membrane oxygenation management; (6) Critical ultrasonography contributes to the management of organs besides based on cardiopulmonary oxygen transport; (7) Critical ultrasonography can help to improve the success of operation; (8) Critical ultrasonography can help to improve the safety and quality of nursing; (9) When performing critical ultrasonography for patients with COVID-19, it needs to implement three-level protection standard, pay attention to disinfect the machine and strictly obey the rules from nosocomial infection. (10) Telemedicine and artificial intelligence centered on critical ultrasonography may help to improve the efficiency of treatment for the patients with COVID-19. In the face of the global spread of the epidemic, all we can do is to share experience, build a defense line, We hope this recommendations can help COVID-19 patients therapy.

[Technical specification for clinical application of critical ultrasonography].

Critical ultrasonography(CUS) is different from the traditional diagnostic ultrasound, the examiner and interpreter of the image are critical care medicine physicians. The core content of CUS is to evaluate the pathophysiological changes of organs and systems and etiology changes. With the idea of critical care medicine as the soul, it can integrate the above information and clinical information, bedside real-time diagnosis and titration treatment, and evaluate the therapeutic effect so as to improve the outcome. CUS is a traditional technique which is applied as a new application method. The consensus of experts on critical ultrasonography in China released in 2016 put forward consensus suggestions on the concept, implementation and application of CUS. It should be further emphasized that the accurate and objective assessment and implementation of CUS requires the standardization of ultrasound image acquisition and the need to establish a CUS procedure. At the same time, the standardized training for CUS accepted by critical care medicine physicians requires the application of technical specifications, and the establishment of technical specifications is the basis for the quality control and continuous improvement of CUS. Chinese Critical Ultrasound Study Group and Critical Hemodynamic Therapy Collabration Group, based on the rich experience of clinical practice in critical care and research, combined with the essence of CUS, to learn the traditional ultrasonic essence, established the clinical application technical specifications of CUS, including in five parts: basic view and relevant indicators to obtain in CUS; basic norms for viscera organ assessment and special assessment; standardized processes and systematic inspection programs; examples of CUS applications; CUS training and the application of qualification certification. The establishment of applied technology standard is helpful for standardized training and clinical correct implementation. It is helpful for clinical evaluation and correct guidance treatment, and is also helpful for quality control and continuous improvement of CUS application.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo.

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER-) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER--breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER--breast cancer cells in vitro. Treatment of ER--breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER- breast cancer treatment.

Cloning, expression of Vibrio alginolyticus outer membrane protein-OmpU gene and its potential application as vaccine in crimson snapper, Lutjanus erythropterus Bloch.

The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein-OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi.

AIMS: The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates. METHODS AND RESULTS: Whole-cell proteins of V. harveyi, strain Li01 and Huang01, were first separated by isoelectric focusing, followed by 2D-PAGE, respectively. Immunogenic proteins were identified by Western blotting, using Epinephelus coioides antisera against V. harveyi strain Li01. Western blot analyses revealed 16 shared immunogenic protein spots in both strains. All of the immunogenic proteins were successfully identified and corresponded to 15 proteins. None of these proteins have been previously reported as immunogenic for V. harveyi. Of the 15 proteins, 11 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) and oligopeptide ATP-binding cassette (ABC) transporter (spot 3) were used as immunogens to immunize E. coioides for investigation of their protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. harveyi Li01 and Huang01. However, vaccination with oligopeptide ABC transporter induces low protective immune response in fish. CONCLUSIONS: Eleven novel specific antigens were found, and OmpN could potentially be used as vaccine candidate for the development of novel vaccine against V. harveyi. SIGNIFICANCE AND IMPACT OF THE STUDY: These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. harveyi, which helps to search for the protective antigens in future.

Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish.

AIMS: The purpose of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. METHODS AND RESULTS: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 degrees C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3.7 x 10(2) CFU ml(-1) (3.7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus-infected fish tissues effectively. CONCLUSIONS: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch).

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.

Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus).

AIMS: The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper. METHODS AND RESULTS: A 410 bp internal fragment of the tdh gene was amplified by touchdown PCR with designed primers. Then its unknown flanking sequences of the 5'- and 3'-ends were finally characterized by inverse PCR and nested PCR. Sequence analysis showed that the tdh gene contain 570 bp ORF which encoded 189 amino acids. The deduced amino acid sequence of the ORF was in significant homology with several Vibrio TDH. The product that the tdh gene expressed in E. coli was purified by Ni(2+)-IDA Sepharose affinity column. The activity of purified TDH was 4651 U mg(-1) protein by hide powder azure digestion. The lethal toxicity test showed that LD(50) values of the purified TDH were 5.68 and 8.34 microg TDH g(-1) body weight for mouse and crimson snapper, respectively. CONCLUSIONS: The complete ORF of tdh gene was obtained by touchdown PCR, inverse PCR and nested PCR. The ORF was perfectly expressed in E. coli. The activity and toxicity assays showed that the N-terminal signal peptide was essential to autocatalyse and fold correctly to obtain the activity and toxicity in the purified TDH. The Native-PAGE analysis showed that the activated tdh gene expressed in E. coli was a dimer with two identical subunits. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the expressed activated TDH can produce the toxicity protein determined on mouse and fish, which will lead to better understandings of the identifying virulence factor that could be considered as a candidate antigen for vaccine and a diagnostic tool for vibriosis. Its use as an immunizing antigen might prevent the ability of V. alginolyticus to infect the marine aquatic animals, as a complementary measure to tick control and appropriate management in countries affected by vibriosis.

Inhibitory effect of erythromycin on P-glycoprotein-mediated biliary excretion of doxorubicin in rats.

The macrolide antibiotic erythromycin has recently been shown to overcome the resistance to anticancer drugs that results from overexpression of P-glycoprotein. The present study, using erythromycin lactobionic acid as a model drug, investigated the inhibitory effects of erythromycin on the efflux of doxorubicin from P388/ADR cells expressing P-glycoprotein and on the biliary excretion mechanism of doxorubicin in rats, which is primarily mediated by P-glycoprotein. Erythromycin lactobionic acid was found to inhibit the efflux of doxorubicin (5 microM) from P388/ADR cells in a concentration-dependent manner. In rats receiving constant-rate infusion of doxorubicin (30 micrograms/min), both the biliary and renal clearance of this drug dramatically decreased and its plasma concentrations increased after an intravenous injection of erythromycin lactobionic acid (100 mg/kg as erythromycin). These results suggest that erythromycin competitively inhibits P-glycoprotein-mediated biliary and renal excretion of doxorubicin. The effect of erythromycin on the biliary secretion of doxorubicin was also analyzed quantitatively by the competitive inhibition model. The computer-estimated values of Vmax/Km, Km and Ki were 8.79 ml/minute, 0.82 microgram/ml and 0.41 microgram/ml, respectively. The findings of these experiments suggest that the inhibitory effect of erythromycin on the P-glycoprotein-mediated biliary excretion of doxorubicin is competitive and that combination chemotherapy of doxorubicin with erythromycin may induce toxicity as a result of increased plasma concentrations of doxorubicin.

[Comparative study of the character of cefazolin pharmacokinetics between Neijiang pig and human].

OBJECTIVE: To study the character of cefazolin (CEZ) pharmacokinetics on Neijiang pig and human. METHODS: The serum concentration of CEZ in 8 normal Chinese adult men, 9 of 8-month male Neijiang pigs and 5 of 4-month male Neijiang pigs were detected by high-performance liquid chromatography (HPLC). RESULTS: The pharmacokinetic parameters suggested that two-compartment model was found in all groups after intramuscular injection of CEZ. In normal men, 8-month pigs and 4-month pigs, the peak time (Tmax) was (58.8 +/- 13.0), (19.7 +/- 9.9) and (18.2 +/- 8.6) min respectively, T1/2 alpha was (42.3 +/- 19.7), (19.0 +/- 7.7) and (9.3 +/- 1.9) min, the peak concentration (Cmax) was (101.6 +/- 14.6), (28.7 +/- 9.0) and (23.5 +/- 4.6) mg/L; Vd was (0.096 +/- 0.016), (0.374 +/- 0.184) and (0.386 +/- 0.211) L/kg; T1/2ka was (22.5 +/- 6.8), (8.6 +/- 4.8) and (10.6 +/- 10.2) min; T1/2 beta was (117.3 +/- 8.6), (84.2 +/- 9.8) and (45.1 +/- 11.5) min; clean rate of plasma Cl was (0.8 +/- 0.1), (6.8 +/- 1.2) and (11.0 +/- 3.0) ml/kg.min; AUC was (21,803 +/- 4,145), (2,407 +/- 443) and (1,636 +/- 685) mg.min/L. CONCLUSION: It could conclude that the Neijiang pigs could eliminate CEZ effectively, but the absorption, distribution and elimination of CEZ in pigs were quicker than that of in human while the absorption from muscle in both pig groups were lower than that in human.

The establishment of rat hybridoma cell lines secreting McAb against strains of potato virus Y and analysis of its stability.

The rat splenocytes immunized with potato virus Y (PVYn) and ratmyeloma (IR983) were fused by PEG (M. W.1450). Three kinds of stable hybridoma cell lines secreting specific monoclonal antibodies (McAbs) were derived. One kind of the cell lines producing McAbs reacts to PVYn specifically. Another reacts to PVYo specifically. The third one reacts to both of the two strains. Tested by the methods of sandwich-ELISA and indirect-ELISA, all kinds of McAbs did not react to seven plant viruses: tobacco mosaic (TMV), cucumber mosaic (CMV), tobacco tech (TEV), alfalfa mosaic (AMV), turnip mosaic (TuMV), potato leaf roll (PLRV), potato virus X (PVX). The biological properties of the hybridoma cell lines and the McAbs were tested.

Establishment of hybridoma cell lines secreting MCAs against potato virus X and determination of their physical and chemical characteristics.

Splenocytes from BALB/c mouse immunized with potato virus X (PVX) and myeloma cells (SP/2) were fused. After cloning and three screening cycles, three hybridoma cell lines secreting strain-specific monoclonal antibodies (MCAs) against PVX were obtained. These cell lines were stable for 20 generations and after storage in frozen form (in liquid nitrogen) for one year. The chromosome numbers of the three hybridoma cell lines clustered in the 92-102 range. The MCAs all belonged to the IgG3 immunoglobulin subclass. The medium supernatant antibody titer detected by indirect ELISA was 1:320-1:640. The mouse ascites antibody titer was 1:102,400-1:204,800, which was more than 300 times the rabbit antiserum titer (1:320). The MCAs had a neutralizing effect on the antigen, with neutralization titer of 1:10(2). There were no apparent changes in antibody activity after repeated freezing/thawing cycles, ammonium sulfate precipitation, or freeze-drying.