Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

By reviewing the historical materia medica， medical books and modern literature， this paper has systematically sorted out and verified the name， origin， quality and other aspects of Ophicalcitum. After herbal textual research， it is shown that before the Qing dynasty， the mineral medicine was mostly recorded in the name of Huarushi， but now it is called Huaruishi， and there is another mixed name Baiyunshi. The light white spots described in the historical materia medica are consistent with the characteristics of marble with sparkling star-like luster， combined with the color like sulfur， color are green， black spots and other serpentine features， it is deduced that it is serpentine marble， consistent with the present-day Ophicalcitum， and Ophicalcitum in the Song dynasty has a high content of serpentine. The main producing areas are Henan， Shaanxi， Shanxi and Sichuan， Jiangsu， Zhejiang， Hebei and other places are also available. Successive generations of materia medica on the quality evaluation of Ophicalcitum is less， the modern to neat and firm in the texture， sandwiched with yellow-green mottled for the best. Ophicalcitum is acidic， astringent and neutral in nature， belonging to the liver meridian， with the efficacy of treatment of gold sores and blood flow， internal leakage of cataracts， dropping afterbirth， now describing its efficacy as removing blood stasis and stopping bleeding. In ancient times， the earliest processing method was burning， followed by calcination by sulphur， calcination， quenching with vinegar and other methods. In modern times， it has been simplified to open calcination， processing with vinegar and the addition of water quenching. The gravimetric method and ethylenediaminetetraacetic acid titration were used to detect the contents of CO₃^2- and CaCO₃ in Ophicalcitum， respectively， and it was found that the gap in CaCO₃ content among commercially available products was wide， and the content of CaCO₃ in sample S13 and sample S18 was the same， but their compositions were different， and according to the contents of CO₃^2- and CaCO₃， the dolomite and calcite contents could be calculated， of which the higher the calcite content the more obvious the sparkling star-like luster. Raman spectroscopy and X-ray diffraction（XRD） were used to detect the physical phase composition of the powder of the samples， and Raman spectroscopy was used for the rapid non-destructive testing of the striped part， which showed that Ophicalcitum was mainly composed of dolomite， calcite， serpentine， olivine and pyroxene， with serpentine dominanting the striped part. In summary， the 2020 edition of Chinese Pharmacopoeia stipulates that the content of CaCO₃ in Ophicalcitum is not less than 40%， which is difficult to control its quality， and it is suggested to increase the detection of CO₃^2- content. This study can provide a scientific basis for the traceability of Ophicalcitum and better guide the clinical medication and rational utilization of resources.

As a microbial therapy method, fecal microbiota transplantation (FMT) has attracted the attention of researchers in recent years. As one of the most direct and effective methods to improve gut microbiota, FMT achieves therapeutic benefits by transplanting functional gut microbiota from healthy human feces into the intestines of patients to reconstruct new gut microbiota. FMT has been proven to be an effective treatment for gastrointestinal diseases such as Clostridium difficile infection, irritable bowel syndrome, and inflammatory bowel disease. In addition, the clinical and basic research of FMT outside the gastrointestinal system is also emerging. It is worth noting that there is bidirectional communication between the gut microbial community and the central nervous system (CNS) through the gut-brain axis. Some gut bacteria can synthesize and release neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and dopamine. Imbalanced gut microbiota may interfere with the normal levels of these neurotransmitters, thereby affecting brain function. Gut microbiota can also produce metabolites that may cross the blood-brain barrier and affect CNS function. FMT may affect the occurrence and development of CNS and its related diseases by reshaping the gut microbiota of patients through a variety of pathways such as nerves, immunity, and metabolites. This article introduces the development of FMT and the research status of FMT in China, and reviews the basic and clinical research of FMT in neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease), neurotraumatic diseases (spinal cord injury, traumatic brain injury) and stroke from the characteristics of three types of nervous system diseases, the characteristics of intestinal flora, and the therapeutic effect and mechanism of fecal microbiota transplantation, summarize the common mechanism of fecal microbiota transplantation in the treatment of CNS diseases and the therapeutic targets. We found that the common mechanisms of FMT in the treatment of nervous system diseases may include the following 3 categories through summary and analysis. (1) Gut microbiota metabolites, such as SCFAs, TMAO and LPS. (2) Inflammatory factors and immune inflammatory pathways such as TLR-MyD88 and NF-κB. (3) Neurotransmitter 5-HT. In the process of reviewing the studies, we found the following problems. (1) In basic researches on the relationship between FMT and CNS diseases, there are relatively few studies involving the autonomic nervous system pathway. (2) Clinical trial studies have shown that FMT improves the severity of patients’ symptoms and may be a promising treatment for a variety of neurological diseases. (3) The improvement of clinical efficacy is closely related to the choice of donor, especially emphasizing that FMT from healthy and young donors may be the key to the improvement of neurological diseases. However, there are common challenges in current research on FMT, such as the scientific and rigorous design of FMT clinical trials, including whether antibiotics are used before transplantation or different antibiotics are used, as well as different FMT processes, different donors, different functional analysis methods of gut microbiota, and the duration of FMT effect. Besides, the safety of FMT should be better elucidated, especially weighing the relationship between the therapeutic benefits and potential risks of FMT carefully. It is worth mentioning that the clinical development of FMT even exceeds its basic research. Science and TIME rated FMT as one of the top 10 breakthroughs in the field of biomedicine in 2013. FMT therapy has great potential in the treatment of nervous system diseases, is expected to open up a new situation in the medical field, and may become an innovative weapon in the medical field.

Sanghuang, a famous ethnomedicine widely used in China, Japan, Korea and other countries for a long history, is produced from the dried fruiting bodies of the medical fungi belonging to Sanghuangporus. With abundant bioactive natural chemicals including polysaccharides, flavonoids, triterpenoids, and polyphenols, Sanghuang exhibits anticancer, antioxidant, blood glucose-and lipid-lowering, liver protecting, anti-inflammatory, antimicrobial, and gout symptom-relieving effects, thus demonstrating broad application and development prospects in the pharmaceutical and food fields. However, the sustainable development of Sanghuang resources is limited by the scarce stock of wild resources, the diverse original fungi of cultivated Sanghuang, the inconsistency of local standards of Sanghuang materials or products, and the lagging application of Sanghuangporus mycelia. In view of the shortages mentioned above, this article reviews the research progress in the current situation of Sanghuang resources, the chemical composition, bioactivities, and fermentation process regulation of Sanghuangporus strains, and the related biosynthesis regulating strategies for enhancing the production of bioactive metabolites. This review is expected to provide comprehensive information for the sustainable development and utilization of Sanghuang resources.
Fermentation ; Medicine, East Asian Traditional ; Basidiomycota

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

In order to strengthen the supervision of the use of drugs in hospitals，the Sichuan Academy of Medical Sciences· Sichuan Provincial People’s Hospital took the lead in compiling the Principles for the Rational Use of National Key Monitoring Drugs （the Second Batch） with a number of experts from multiple medical units in accordance with the Second Batch of National Key Monitoring Rational Drug Use List （hereinafter referred to as “the List”） issued by the National Health Commission. According to the method of the WHO Guidelines Development Manual， the writing team used the Delphi method to unify expert opinions by reading and summarizing the domestic and foreign literature evidence of related drugs， and applied the evaluation， formulation and evaluation method of recommendation grading （GRADE） to evaluate the quality of evidence formed， focusing on more than 30 drugs in the List about the evaluation of off-label indications of drugs， key points of rational drug use and key points of pharmaceutical monitoring. It aims to promote the scientific standardization and effective management of clinical medication， further improve the quality of medical services， reduce the risk of adverse drug reactions and drug abuse， promote rational drug use， and improve public health.