Single molecule fluorescence in situ hybridization (smFISH) is a method for imaging single mRNA molecule in fixed cell or tissue using oligonucleotide probes coupled with fluorophores. It can realize real-time study of interested transcripts by RNA localization and quantification. smFISH is widely suitable for many types of biological samples such as cell and tissue sections. It was invented in 1982 which opened up the application of visualizing single molecules. However, due to its shortcomings such as poor binding specificity, Raj et al. optimized this technique in 2008, using 48 independent probes that were separately coupled with fluorophores to locate transcripts. In contrast, methods using multiple labeled probes can distinguish false positive or false negative results due to a single probe misbinding or unbinding event. However, with the continuous application of the technique, it was found that the scheme still has many technical defects, such as low probe specificity, weak fluorescence intensity, low hybridization efficiency, and high background fluorescence. Since then, a series of derivative technologies have been developed. For example, HCR-FISH is a multi-fluorescence in situ hybridization method based on orthogonal amplification and hybridization chain reaction, which significantly improves the problem of weak signal. SeqFISH amplifies the signal and reduces nonspecific binding by continuously hybridizing the mRNA in the cell, imaging it, and stripping the probe in order to barcode RNA. MERFISH utilizes combination labeling, continuous imaging and other technologies to increase detection throughput, and uses binary barcodes to offset single-molecule labeling and detection errors, with more advanced built-in error correction functions to effectively improve the accuracy of results. ClampFISH uses biological orthogonal click chemistry to effectively lock the probe around the target and prevent the probe from disengaging in amplification microscopy. RNAscope amplifies its own signal while simultaneously suppressing the background by using novel probe design strategy and hybridization-based signal amplification system. Split-FISH uses splitting probes for signal enhancement to accurately detect single RNA molecule in complex tissue environments. AmpFISH achieves imaging of short RNA molecules by preparing long single-strand DNA concatemers through controlled rolling circle amplification. CircFISH uses two unique sets of probes (PC probes and PL probes) to distinguish between linear and circular RNAs. π-FISH rainbow enables simultaneous detection of DNA, RNA, and proteins at the single-molecule level with π-FISH target probes. HT-smFISH is more suitable for large or high throughput form of systematic experiments. With the development of technology, the subsequent data analysis process is particularly important. Different analysis software, such as dotdotdot and FISH-quant v2, also improve the process of smFISH. The excellent ability of smFISH to visualize single molecule of RNA makes that it is widely used in basic biological disciplines such as tumor biology, developmental biology, neurobiology, botany, virology. In this paper, we reviewed the basic principle of smFISH technology, its development process and improvement, limitations of smFISH technology and how to avoid them, its derivative technologies include HCR-FISH, SeqFISH, MERFISH, ClampFISH, RNAscope, Split-FISH, AmpFISH, CircFISH, π-FISH rainbow and HT-smFISH. The application progress of smFISH in different biological disciplines, such as developmental biology, tumor biology, neurobiology. Finally, the development prospect of smFISH technology is prospected.

Objective:This study aimed to investigate the correlation between the levels of serum amyloid A protein (SAA) and apolipoprotein A-Ⅰ (ApoA-Ⅰ) with the severity and prognosis of septic patients, in order to find new clinical prognostic markers for sepsis patients.Methods:This study prospectively included patients admitted to the intensive care unit of Northern Jiangsu People's Hospital from September 2021 to February 2022. Patients were diagnosed with sepsis according to the Sepsis-3 criteria and aged between 18 and 80 years old. Peripheral venous blood samples were collected at 0 h, 24 h, and 72 h after inclusion in the study, measured the levels of ApoA-Ⅰ and SAA, and the 72 h ΔSAA and 72 h ΔApoA-Ⅰwere calculated.. Patient demographics, laboratory parameters, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) scores, sequential organ failure assessment scores, etc., were recorded. Patients were divided into survival and death groups based on outcomes, and were divided into shock and non-shock groups based on the presence of shock. Logistic regression was used to combine ApoA-I and SAA to establish a new combined index. Receiver Operating Characteristic curve analysis was performed to evaluate the predictive value of SAA, ApoA-Ⅰ, 72 h ΔApoA-Ⅰ, 72 h ΔSAA and the combined SAA and ApoA-Ⅰ for the prognosis of sepsis patients.Results:A total of 108 patients were included in the analysis, with 48 cases in the non-septic shock group and 60 cases in the septic shock group; 77 cases in the survival group and 31 cases in the death group. There were statistically significant differences in SAA and ApoA-Ⅰ levels at each time point between the shock and non-shock groups (all P<0.05), as well as between the death and survival groups (all P<0.05). SAA levels at each time point were positively correlated with APACHEⅡ scores (all P<0.001), while ApoA-Ⅰ levels at each time point were negatively correlated with APACHEⅡ scores (all P<0.01). SAA levels could predict the risk of death in sepsis patients, with the highest area under curve (AUC) value at 24 h SAA (AUC=0.713, P=0.001), sensitivity was 65.3%, and specificity was 72.7% for predicting 28-day mortality in sepsis. ApoA-Ⅰ levels at each time point could also predict the risk of death in sepsis patients, with the highest AUC value at 72 h ApoA-Ⅰ (AUC=0.743, P<0.001), sensitivity was 69.4%, and specificity was 77.1% for predicting 28-day survival in sepsis. The combined detection of 24 h SAA and 72 h ApoA-Ⅰ increased the AUC value (AUC=0.758, P<0.05), but the Z test showed that the prediction of death risk in patients with sepsis was not significantly higher than that of a single index ( P>0.05). Conclusions:Serum levels of SAA and ApoA-Ⅰ could reflect the severity of sepsis in patients and serve as independent indicators for predicting the prognosis of sepsis patients. The overall diagnostic efficacy of the combined SAA and ApoA-Ⅰ was not significantly different from that of a single index.

Cancer-related fatigue (CRF) belongs to the category of "consumptive disease" in TCM, and its occurrence is based on "internal deficiency" of the body causing by the tumor. Its nature is intermingled deficiency and excess. Its pathogenesis is the deficiency of qi, blood, yin and yang and zang-fu viscera dysfunction caused by disorders of "rise and fall of middle qi" and kidney origin depletion. The theory of "treating overstrain syndrome with warming methods" originates from Huang Di Nei Jing, which proposes that warming methods are the basic methods of treating consumptive disease. Therefore, starting from the cause and pathogenesis of CRF, this article sorted out the theoretical origin of "treating overstrain syndrome with warming methods", and discussed the clinical application of warming methods for the treatment of CRF combining with modern clinical research, with the purpose to provide references for clinical practice.

Objective:To investigate the effect of the interaction between metabolic syndrome and smoking on the risk of subsequent cardiovascular events.Methods:Urban residents aged 40 and above in the Yunyan District of Guiyang City were selected from " Risk Evaluation of cAncers in Chinese diabeTic Individuals: A lONgitudinal(REACTION) Study". The baseline survey started in 2011 and general information including gender, age, medical history, lifestyle habits, and smoking status were collected. Additionally, biochemical indicators related to metabolic syndrome(MS) were measured. The study participants were then followed up, and the first cardiovascular events occurring after the initial survey were recorded. The average follow-up period was 10.07±1.49 years. The interaction between metabolic syndrome and smoking on subsequent cardiovascular events was analyzed using Cox proportional hazards models.Results:The study included a total of 7 275 individuals, among whom 639 experienced cardiovascular events. After adjusting for multiple variables, compared to non-smokers without metabolic syndrome(MS), smokers with MS showed a higher risk of cardiovascular events, with a hazard ratio( HR) of 6.54(95% CI 4.88, 8.78). This risk was higher than that of individuals with MS who never smoked [ HR 1.39(95% CI 1.11, 1.75)] and non-MS smokers [ HR 2.48(95% CI 1.77, 3.49)]. There was an additive interaction between MS and smoking on the occurrence of cardiovascular events, with a relative excess risk due to interaction(RERI) of 3.30(95% CI 1.89, 4.70), an attributable proportion(AP) of 0.55(95% CI 0.43, 0.59), and a synergy index(S) of 3.07(95% CI 1.94, 4.84). Furthermore, when stratifying the duration of smoking cessation, long-term quitters(≥8 years) showed a lower risk of cardiovascular events compared to current smokers, regardless of whether they had MS. The hazard ratios were 0.45(95% CI 0.26, 0.78) for individuals with MS and 0.42(95% CI 0.19, 0.95) for individuals without MS. Conclusions:There is an additive interaction between smoking and MS on the risk of cardiovascular events. The coexistence of both factors significantly increases the risk of cardiovascular events.

Objective:To study the characteristics of clinical, laboratory, imaging, genetic and differential diagnosis of McLeod syndrome.Methods:The clinical characteristics of 2 cases of McLeod syndrome confirmed by gene detection in Qilu Hospital (Qingdao) on June 27, 2018 and in Qilu Hospital of Shandong University on September 11, 2019 were analyzed retrospectively. And the characteristics of patients of McLeod syndrome reported in China were analyzed in combination with literature review.Results:Both of the 2 patients were adult male, aged 57 and 61 years, respectively, with a slowly progressive course, beginning with gradually involuntary movement of trunk and extremities, involving involuntary biting of the tongue and dysphagia. Two patients had mild cognitive impairment; one patient had emotional agitation. Imaging study showed atrophy of caput nuclei caudate. Neuroelectrophysiological examination of case 1 showed sensory axon neuropathy in both upper limbs with severe damage to the left ulnar nerve. Creatine kinase (CK) was mildly elevated in 2 patients. The peripheral blood smear of 1 patient showed increased acanthocytes, accounting for 13%, the other patient showed no increased acanthocyte. McLeod syndrome related gene was tested in the 2 patients, case 1 with deletion mutation of exon 2 of XK gene, and case 2 with hemizygotic mutation of XK gene c.898delC p.L300 *. Conclusions:The clinical manifestations of McLeod syndrome are various and the differential diagnosis is crucial. For elderly male with cephalic facial chorea, elevated CK level and neuromuscular diseases, the possibility of McLeod syndrome should be screened.

Abstract: Objective　To analyze the association between drug resistance and the risk of latent tuberculosis infection and disease among household contacts of patients with pulmonary tuberculosis, and to explore whether the compensatory mutation of drug-resistant Mycobacterium tuberculosis will enhance its pathogenicity or transmission ability. Methods　The English and Chinese databases, including PubMed, web of science, EMBASE, Cochrane library database, CNKI and Wanfang database, were searched by computer from the time of establishment of the database to January 2022. Cohort studies on the risk of infection and disease among household contacts of patients with drug-resistant and sensitive pulmonary tuberculosis were searched and screened according to the inclusion and exclusion criteria. The data were extracted and evaluated by NOS scale, using stata16.0 software meta-analysis to calculate the combined effect of tuberculosis infection and disease risk of family contacts, and carry out heterogeneity test, subgroup analysis and sensitivity analysis. Results　A total of 7 cohort studies involving 9653 TB index cases and 29, 734 house contacts were included. The results of meta-analysis showed that compared with drug-sensitive pulmonary tuberculosis patients, the risk of tuberculosis infection in house contacts of drug-resistant pulmonary tuberculosis patients was increased (OR=1.56, 95%CI=1.25-1.96, P<0.001), but there was no difference in the risk of incidence (RR=1.06, 95%CI=0.80-1.41, P=0.67>0.05). Subgroup analysis showed that the risk of latent tuberculosis infection in house contacts was affected by the study area, and the size of family contacts had an impact on the risk of TB . Sensitivity analysis showed that the results of meta-analysis were robust. Conclusion　Compared with drug sensitive TB patients, household contacts with drug-resistant TB patients had a higher risk of tuberculosis, but there was no difference in the risk of TB among the two groups.

OBJECTIVE: To understand the current state and problem of screening and management of chronic kidney disease (CKD) in the community, and to explore the improving strategies. METHODS: We established a community-CKD integrated data science platform based on medical information from 79 community health centers, in Xicheng District, Beijing. Patients who referred to 79 community health centers from 21 June 2015 to 20 November 2021 were retrospectively included in this study using the CKD data platform. The monitoring of the indicator of kidney injury, risk factor control, medicine use and device configuration in community were assessed in the study. RESULTS: In the study, 70.6% of the population were identified with high risk of CKD in the total 374 498 individuals who referred to the community health centers. Hypertension (62.3%), coronary heart disease (43.3%) and diabetes (30.4%) were the most common risk factors in high-risk CKD population. Only 17.2% of the patients with high risk of CKD were screened for kidney injury including at least one serum creatine (Scr) or albuminuria test, among which 10 992 (24.2%) individuals were defined as CKD. 22.7% (11 338/49 908) of the total patients with kidney screening in community were defined as CKD, of whom, 42.6% and 46.1% were identified by estimated glomerular filtration rate (eGFR) < 60 mL/(min·1.73 m²) and abnormalities of urinary proteins, respectively. The overall CKD detection rate in the community was 5.2% (19 299/374 498), and the miss-diagnosis rate of CKD was 38.1%. Of the 79 community health centers, 13 (16.5%) were equipped with ACR testing device, and eGFR was reported directly in 66 (83.5%) centers. Altogether 60.3% and 99.7% of the community CKD patients achieved glucose control and blood pressure control, respectively, and 59.3% of the CKD patients who had proteinuria was treated with renin-angiotensin-aldosterone system (RAAS) inhibitors. CONCLUSION High-risk CKD population account for a substantial proportion of patients who refer to the community. Early screening, prevention and management of CKD in the community are of great importance to improve the prognosis and decrease the burden of CKD. It's essential to establish a screening and monitoring system, strengthen standardized management and clinician training for improving the ability of CKD management in the community.
Albuminuria/epidemiology* ; Blood Glucose ; Creatine ; Glomerular Filtration Rate ; Humans ; Renal Insufficiency, Chronic/therapy* ; Retrospective Studies

Background/Aims: Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. We aimed to investigate the usefulness of a key biomarker, lipocalin-2 (LCN2), for the detection of NASH progression. Methods: A mouse NASH model was established using a high-fat diet and a high-sugar drinking water. Gene expression profile of the NASH model was analyzed using RNA sequencing. Moreover, 360 NAFLD patients (steatosis, 83; NASH, 277), 40 healthy individuals, and 87 patients with alcoholic fatty liver disease were recruited. Results: Inflammatory infiltration, focal necrosis in the leaflets, steatosis, and fibrosis were documented in the mouse liver. In total, 504 genes were differentially expressed in the livers of NASH mice, and showed significant functional enrichment in the inflammation-related category. Upregulated liver LCN2 was found to be significantly interactive with various interleukins and toll-like receptors. Serum LCN2 levels were significantly increased in NAFLD patients. Serum LCN2 levels were correlated with steatosis, intralobular inflammation, semiquantitative fibrosis score, and nonalcoholic fatty liver disease activity score. The area under the curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis. Conclusions LCN2 might be involved in the transition from NAFL to NASH by mediating inflammation. Serum LCN2 levels might be a novel biomarker for the diagnosis of NASH.

Polyphyllin I (PPI) purified from Polyphyllarhizomes displays puissant cytotoxicity in many kinds of cancers. Several researches investigated its anti-cancer activity. But novel mechanisms are still worth investigation. This study aimed to explore PPI-induced endoplasmic reticulum (ER) stress as well as the underlying mechanism in non-small cell lung cancer (NSCLC). Cell viability or colony-forming was detected by MTT or crystal violet respectively. Cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by flow cytometry. Gene and protein levels were evaluated by qRT-PCR and immunoblotting respectively. Protein interaction was determined by immunoprecipitation or immunofluorescence assay. Gene overexpression or silencing was carried out by transient transfection with plasmids or small interfering RNAs. The Cancer Genome Atlas (TCGA) database was used for Gene Set Enrichment Analysis (GSEA), survival analysis, gene expression statistics or pathway enrichment assay. PPI inhibited the propagation of NSCLC cells, increased non-viable apoptotic cells, arrested cell cycle at G2/M phase, induced ROS levels but failed to decrease mitochondrial membrane potential. High levels of GRP78 indicates poor prognosis in NSCLC patients. PPI selectively suppressed unfolded protein response (UPR)-induced GRP78 expression, subsequently protected CHOP from GRP78-mediated ubiquitination and degradation. We demonstrated that the natural product PPI, obtained from traditional herbal medicine, deserves for further study as a valuable candidate for lead compound in the chemotherapy of NSCLC.

Background/Aims: Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. We aimed to investigate the usefulness of a key biomarker, lipocalin-2 (LCN2), for the detection of NASH progression. Methods: A mouse NASH model was established using a high-fat diet and a high-sugar drinking water. Gene expression profile of the NASH model was analyzed using RNA sequencing. Moreover, 360 NAFLD patients (steatosis, 83; NASH, 277), 40 healthy individuals, and 87 patients with alcoholic fatty liver disease were recruited. Results: Inflammatory infiltration, focal necrosis in the leaflets, steatosis, and fibrosis were documented in the mouse liver. In total, 504 genes were differentially expressed in the livers of NASH mice, and showed significant functional enrichment in the inflammation-related category. Upregulated liver LCN2 was found to be significantly interactive with various interleukins and toll-like receptors. Serum LCN2 levels were significantly increased in NAFLD patients. Serum LCN2 levels were correlated with steatosis, intralobular inflammation, semiquantitative fibrosis score, and nonalcoholic fatty liver disease activity score. The area under the curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis. Conclusions LCN2 might be involved in the transition from NAFL to NASH by mediating inflammation. Serum LCN2 levels might be a novel biomarker for the diagnosis of NASH.