ObjectiveTo investigate the molecular mechanisms by which Wenyang Jiedu granules （WYJD） alleviate influenza A virus （IAV）-induced pneumonia based on single-cell transcriptome sequencing. MethodsThirty female BALB/c mice were randomly divided into a blank control group （Control）， IAV group， and WYJD low-， medium-， and high-dose groups （WYJD-L， WYJD-M， WYJD-H； 2.925， 5.85， 11.7 g·kg^-1， n=6）. Except for the Control group， all other groups were intranasally inoculated with IAV subtype H1N1 （A/PR/8/34） to establish an infection model. Two hours after modeling， drug administration was initiated and continued for 5 consecutive days， with daily monitoring of body weight and general condition. On day 6， mice were sacrificed and samples were collected. Lung index was calculated， and histopathological examination of lung tissue was performed. Lung tissues from the Control， IAV， and WYJD-H groups were subjected to single-cell transcriptome sequencing （n=3）， focusing on type I alveolar epithelial cells （AT1） to analyze changes in gene expression and signaling pathways. Western blot was used to detect the expression changes of relevant proteins to validate the single-cell sequencing results. ResultsCompared with the Control group， the IAV group exhibited significantly decreased body weight （P<0.05） and significantly increased lung index （P<0.05）. Compared with the IAV group， all WYJD-treated groups exhibited significantly increased body weight （P<0.01） and significantly decreased lung index （P<0.01）. Single-cell sequencing analysis revealed that WYJD inhibited overactivation of interferon and inflammatory signaling pathways in AT1 cells after IAV infection， including interferon-γ response， interferon-α response， tumor necrosis factor-α/nuclear factor-κB （TNF-α/NF-κB）， and interleukin-6/Janus kinase/signal transducer and activator of transcription 3 （IL-6/JAK/STAT3） pathways. Compared with the Control group， the number of AT1 cells in the IAV group showed a decreasing trend. Compared with the IAV group， the WYJD-H group showed an increasing trend， although neither difference was statistically significant. Further analysis of AT1 cell subpopulation gene expression showed that， compared with the Control group， the IAV group exhibited increased expression of pro-apoptotic genes FAS cell surface death receptor （FAS） and cyclin-dependent kinase inhibitor 1A （CDKN1A）， a significant increase in tumor protein p53 （Tp53） expression （P<0.05）， and significant decreases in expression of the AT1 marker gene advanced glycosylation end-product-specific receptor （AGER） and membrane structural gene caveolin1 （CAV1） （P<0.05， P<0.01）. Compared with the IAV group， the WYJD-H group showed significantly decreased expression of FAS， CDKN1A， and Tp53 （P<0.05， P<0.01）， and significantly increased expression of AGER and CAV1 （P<0.05， P<0.01）. Regarding interferon response-related genes， compared with the Control group， the IAV group showed increased expression of interferon-stimulated gene 15 （ISG15）， interferon-induced protein with tetratricopeptide repeats 3 （IFIT3）， signal transducer and activator of transcription 2 （STAT2）， bone marrow stromal cell antigen 2 （BST2）， and C-X-C motif chemokine ligand 10 （CXCL10）， with a significant increase in 2′，5′-oligoadenylate synthetase-like protein 1 （OASL1） （P<0.05）. Compared with the IAV group， the WYJD-H group showed significantly decreased expression of all the above genes， with highly significant differences for ISG15， IFIT3， STAT2， BST2， and OASL1 （P<0.01）， and a significant difference for CXCL10 （P<0.05）. Among inflammation-related genes， compared with the Control group， the IAV group showed significantly increased expression of intercellular adhesion molecule 1 （ICAM1）， tumor necrosis factor alpha-induced protein 3 （TNFAIP3）， keratin 8 （KRT8）， tumor necrosis factor receptor superfamily member 1A （TNFRSF1A）， and TNFRSF1B （P<0.01）， and increased expression of NFKBIA， a negative regulator of NF-κB （P<0.05）. Compared with the IAV group， the WYJD-H group showed significantly decreased expression of KRT8 and TNFRSF1B （P<0.05）， while ICAM1， NFKBIA， TNFAIP3， and TNFRSF1A showed decreasing trends without statistical significance. Western blot validation showed that， compared with the Control group， protein expression levels of ISG15， FAS， p53， and phosphorylated p65 （p-p65） in lung tissue of the IAV group were significantly increased （P<0.05， P<0.01）. Compared with the IAV group， the WYJD-H group showed significantly decreased expression of these proteins （P<0.05， P<0.01）. ConclusionWYJD may alleviate viral pneumonia by targeting gene expression in AT1 cells， inhibiting overactivated interferon and inflammatory signaling pathways after IAV infection， and downregulating pro-apoptotic signaling， thereby reducing alveolar epithelial injury.

Epigenetics is the link between the genome and environment, which can respond to physiological (such as age) or environmental factors (such as diet, stress, and pollution) and induce changes in epigenetic modifications (such as DNA methylation, non-coding RNA, and histone modifications). It can also serve as cellular memory transmitted from generation to generation. Sperm is highly responsive to such environmental changes and has unique epigenetic profiles. The paternal inter-/trans-generational inheritance mediated by sperm epigenetic changes is closely related to the health of offspring, which is an issue of great concern. This review has summarized the epigenetic mechanisms of paternal inter-/trans-generational inheritance and recent studies on the paternal inheritance mediated by sperm epigenetic changes in human and mice, which may facilitate understanding of the relationship between paternal epigenetic changes and the health of offspring caused by physiological or environmental changes and provide a basis for genetic counseling and clinical intervention.
Epigenesis, Genetic ; Humans ; Male ; Animals ; Spermatozoa/metabolism* ; DNA Methylation ; Paternal Inheritance ; Mice

Epigenetics is the link between the genome and environment, which can respond to physiological (such as age) or environmental factors (such as diet, stress, and pollution) and induce changes in epigenetic modifications (such as DNA methylation, non-coding RNA, and histone modifications). It can also serve as cellular memory transmitted from generation to generation. Sperm is highly responsive to such environmental changes and has unique epigenetic profiles. The paternal inter-/trans-generational inheritance mediated by sperm epigenetic changes is closely related to the health of offspring, which is an issue of great concern. This review has summarized the epigenetic mechanisms of paternal inter-/trans-generational inheritance and recent studies on the paternal inheritance mediated by sperm epigenetic changes in human and mice, which may facilitate understanding of the relationship between paternal epigenetic changes and the health of offspring caused by physiological or environmental changes and provide a basis for genetic counseling and clinical intervention.

Epigenetics is the link between the genome and environment, which can respond to physiological (such as age) or environmental factors (such as diet, stress, and pollution) and induce changes in epigenetic modifications (such as DNA methylation, non-coding RNA, and histone modifications). It can also serve as cellular memory transmitted from generation to generation. Sperm is highly responsive to such environmental changes and has unique epigenetic profiles. The paternal inter-/trans-generational inheritance mediated by sperm epigenetic changes is closely related to the health of offspring, which is an issue of great concern. This review has summarized the epigenetic mechanisms of paternal inter-/trans-generational inheritance and recent studies on the paternal inheritance mediated by sperm epigenetic changes in human and mice, which may facilitate understanding of the relationship between paternal epigenetic changes and the health of offspring caused by physiological or environmental changes and provide a basis for genetic counseling and clinical intervention.

AIM:To investigate the effect of ankyrin repeat domain 49(ANKRD49)on epithelial-mesenchy-mal transition(EMT)in NCI-H1299 cells,and to explore its mechanism.METHODS:The ANKRD49 was over-ex-pressed in NCI-H1299 cells.The morphological changes of ANKRD49-overpressing NCI-H1299 cells were observed under microscope.The mRNA and protein expression levels of EMT-related markers[E-cadherin,transforming growth factor-β1(TGF-β1),vimentin and α-smooth muscle actin(α-SMA)]and EMT-related transcription factors(Snail1,Slug,Twist and ZEB1)were detected by RT-qPCR Western blot.Immunofluorescence staining was performed to observe the localiza-tion and expression of E-cadherin and vimentin in ANKRD49-overexpressing cells or control cells.Immunohistochemical method was performed to examine the levels of E-cadherin,α-SMA,Snail,Slug and ZEB1 in lung tissues of nude mice in-oculated with ANKRD49-overexpressing H1299 cells or control cells.RESULTS:Compared with the control group,the ANKRD49 overexpressing cells showed mesenchymal cell morphology(fusiform and less tight connections).RT-qPCR and Western blot results showed that the mRNA and protein levels of mesenchymal markers vimentin and α-SMA in ANKRD49 overexpressing cells were significantly higher than those in cells of control group,while the mRNA and protein levels of epithelial marker E-cadherin were lower than those in cells of control group.Compared with control group,the im-munofluorescence intensity of E-cadherin of H1299 cells decreased in after ANKRD49 overexpression,while that of vimen-tin increased significantly.Snail,Slug and ZEB1 expression were significantly elevated in ANKRD49 overexpressing cells compared with control group.The levels of E-cadherin in lung tissues of nude mice inoculated with ANKRD49-overexpressing H1299 cells declined,while the levels of α-SMA,Snail,Slug and ZEB1 increased compared with those in control mice.CONCLUSION:ANKRD49 promoted EMT of NCI-H1299 cells by increasing the expression of Snail1,Slug and ZEB1 and consequent downreguation of E-cadherin and upregulation of vimentin and α-SMA.

Abstract： Objective　To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods　In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results　The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions　In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein.

OBJECTIVE To establish the fingerp rint of decoction pi eces and dispensing granules of Gardenia jasminoides ，to determine the contents of 6 components，so as to evaluate its quality combined with chemical pattern recognition. METHODS High performance liquid chromatography （HPLC）was used. Using geniposide as the reference ，Similarity Evaluation System for Chromatographic Fingerprint of TCM （2012 edition）was used to draw the fingerprints of 20 batches of G. jasminoides decoction pieces and 10 batches of G. jasminoides dispensing granules. Similarity evaluation and common peaks identification were conducted. The same HPLC method was adopted to determine the contents of deacetyl asperulosidic acid methyl ester ，geniposide， picrocrocin，rutin，crocin-Ⅰ and crocin- Ⅱ. ORIGIN 9.1 software was used for hierarchical clustering analysis ，and SIMCA 16.0 software was used for principal component analysis （PCA） and partial least squares-discriminant analysis. The differential components affecting the quality of decoction pieces and dispensing granules were screened by taking the variable importance in projection（VIP）value＞1 as the standard. RESULTS There were 24 common peaks for both 20 batches of G. jasminoides decoction piece and 10 batches of G. jasminoides dispensing granules ；a total of 22 common peaks were found in the fingerprints of 30 batches of samples ，and the similarity was not lower than 0.96；six common peaks were identified ，i.e. deacetyl asperulosidic acid methyl ester （peak 2），geniposide（peak 6），picrocrocin（peak 9），rutin（peak 11），crocin-Ⅰ（peak 15），crocin-Ⅱ（peak 17）. Average contents of above 6 components in G. jasminoides decoction pieces were 1.04，57.00，1.30，1.03，9.63 and 0.99 mg/g， respectively；those of G. jasmin oides dispensing granules were 0.96，17.04，0.37，0.27，0.73 and 0.04 mg/g，respectively. PCA results showed that G. jasminoides decoction pieces and G. jasminoides dispensing granules were clustered into respective one category ，which was consistent with results of cluster analysis. There were 9 common peaks with VIP value ＞1， which were 16，14，3，17（crocin-Ⅱ），15（crocin-Ⅰ），18， 22， 2 （deacetyl asperulosidic acid methyl ester） and 21. CONCLUSIONS The estab lished fingerprint and content determination method are simple and reproducible. Combined with chemical pattern recognition ，it can be used to evaluate the quality of decoction pieces and dispensing granules of G. jasminoides . Nine corresponding components represented by peak 16 and so on are the differential components that affect the quality of them.

Objective:To evaluate the application of a medical image software (RadiAnt) in anatomical measurements and precision craniotomy via suboccipital retrosigmoid sinus approach.Methods:A total of 43 inpatients who underwent craniocerebral CT venography (CTV) in our hospital from June 2020 to June 2021 were selected for the study; the CTV data of 35 patients was used to measure the spatial relations between transverse sigmoid sinus junction (TSSJ) and asterion; the preoperative planning in suboccipital retrosigmoid sinus craniotomy with the software was performed in the left 8 patients. Craniotomy time (subjected to exposure of venous sinus margin), venous sinus injury and incidence of complications within 2 weeks of craniotomy in these 8 patients were recorded.Results:(1) Anatomic measurement: for the left side, TSSJ was located at (0.89±0.33) cm lateral and (0.63±0.46) cm inferior to the asterion, and their direct distance was (1.15±0.42) cm; TSSJ was located at (0.76±0.49) cm interior and (1.97±0.52) cm superior to the starting point of the mastoid notch, and their direct distance was (2.18±0.49) cm; about 29% asterion were located superior to the transverse sinus, 37% were located on the surface of the transverse sinus, and 34% were located inferior to the transverse sinus. For the right side, TSSJ was located at (0.88±0.39) cm lateral and (0.64±0.43) cm inferior to the asterion, and their direct distance was (1.12±0.54) cm; TSSJ was located at (0.74±0.40) cm interior and (1.93±0.45) cm superior to the starting point of the mastoid notch, and their direct distance was (2.16±0.43) cm; about 26% asterion were located superior to the transverse sinus, 40% were located on the surface of the transverse sinus, and 34% were located inferior to the transverse sinus. (2) Preoperative planning and surgeries: in these 8 patients, the key-hole was located at (0.96±0.49) cm lateral and (0.53±0.18) cm inferior to the asterion, and (0.46±0.35) cm interior and (1.76±0.47) superior to the starting point of mastoid notch. The interior of sigmoid sinus was located (0.13±0.51) cm interior and (0.21±0.46) cm superior to the starting point of mastoid notch. The inferior of the transverse sinus was located (2.17±0.45) cm interior and (0.53±0.35) cm inferior to the asterion. An accurate localization of the real position of TSSJ, inferior of transverse sinus and interior of sigmoid sinus was performed in all 8 surgical patients. The distance between the margin of the bone window and the interior of sigmoid sinus was (3.5±1.0) mm, and the craniotomy time was (25.7±4.1) min; no sinus injury was noted. Post-operative CT showed good reposition of the bone flaps and less bone defect. There was no cerebrospinal fluid leakage or subcutaneous effusion during the 2 weeks of follow-up.Conclusion:Anatomical measurements and preoperative planning can be quickly finished with low cost with Radiant ?, which provides an efficient and safe method for accurate craniotomy via suboccipital retrosigmoid approach.

Objective:To construct and evaluate a decision tree based on biomarkers for predicting severe acute kidney injury (AKI) in critical patients.Methods:A prospectively study was conducted. Critical patients who had been admitted to the department of critical care medicine of Xiaolan Hospital of Southern Medical University from January 2017 to June 2018 were enrolled. The clinical data of the patients were recorded, and the biomarkers, including serum cystatin C (sCys C) and urinary N-acetyl-β-D-glucosaminidase (uNAG) were established immediately after admission to intensive care unit (ICU), and the end points were recorded. The test cohort was established with patient data from January to December 2017. The decision tree classification and regression tree (CART) algorithm was used, and the best cut-off values of biomarkers were used as the decision node to construct a biomarker decision tree model for predicting severe AKI. The accuracy of the decision tree model was evaluated by the overall accuracy and the receiver operating characteristic (ROC) curve. The validation cohort, established on patient data from January to June 2018, was used to further validate the accuracy and predictive ability of the decision tree.Results:In test cohort, 263 patients were enrolled, of whom 57 developed severe AKI [defined as phase 2 and 3 of Kidney Disease: Improving Global Outcomes (KDIGO) criterion]. Compared with patients without severe AKI, severe AKI patients were older [years old: 64 (49, 74) vs. 52 (41, 66)], acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score were higher [23 (19, 27) vs. 15 (11, 20)], the incidence of hypertension, diabetes and other basic diseases and sepsis were higher (64.9% vs. 40.3%, 28.1% vs. 10.7%, 63.2% vs. 29.6%), the levels of sCys C and uNAG were higher [sCys C (mg/L): 1.38 (1.12, 2.02) vs. 0.79 (0.67, 0.98), uNAG (U/mmol Cr): 5.91 (2.43, 10.68) vs. 2.72 (1.60, 3.90)], hospital mortality and 90-day mortality were higher (21.1% vs. 4.4%, 52.6% vs. 13.1%), the length of ICU stay was longer [days: 6.0 (4.0, 9.5) vs. 3.0 (1.0, 6.0)], and renal replacement therapy requirement was higher (22.8% vs. 1.9%), with statistically significant differences (all P < 0.05). ROC curve analysis showed that the areas under ROC curve (AUC) of sCys C and uNAG in predicting severe AKI were 0.857 [95% confidence interval (95% CI) was 0.809-0.897) ] and 0.735 (95% CI was 0.678-0.788), and the best cut-off values were 1.05 mg/L and 5.39 U/mmol Cr, respectively. The structure of the biomarker decision tree model constructed by biomarkers were intuitive. The overall accuracy in predicting severe AKI was 86.0%, and AUC was 0.905 (95% CI was 0.863-0.937), the sensitivity was 0.912, and the specificity was 0.796. In validation cohort of 130 patients, this decision tree yielded an excellent AUC of 0.909 (95% CI was 0.846-0.952), the sensitivity was 0.906, and the specificity was 0.816, with an overall accuracy of 81.0%. Conclusion:The decision tree model based on biomarkers for predicting severe AKI in critical patients is highly accurate, intuitive and executable, which is helpful for clinical judgment and decision.

The oral colon-specific drug delivery system (OCDDS) has gained more attention from investigators in recent years since it can increase local drug concentration and reduce dosage and side effects.The type of colonspecific drug delivery system consists of enzyme dependent,pH dependent,time dependent,pressure dependent system and CODEDSTM.The development of many new materials and technology is very important for the preparation of new type of precise positioning colon-specific preparation.This article summarizes the advances in excipients and technique for oral colon-specific drug delivery system in recent years.It may provide a reference and a basis for the researchers concerned.