Detection of multiple biomarkers associated with satellite cell fate in the contused skeletal muscle of rats for wound age estimation.

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7 days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7 days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3 days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.

The roles of AMPK/mTOR autophagy pathway in the acute kidney injury-induced acute lung injury.

Acute kidney injury (AKI) is one of the most challenging clinical problems in kidney disease due to serious complications and high mortality rate, which can lead to acute lung injury (ALI) through inflammatory reactions and oxidative stress. Adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has been reported to be involved in the development of renal ischemia-reperfusion through autophagy and it remains unclear whether AMPK/mTOR pathway has an effect on the AKI-induced ALI. In this study, we aimed to investigate the effects of autophagy-related AMPK/mTOR signaling pathway on inflammatory factors and oxidative stress in an AKI-induced ALI model. The 48 male Sprague-Dawley rats were divided into four groups randomly: (i) sham, (ii) ischemia/reperfusion injury (IRI), (iii) IRI + rapamycin (RA), and (iv) IRI + 3-methyladenine (3-MA). Unilateral flank incisions were made and right kidneys were excised. The left kidney was subjected to 60 min of ischemia followed by 12, 24, 48, and 72 h of reperfusion. The levels of Scr, blood urea nitrogen (BUN), Wet/Dry ratio, indexes of inflammation, and oxidative stress were assayed. Histological examinations were performed. The protein expression of AMPK, mTOR, LC3-II/LC3-I ratio, and Beclin-1, ULK1 was evaluated by western blotting and immunohistochemistry. Compared to the rats from the sham group, IRI rats showed significantly pulmonary damage after AKI with increased Scr, BUN, Wet/Dry ratio, indexes of inflammation, and oxidative stress. The expression of AMPK, LC3-II/LC3-I ratio, Beclin-1, and ULK1 and were increased, while p62 and mTOR were decreased. In addition, RA treatment significantly attenuated lung injury by promoting autophagy through the activation of the AMPK/mTOR pathway, and 3-MA treatment exhibited adverse effects inversely. Therefore, the activation of the AMPK/mTOR pathway after renal IRI induction could significantly attenuate kidney injury and following AKI-induced ALI by inducing autophagy, which alienates inflammation, oxidative stress, and apoptosis.

Comparison of CT Values between Thrombus and Postmortem Clot Based on Cadaveric Pulmonary Angiography.

OBJECTIVES: To explore the difference in CT values between pulmonary thromboembolism and postmortem clot in postmortem CT pulmonary angiography (CTPA) to further improve the application value of virtual autopsy. METHODS: Postmortem CTPA data with the definite cause of death from 2016 to 2019 were collected and divided into pulmonary thromboembolism group (n=4), postmortem clot group (n=5), and control group (n=5). CT values of pulmonary trunk and left and right pulmonary artery contents in each group were measured and analyzed statistically. RESULTS: The average CT value in the pulmonary thromboembolism group and postmortem clot group were (168.4±53.8) Hu and (282.7±78.0) Hu, respectively, which were lower than those of the control group (1 193.0±82.9) Hu (P<0.05). The average CT value of the postmortem clot group was higher than that of the pulmonary thromboembolism group (P<0.05). CONCLUSIONS: CT value is reliable and feasible as a relatively objective quantitative index to distinguish pulmonary thromboembolism and postmortem clot in postmortem CTPA. At the same time, it can provide a scientific basis to a certain extent for ruling out pulmonary thromboembolism deaths.

Individualized management of immunosuppressants in liver transplant recipients by using a novel immune score system.

BACKGROUND: There is no reliable means to evaluate the immune status of liver transplant recipients. We proposed a novel score model, namely Mingdao immune cell analysis and Mingdao immune score system, to quantify the immunity. METHODS: Data from those who underwent a single liver transplant between January 2017 and June 2020 at Beijing Chaoyang Hospital, were collected. In addition, healthy volunteers were also enrolled. The score model was based on the immune cell populations determined by flow cytometry. RESULTS: There were a total of 376 healthy controls with 376 tests and 148 liver transplant recipients with 284 tests in this study. Evaluated by Mingdao immune cell analysis and Mingdao immune score system, the mean scores of healthy controls were near zero suggesting a balanced immune system. In contrast, the mean scores of liver transplant recipients were negative both before and after surgery indicating a compromised immune system. When liver transplant recipients were given a reduced or routine first dose according to their preoperative score, they had similar recovery of liver function. Moreover, liver transplant recipients with increased scores ≥ 5 were associated with elevated aspartate transaminase and alanine amiotransferase. Finally, on multivariate analysis the score model was the only significant independent risk factor for clinical acute rejection (P = 0.021; Odds ratio, 0.913; 95% confidence interval, 0.845-0.987). CONCLUSION: The novel score model could be used as an indicator to reflect immunity and to regulate immunosuppressants in liver transplant recipients after surgery.

Practical oxazole synthesis mediated by iodine from α-bromoketones and benzylamine derivatives.

The reagent system of I2/K2CO3 could efficiently promote the oxazole synthesis from α-bromoketones and benzylamine derivatives in DMF. This method was not only suitable for 2,5-diaryl oxazole synthesis but also for 2,4,5-trisubstituted oxazole and 5-alkyl/alkenyl oxazole synthesis. Furthermore, this method was successfully applied to a one-step synthesis of a natural product halfordinol in 62% yield.

Protective Mechanism of Cordyceps sinensis Treatment on Acute Kidney Injury-Induced Acute Lung Injury through AMPK/mTOR Signaling Pathway.

OBJECTIVE: To investigate protective effect of Cordyceps sinensis (CS) through autophagy-associated adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in acute kidney injury (AKI)-induced acute lung injury (ALI). METHODS: Forty-eight male Sprague-Dawley rats were divided into 4 groups according to a random number table, including the normal saline (NS)-treated sham group (sham group), NS-treated ischemia reperfusion injury (IRI) group (IRI group), and low- (5 g/kg·d) and high-dose (10 g/kg·d) CS-treated IRI groups (CS1 and CS2 groups), 12 rats in each group. Nephrectomy of the right kidney was performed on the IRI rat model that was subjected to 60 min of left renal pedicle occlusion followed by 12, 24, 48, and 72 h of reperfusion. The wet-to-dry (W/D) ratio of lung, levels of serum creatinine (Scr), blood urea nitrogen (BUN), inflammatory cytokines such as interleukin- ß and tumor necrosis factor- α, and biomarkers of oxidative stress such as superoxide dismutase, malonaldehyde (MDA) and myeloperoxidase (MPO), were assayed. Histological examinations were conducted to determine damage of tissues in the kidney and lung. The protein expressions of light chain 3 II/light chain 3 I (LC3-II/LC3-I), uncoordinated-51-like kinase 1 (ULK1), P62, AMPK and mTOR were measured by Western blot and immunohistochemistry, respectively. RESULTS: The renal IRI induced pulmonary injury following AKI, resulting in significant increases in W/D ratio of lung, and the levels of Scr, BUN, inflammatory cytokines, MDA and MPO (P<0.01); all of these were reduced in the CS groups (P<0.05 or P<0.01). Compared with the IRI groups, the expression levels of P62 and mTOR were significantly lower (P<0.05 or P<0.01), while those of LC3-II/LC3-I, ULK1, and AMPK were significantly higher in the CS2 group (P<0.05 or P<0.01). CONCLUSION: CS had a potential in treating lung injury following renal IRI through activation of the autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI.

Granzyme B-Producing B Cells Function as a Feedback Loop for T Helper Cells in Liver Transplant Recipients with Acute Rejection.

Granzyme B-producing B cells have been reportedly reported to be an important regulatory B cell subset in the pathogenesis of many diseases. However, its role in liver transplant recipients with acute rejection has never been well elucidated. Seventeen liver transplant recipients with acute rejection and 25 controls with stable liver function were enrolled in this study to determine the function of granzyme B-producing B cells via flow cytometry. Liver transplant recipients with acute rejection had higher expression of granzyme B in CD19+B cells when compared with controls. Following rejection, the granzyme B production was even elevated although comparison failed to reach significance. The percentages of CD27+granzyme B+CD19+B cells and CD138+granzyme B+CD19+B cells were lower than those of CD27-granzyme B+CD19+B cells and CD138-granzyme B+CD19+B cells in patients with acute rejection, respectively. While the production of CD27 and CD138 was not different between liver transplant recipients with and without acute rejection but increased expression of the two surface markers was observed following rejection. Furthermore, the frequency of granzyme B+CD19+B cells correlated with the level of alanine aminotransferase instead of tacrolimus. CD19+B cells could produce granzyme B when stimulated with IgG + IgM and CpG in the presence of the supernatant of activated CD4+CD25-T cells. In return, granzyme B+CD19+B cells could suppress the proliferation of CD4+CD25-T cells in a granzyme B- and contact-dependent manner. The increased expression of granzyme B in CD19+B cells from liver transplant recipients with acute rejection might function as a feedback loop for the activation of the CD4+CD25-T cells.

The Model for End-Stage Liver Disease Score and the Follow-Up Period Can Cause the Shift of Circulating Lymphocyte Subsets in Liver Transplant Recipients.

Little is known about the shift of lymphocytes under the condition of the model for end-stage liver disease score and the follow-up period. Then, we detected the peripheral blood from liver transplant recipients by flow cytometry and compared the results. The model for end-stage liver disease score affected the percentages of T-cell subsets and B cells during the short-term follow-up period, but failed to influence the lymphocyte subsets during the long-term follow-up period. In contrast, the follow-up period not only affected the absolute counts of T-cell subsets and natural killer (NK) cells in patients with the low model for end-stage liver disease scores, but also influenced the percentages and absolute counts of T-cell subsets in patients with the high model for end-stage liver disease scores. In the two-way ANOVA, we further revealed that the model for end-stage liver disease score was associated with the percentages of T cells and CD4+ T cells and the absolute numbers of T-cell subsets and B cells, while the follow-up period was associated with the percentages of T-cell subsets and the absolute numbers of lymphocyte subsets. Therefore, patients with either the low model for end-stage liver disease scores or the long-term follow-up period are in a relatively activated immune condition.

Decreased granzyme B+CD19+B cells are associated with tumor progression following liver transplantation.

Lymphocytes play an important role in antitumor immunity following organ transplantation. However, the function of granzyme B+CD19+B cells on the hepatocellular carcinoma cells from liver transplant recipients remains largely unknown; we aimed to analyze the function and elucidate the mechanisms behind it. Blood samples and clinical data from liver transplant recipients and healthy controls at Beijing Chaoyang Hospital as well as from a validation cohort were collected and analyzed. In this study, we found decreased granzyme B+CD19+B cells were correlated with early hepatocellular carcinoma recurrence and could further identify liver transplant recipients with poor tumor differentiation, microvascular invasion, increased total tumor diameter, and tumor beyond Milan criteria. Notably, granzyme B+CD19+B cells directly inhibited the proliferation, migration, and invasion of hepatocellular carcinoma cells. Upon activation regulatory B cells from liver transplant recipients with hepatocellular carcinoma recurrence displayed a CD5+CD38+CD27+CD138+CD19+ granzyme B+ phenotype, but the increased expression of CD5, CD38, and CD138, and the decreased protein level and transcriptional level requiring JAK/STAT signaling. In an independent validation cohort, liver transplant recipients with decreased granzyme B+CD19+B cells had not only early hepatocellular carcinoma cell recurrence but also shorter survival. Our study provides comprehensive data from liver transplant recipients with hepatocellular carcinoma, indicating a critical role of granzyme B+CD19+B cells in preventing cancer progression. Our findings warrant further investigations for the design of future immunotherapies leading to immune responses and improved patient survival.

Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems.

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpf1) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.

Distribution of Avian Influenza A Viruses in Poultry-Related Environment and Its Association with Human Infection in Henan, 2016 to 2017.

OBJECTIVE: To survey avian influenza A viruses (AIVs) in the environment and explore the reasons for the surge in human H7N9 cases. METHODS: A total of 1,045 samples were collected from routine surveillance on poultry-related environments and 307 samples from human H7N9 cases-exposed environments in Henan from 2016 to 2017. The nucleic acids of influenza A (Flu A), H5, H7, and H9 subtypes were detected by real-time polymerase chain reaction. RESULTS: A total of 27 H7N9 cases were confirmed in Henan from 2016 to 2017, 24 had a history of live poultry exposure, and 15 had H7N9 virus detected in the related live poultry markets (LPMs). About 96% (264/275) Flu A positive-environmental samples were from LPMs. H9 was the main AIV subtype (10.05%) from routine surveillance sites with only 1 H7-positive sample, whereas 21.17% samples were H7-positive in H7N9 cases-exposed environments. Samples from H7N9 cases-exposed LPMs (47.56%) had much higher AIVs positive rates than those from routine surveillance sites (12.34%). The H7+H9 combination of mixed infection was 78.18% (43/55) of H7-positive samples and 41.34% (43/104) of H9-positive samples. CONCLUSION: The contamination status of AIVs in poultry-related environments is closely associated with the incidence of human infection caused by AIVs. Therefore, systematic surveillance of AIVs in LPMs in China is essential for the detection of novel reassortant viruses and their potential for interspecies transmission.

Iodine-mediated intramolecular amination of ketones: the synthesis of 2-acylindoles and 2-acylindolines by tuning N-protecting groups.

A general method for constructing both 2-acylindoles and 2-acylindolines via I2-mediated intramolecular C-N bond formation is presented, and the selective formation of either 2-acylindoles or 2-acylindolines just depends on the nitrogen protecting groups used in the same substrate skeletons.