Next generation sequencing predicting clinical effect of immunotherapy on adult rhabdomyosarcoma patient: A case report.

RATIONALE: Rhabdomyosarcoma (RMS) is a rare sarcoma that rarely occurs in adults and accounts for only 1% of all adult tumors. The standard treatment for RMS is surgical resection, radiotherapy, and chemotherapy. PATIENT CONCERNS: Adult patients often present with an aggressive course and poor prognosis. DIAGNOSES: The patient was diagnosed with RMS in September 2019 and was confirmed by hematoxylin-eosin staining and immunohistochemistry after surgical resection. INTERVENTIONS: The patient received surgical resection in September 2019. He was admitted to another hospital after the first recurrence in November 2019. After the second routine surgical resection, the patient underwent chemotherapy, radiotherapy, and anlotinib maintenance treatment. He relapsed again in October 2020 and was admitted to our hospital. Next-generation sequencing was performed on the punctured tissue of the patient's lung metastatic lesion, and high tumor mutational burden (TMB-H), high microsatellite instability (MSI-H), and positive programmed death-ligand 1 (PD-L1) were observed. The patient then received toripalimab and anlotinib combined therapy and was evaluated for a partial response after 2 months. OUTCOMES: This benefit has persisted for more than 17 months. LESSONS: This is the longest progression-free survival for PD-1 inhibitors in RMS, and there is a trend of continued extension of progression-free survival in this patient. This case supports the hypothesis that positive PD-L1, TMB-H, and MSI-H could be beneficial biomarkers for immunotherapy in adult RMS.

Designing a Light-Induced Glycosylation Reaction for Poststage Synthesis of ADP-2″-Deoxyribosyl Derivatives.

Modifications on the hydroxyl groups of ADP-ribosyl units can provide valuable tools for investigating ADP-ribosylation-related molecular interactions, but the chemical syntheses of these compounds are usually difficult due to their inherent complex structures. In this study, we report a poststage synthetic protocol for accessing novel ADP-2″-deoxyribosyl derivatives through designing a light-induced biomimetic reaction, and SPR assays revealed effective binding of ADP-2″-deoxyribosyl peptides to MacroH2A1.1 with a high affinity (KD = 3.75 × 10-6 M).

Clinical efficacy of Jingyin granules, a Chinese patent medicine, in treating patients infected with coronavirus disease 2019.

BACKGROUND: Jingyin granules (JY), one patented Chinese herbal formula, have been advised for treating coronavirus disease 2019 (COVID-19) in China. As of now, the safety and effectiveness of JY in treating COVID-19 patients were still to be evaluated. PURPOSE: To investigate the safety and clinical effectiveness of JY in treating mild COVID-19 patients. STUDY DESIGN: We carried out a prospective cohort study, as the highly infectious COVID-19 omicron variant ranged in Shanghai (ClinicalTrial.gov registration number: ChiCTR2200058692). METHODS: Participants infected with COVID-19, who were diagnosed as mild cases, were assigned to receive either JY (JY group) or traditional Chinese medicine placebo (placebo group) orally for 7 days. The primary clinical indicators were the RNA negative conversion rate (NCR) and the incidence of severe cases. The secondary clinical indicators were the negative conversion time (NCT), inpatient length of stay (ILOS), and the disappearance rates of clinical symptoms. RESULTS: Nine hundred participants were recruited in this clinical trial study, and 830 patients met the eligibility criteria. Seven hundred and ninety-one patients, accomplished the following-up assessment, including 423 cases of JY group and 368 cases of placebo group. NCR in JY group at 7-day posttreatment was considerably greater compared with placebo group (89.8% [380/423] vs 82.6% [304/368], P = 0.003). None of the patients with mild COVID-19 developed into severe cases. The median NCT of SARS-CoV-2 and ILOS in JY group were lesser than that in placebo group (4.0 [3.0,6.0]vs 5.0 [4.0,7.0] days, P < 0.001; 6.0 [4.0, 8.0] vs 7.0 [5.0, 9.0] days, P < 0.001). In both groups, the obvious improvement in clinical symptoms was observed, but the difference was not significant. In the subgroup of age ≤ 60 years, JY promoted SARS-CoV-2 RNA negative conversion (HR=1.242; 95% CI: 1.069-1.444, P < 0.001). No patients in both groups were reported as the case of serious adverse event. CONCLUSION: JY maybe the potential medicine for treating mild COVID-19 patients, which had beneficial effects on increasing NCR, and shortening NCT and ILOS.

Traditional Chinese Medicine JingYinGuBiao Formula Therapy Improves the Negative Conversion Rate of SARS-CoV2 in Patients with Mild COVID-19.

Background: Traditional Chinese Medicine (TCM) JingYinGuBiao formula (JYGB) was recommended by the Expert consensus on Traditional Chinese Medicine diagnosis and treatment of COVID-19 infection in Shanghai. We evaluated the safety and efficacy of JYGB in treating mild COVID-19 patients. Methods: A prospective, double-blind, randomized, controlled trial was conducted (ClinicalTrial.gov registration number: ChiCTR2200058695). A total of 885 patients were randomized into the treatment group (administration of JYGB,n=508) or the control group (administration of TCM placebo, n=377) with 7-day treatment. The primary outcomes were the negative conversion rate and negative conversion time of SARS-CoV2 RNA. Secondary outcomes included the hospitalized days and symptom improvement. Results: A total of 490 and 368 patients in the treatment and control groups completed the study. The cumulative negative conversion rates at 2 days, 3 days, 4 days, and 6 days post randomization in the treatment group were all markedly higher than those in the control group (13.88% vs. 9.24%, P=0.04; 32.24% vs. 16.58%, P<0.001; 51.43% vs. 36.14%, P <0.001; 77.76% vs. 69.84%, P=0.008). Compared with the control group, after JYGB treatment, the median negative conversion time (4.0 [3.0-6.0] vs. 5.0 [4.0-7.0] days, P<0.001) and hospitalized days (6.0 [4.0-8.0] vs. 7.0 [5.0-9.0] days, P<0.001) were reduced. While the symptoms were improved, there were no significant differences in symptom disappearance rates between both groups. In addition, further sub-group analysis showed that for patients with interval time ≤4 days or patients≤ 60 years, the clinical effects of JYGB were more remarkable with an increase in cumulative negative conversion rates, a decrease in negative conversion time and hospitalized days. JYGB was well tolerated without any severe side effects. Conclusion: JYGB, a TCM prescription, improves the negative conversion rate of SARS-CoV2 in mild COVID-19 patients.

Guben Tongluo Formula Protects LPS-induced Damage in Lamina Propria B Lymphocytes Through TLR4/MyD88/NF-κB Pathway.

OBJECTIVE: The main pathological feature of immunoglobulin A nephropathy (IgAN), an autoimmune kidney disease, is the deposition of IgA immune complexes, accompanied by mesangial cell proliferation and elevated urine protein. The Guben Tongluo formula (GTF) is a traditional Chinese medicine prescription, which has predominant protective effects on IgAN. However, the therapeutic mechanism of the GTF in IgAN remains elusive. The present study aimed to determine the effects of GTF in treating IgAN via regulating the TLR4/MyD88/NF-κB pathway. METHODS: In the present study, lamina propria B lymphocytes were treated with different concentrations of lipopolysaccharide (LPS) (0, 1, 5, 10 and 20 ng/mL). Flow cytometry was used to define positive CD86+CD19+ cells. CCK-8 assay was used to examine cell proliferation. RNAi was used to induce TLR4 silencing. qRT-PCR and Western blotting were used to determine gene expression. RESULTS: It was found that the LPS dose-dependently increased the content of IgA and galactose-deficient IgA1 (Gd-IgA), the levels of TLR4, Cosmc, MyD88 and phosphorylated (p)-NF-κB, and the ratio of CD86+CD19+ and IgA-producing B cells. However, the TLR4 knockdown reversed the role of LPS. This suggests that TLR4 mediates the effects of LPS on lamina propria B lymphocytes. Furthermore, the GTF could dose-dependently counteract the effects of LPS and TLR4 overexpression on lamina propria B lymphocytes through the TLR4/MyD88/NF-κB pathway. CONCLUSION: Collectively, these results demonstrate that the GTF can regulate the TLR4/MyD88/NF-κB pathway to treat IgAN model lamina propria B lymphocytes stimulated by LPS.

Sorafenib inhibits LPS-induced inflammation by regulating Lyn-MAPK-NF-kB/AP-1 pathway and TLR4 expression.

Sorafenib is an anti-tumor drug widely used in clinical treatment, which can inhibit tyrosine kinase receptor on cell surface and serine/threonine kinase in downstream Ras/MAPK cascade signaling pathway of cells. Tyrosine kinase phosphorylation plays an important role in inflammatory mechanism, such as TLR4 tyrosine phosphorylation, MAPK pathway protein activation, and activation of downstream NF-кB. However, the effects of sorafenib on LPS-induced inflammatory reaction and its specific mechanism have still remained unknown. We found that sorafenib inhibited the phosphorylation of tyrosine kinase Lyn induced by LPS, thereby reducing the phosphorylation level of p38 and JNK, inhibiting the activation of c-Jun and NF-κB, and then inhibiting the expression of inflammatory factors IL-6, IL-1ß, and TNF-α. Furthermore, sorafenib also decreased the expression of TLR4 on the macrophage membrane to inhibit the expression of inflammatory factors latterly, which may be related to the inactivation of Lyn. These results provide a new perspective and direction for the clinical treatment of sepsis.

Propofol Upregulates MicroRNA-30b to Inhibit Excessive Autophagy and Apoptosis and Attenuates Ischemia/Reperfusion Injury In Vitro and in Patients.

Evidence reveals that propofol protects cells via suppressing excessive autophagy induced by hypoxia/reoxygenation (H/R). Previously, we found in a genome-wide microRNA profile analysis that several autophagy-related microRNAs were significantly altered during the process of H/R in the presence or absence of propofol posthypoxia treatment (P-PostH), but how these microRNAs work in P-PostH is still largely unknown. Here, we found that one of these microRNAs, microRNA-30b (miR-30b), in human umbilical vein endothelial cells (HUVECs) was downregulated by H/R treatment but significantly upregulated by 100 M propofol after H/R treatment. miR-30b showed similar changes in open heart surgery patients. By dual-luciferase assay, we found that Beclin-1 is the direct target of miR-30b. This conclusion was also supported by knockdown or overexpression of miR-30b. Further studies showed that miR-30b inhibited H/R-induced autophagy activation. Overexpression or knockdown of miR-30b regulated autophagy-related protein gene expression in vitro. To clarify the specific role of propofol in the inhibition of autophagy and distinguish the induction of autophagy from the damage of autophagy flux, we used bafilomycin A1. LC3-II levels were decreased in the group treated with propofol combined with bafilomycin A1 compared with the group treated with bafilomycin A1 alone after hypoxia and reoxygenation. Moreover, HUVECs transfected with Ad-mCherry-GFP-LC3b confirmed the inhibitory effect of miR-30b on autophagy flux. Finally, we found that miR-30b is able to increase the cellular viability under the H/R condition, partially mimicking the protective effect of propofol which suppressed autophagy via enhancing miR-30b and targeting Beclin-1. Therefore, we concluded that propofol upregulates miR-30b to repress excessive autophagy via targeting Beclin-1 under H/R condition. Thus, our results revealed a novel mechanism of the protective role of propofol during anesthesia. Clinical Trial Registration Number. This trial is registered with ChiCTR-IPR-14005470. The name of the trial register: Propofol Upregulates MicroRNA-30b to Repress Beclin-1 and Inhibits Excessive Autophagy and Apoptosis.

Remimazolam Protects Against LPS-Induced Endotoxicity Improving Survival of Endotoxemia Mice.

Remimazolam is a new benzodiazepine of sedative drugs with an ultra-short-acting anesthetic effect, commonly used for critically ill patients (especially septic patients) in intensive care units (ICUs). Although some anesthetics have been reported to show certain anti-inflammatory effects, the role of remimazolam in inflammation is still remained unknown. Here, we studied the effects of remimazolam on macrophage in response to LPS both in vivo and in vitro. Interestingly, compared with LPS treatment group, remimazolam remarkably improved survival rate of endotoxemia mice and decreased the release of LPS-induced inflammatory mediators (such as TNF-α, IL-6, and IL-1ß). We further found that remimazolam not only inhibited the activation of MAPK signal pathway at 15 min after LPS treatment but also disturbed Rab5a related TLR4 expression at cell surface in response to LPS at a later time. Such evidence suggests that remimazolam might be beneficial to septic patients who are suffering from uncontrolled inflammatory responses.

TRIM40 inhibits IgA1-induced proliferation of glomerular mesangial cells by inactivating NLRP3 inflammasome through ubiquitination.

IgA nephropathy, as the most common type of glomerulonephritis, causes chronic renal disease and progresses into kidney failure. Aberrant IgA deposition in the glomerular mesangium induces NLRP3 inflammasome activation for massive local inflammation, and is recognized as the primary pathogenesis in IgA nephropathy. Tripartite motif (TRIM)-containing proteins are E3 ubiquitin ligases that possess crucial regulatory functions in innate immunity, but their functional roles in IgA nephropathy are still unclear. Here, we aimed to identify TRIM-containing proteins that regulate IgA nephropathy and their underlying mechanisms. An in vitro IgA1-induction model was established in glomerular mesangial cells (GMCs) and showed that IgA1 could promote GMC proliferation by activating NLRP3 inflammasome. TRIM40, which was downregulated by IgA1 and interacted with NLRP3, was recognized as a promising candidate. In addition, TRIM40 could suppress IgA1-induced GMC proliferation by inhibiting the activation of NLRP3 inflammasome. Based on coimmunoprecipitation and ubiquitination assays, we confirmed that TRIM40 could mediate the ubiquitination of NLRP3, which explained its regulatory effects on NLRP3 inflammasome and GMC proliferation. More importantly, a dominant-negative mutant of TRIM40 lacking the RING domain (ΔRING) did not affect NLRP3 ubiquitination, and had no effects on IgA1-induced GMC proliferation or NLRP3 inflammasome activation. This study revealed the biological functions of TRIM40 in IgA nephropathy, facilitating its application as therapeutic target for IgA nephropathy and other NLRP3 inflammasome-relevant diseases.

HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus.

BACKGROUND: Woronin bodies are fungal-specific organelles whose formation is derived from peroxisomes. The former are believed to be involved in the regulation of mycotoxins biosynthesis, but not in their damage repair function. The hexagonal peroxisome protein (HexA or Hex1) encoded by hexA gene in Aspergillus is the main and the essential component of the Woronin body. However, little is known about HexA in Aspergillus flavus. RESULTS: In this study, hexA knock-out mutant (ΔhexA) and complementation strain (ΔhexAC) were produced using homologous recombination. The results showed that, ΔhexA and ΔhexAC were successfully constructed. And the data analysis indicated that the colony diameter, stress sensitivity and the sclerotia formation of A. flavus were nearly not affected by the absence of HexA. Yet, the deletion of hexA gene reduced the production of asexual spores and lessened virulence on peanuts and maize seeds markedly. In addition, it was also found that there was a significant decrease of Aflatoxin B1 production in deletion mutant, when compared to wild type. CONCLUSIONS: Therefore, it suggested that the hexA gene has an essential function in conidia production and secondary metabolism in A. flavus. The gene is also believed to be playing an important role in the invasion of A. flavus to the host.

Osmotic-Adaptation Response of sakA/hogA Gene to Aflatoxin Biosynthesis, Morphology Development and Pathogenicity in Aspergillus flavus.

Aspergillus flavus is one of the fungi from the big family of Aspergillus genus and it is capable of colonizing a large number of seed/crops and living organisms such as animals and human beings. SakA (also called hogA/hog1) is an integral part of the mitogen activated protein kinase signal of the high osmolarity glycerol pathway. In this study, the AfsakA gene was deleted (∆AfsakA) then complemented (∆AfsakA::AfsakA) using homologous recombination and the osmotic stress was induced by 1.2 mol/L D-sorbital and 1.2 mol/L sodium chloride. The result showed that ∆AfsakA mutant caused a significant influence on conidial formation compared to wild-type and ∆AfsakA::AfsakA strains. It was also found that AfsakA responds to both the osmotic stress and the cell wall stress. In the absence of osmotic stress, ∆AfsakA mutant produced more sclerotia in contrast to other strains, whereas all strains failed to generate sclerotia under osmotic stress. Furthermore, the deletion of AfsakA resulted in the increase of Aflatoxin B1 production compared to other strains. The virulence assay on both maize kernel and peanut seeds showed that ∆AfsakA strain drastically produced more conidia and Aflatoxin B1 than wild-type and complementary strains. AfSakA-mCherry was located to the cytoplasm in the absence of osmotic stress, while it translocated to the nucleus upon exposure to the osmotic stimuli. This study provides new insights on the development and evaluation of aflatoxin biosynthesis and also provides better understanding on how to prevent Aspergillus infections which would be considered the first step towards the prevention of the seeds damages caused by A. flavus.

PbsB Regulates Morphogenesis, Aflatoxin B1 Biosynthesis, and Pathogenicity of Aspergillus flavus.

As an opportunistic pathogen, Aspergillus flavus is one of the major causes of food contamination around the world. In this study, pbsB gene knockout mutant (ΔpbsB) and pbsB overexpression strain (OE) of A. flavus were constructed by homologous recombination. The results showed that the mycelia growth, conidiation, and the formation of sclerotia in ΔpbsB mutant were significantly suppressed, and up-regulated in OE strian compared to wild-type strain (WT). Q-PCR analysis showed that PbsB regulated the sclerotia formation through sclerotia related gene nsdC. With TLC and qRT-PCR analysis, it was found that PbsB up-regulated the bio-synthesis of aflatoxin B1 (AFB1) through regulatory gene aflR and structural gene aflC, aflD, aflK, and aflQ in the aflatoxin gene cluster. In osmotic stress response analysis, ΔpbsB mutant was significantly more sensitive to osmotic pressure with 1.2 mol/L sorbitol, compared to WT and OE strains. In virulence analysis, the infection capacity of ΔpbsB strain to peanut and maize kernels decreased dramatically, and significantly fewer spores and lesser mycelia were produced in ΔpbsB strain on the surface of peanut and maize kernels, and the infection capacity of OE strain to kernels increased significantly compared with WT strain. The AFB1 bio-synthesis ability of A. flavus in crop invasion models was also found to be coincide with the expression level of pbsB. All the results of the study shows that, as a MAPKK, PbsB is critical for growth and virulence in A. flavus, and lay a theoretical foundation for the prevention and control of A. flavus contamination.

A system biology approach to understanding the molecular mechanisms of Gubentongluo decoction acting on IgA Nephropathy.

BACKGROUND: Traditional Chinese medicine (TCM) has been widely used in treating various diseases in eastern Asia for several thousand years, and is becoming increasingly popular in western countries. Gubentongluo (GBTL) decoction, as a classic TCM formula, is commonly applied to treat IgA Nephropathy (IgAN) in China. To date, however, the pharmacological/molecular mechanisms of GBTL have not been fully elucidated. METHOD: In the present study, we used a system biological approach to explore these mechanisms acting on IgAN. RESULTS: First, we found 3876 potential target proteins for GBTL (based on TCMID) and 25 known IgAN associated biomarkers (based on the OMIM or IPA database).16 of the latter biomarkers were direct targets of 6 of the 9 herbs in GBTL, suggesting that these components play a vital role in treating IgAN. Second, we showed that these 6 herbs mainly regulate the immune system and renin-angiotensin system, imbalance in which is the main factor leading to IgAN. Importantly, HUANG QI links with 14 biomarkers, indicating that it is the most important herb in GBTL for treating IgAN. Also, relationships of other herbs with IgAN were explored. Third, we demonstrated that the remaining 9 IgAN associated proteins are responses to biological processes, such as antigen processing, protein ubiquitination and cell cycle regulation, which are crucial for IgAN development. Finally, we found that GBTL could induce a significant increase in the levels of two target gene: TNF and NOS2. CONCLUSIONS: Further studies are called to develop/modify the formula of GBTL, in order to enhance its effect on IgAN.

Urinary podocyte can be an indicator for the pathogenetic condition of patients with IgA nephropathy.

BACKGROUND: To evaluate the clinical significance of the urinary podocytes in patients with IgA nephropathy. METHODS: Urine samples from 102 biopsy-confirmed IgA nephropathy patients were collected to detect urinary podocytes using an indirect immunofluorescence staining method with anti-human Podocalyxin (PCX) antibody. Then, correlation analysis was performed between the urinary podocyte counts and the clinicopathological changes. RESULTS: Upon comparison with IgA nephropathy patients with negative urinary podocytes, IgA nephropathy patients with positive urinary podocytes presented a significant reduction in plasma albumin and glomerular filtration rate and remarkable rise in urinary protein excretion, blood cholesterol, and mean arterial pressure. Pathologically, the renal tissues of IgA nephropathy patients with positive urinary podocytes presented less podocytes in the glomerulus (6.03 ± 3.61 cells/glomerulus vs. 12.58 ± 7.23 cells/glomerulus, p < 0.001), more mesangial matrix, and more aggravated interstitial fibrosis and foot process fusion than in IgA nephropathy patients with negative urinary podocytes. In addition, the urinary podocyte counts were positively correlated with serum creatinine and 24-hour urinary protein excretion (r = 0.332, p < 0.05 and r = 0.387, p < 0.05, respectively) and negatively correlated with the number of podocytes in the renal tissues (r = -0.416, p 0 < 0.05). CONCLUSIONS: Detection of urinary podocytes can be a noninvasive indicator for reflecting the severity of IgA nephropathy.

[Simulation model for the crop development stages in sunflower-potato intercropping].

Potato-sunflower intercropping is a prevailing cropping system in the agricultural and pastoral ecotone in China. To precisely simulate the crop phenology in the intercropping system is of significance for the assessment and optimization of intercropping systems. In this paper, the simulation model for the development stages of sunflower and potato in monoculture and intercropping was established, based on the crop's physiological development time, and validated with the field experimental data from 2010 to 2011. A good fitness was observed between the simulated and observed values of the crop's development stages. The root mean square error (RMSE) of the development stages from sowing to emergence, emergence to flowering, flowering to mature, and from sowing to mature was 1.2, 2.9, 2.4 and 2.6 d, respectively, with the prediction error lower than 5%. The model was strong on mechanistic, explanation and adaptability, and could be applied as a good tool in the researches of crop growth and development.