Association of recurrent APOBEC3B alterations with the prognosis of gastric-type cervical adenocarcinoma.

OBJECTIVE: Gastric-type cervical adenocarcinoma (GCA) is a rare and aggressive type of endocervical adenocarcinoma (ECA) with distinct histopathologic features and unfavorable treatment outcomes, but no genomic prognostic factor has been revealed. We aimed to systematically investigate the somatic alterations of GCA at genome-wide level and evaluate their prognostic value. METHODS: We performed whole-exome sequencing (WES) on 25 pairs of tumor and matched normal samples to characterize the genomic features of Chinese patients with GCA and investigated their relations to histopathological characterizations and prognosis. The prognostic value of the genomic alterations was evaluated in a total of 58 GCA patients. RESULTS: Mutations were commonly observed in reported GCA-related driver genes, including TP53 (32%), CDKN2A (20%), SKT11 (20%), BRCA2 (12%), SMAD4 (12%), and ERBB2 (12%). Recurrent novel trunk mutations were also observed in PBRM1 (12%), FRMPD4 (12%), and NOP2 (8%) with high variant allele frequency. Moreover, enrichment of the APOBEC signature was attributed to frequent gain of somatic copy number alteration (SCNA) of APOBEC3B (20%), which perfectly matched the nuclear-positive staining of APOBEC3B through immunohistochemistry. In contrast, APOBEC3B alteration was absent in patients with conventional type of ECA (N = 52). Notably, positive APOBEC3B was consistently enriched in patients with favorable prognosis in both the discovery cohort and an additional 33 GCA patients, thus indicating a significant association with lower relapse risk of GCA independent of cancer stage (P = 0.02). CONCLUSION: Our results can aid understanding of the molecular basis of GCA in the Chinese population by providing genomic profiles and highlighting the potential prognostic value of APOBEC3B for GCA through routine clinical IHC.

Efficacy and safety of nivolumab for advanced/recurrent non-small-cell lung cancer: an up-to-date meta-analysis of large-scale phase III randomized controlled trials.

Aim: To evaluate the efficacy and safety of nivolumab for advanced/recurrent non-small-cell lung cancer (NSCLC). Methods: Electronic databases were searched, and a systematic evaluation was performed. Results: Seven phase III randomized controlled trials and 4705 patients were included. Nivolumab improved overall survival, progression-free survival and overall response rate for advanced/recurrent NSCLC. The efficacy of nivolumab was not correlated with PD-L1 expression levels. Nivolumab combination therapy was superior to nivolumab monotherapy. NSCLC patients may benefit from nivolumab in both first-line and second-line treatment. Nivolumab did not enhance the risk of commonly reported adverse events such as fatigue, hepatotoxicity and gastrointestinal events, but enhanced the risk of immune-related adverse events. Nivolumab-associated pneumonitis is a concern. Conclusion: Nivolumab showed improved efficacy for advanced/recurrent NSCLC but increased the risk of immune-related adverse events.

[Expression and Role of PD-L1 in a Mouse Model of Necrotizing Enterocolitis].

Objective: To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC). Methods: A total of 20 wild-type C57 BL/6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L1 (+/+) mice and 14 PD-L1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors. Results: The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P<0.05). According to the results of the PD-L1 gene mouse experiment, the control groups of PD-L1 (+/+) mice and PD-L1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L1 (+/+) NEC mouse ( P<0.05). In addition, serum interleukin (IL)-10 in PD-L1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1ß all increased by more than 25% (all P<0.05). Conclusion: PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes.

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome-specific painting.

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide-based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.

The bromodomain and extra-terminal domain inhibitor JQ1 synergistically sensitizes human colorectal cancer cells to topoisomerase I inhibitors through repression of Mre11-mediated DNA repair pathway.

Camptothecin (CPT) and its derivatives, irinotecan and topotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs. Mechanistically, they induce DNA double-strand breaks (DSBs). Although CPT is an effective chemotherapeutic agent used in the management of advanced colorectal cancer, there exist associated side effects. Herein, we aimed to establish novel drug combinations that can effectively aid in managing the CPT-related side effects. Besides, bromodomain and extra-terminal domain (BET) inhibitors have proved as promising drugs that target epigenetic mechanisms in various cancers, they alter DNA repair processes, hence are a potential candidate for CPT synthetic lethality. A novel BET inhibitor JQ1 synergized with CPT, exerted antiproliferative effects. Through cell cycle analyses and apoptosis assays, we revealed that a combination of CPT and JQ1 induces subG1-phase arrest and enhances cell apoptosis. This combination increased the intensity of γ-H2AX staining, a specific marker of DSBs. Moreover, colorectal cancer cells highly expressing Top1 showed greater sensitivity to JQ1, which was lowered through the lentiviral shRNA-mediated knockdown of Top1. JQ1, combined with CPT, impeded the recruitment of the Mre11-mediated MRN complex. Finally, JQ1 enhanced the in vivo sensitivity of tumors to CPT without inducing toxicity. These results demonstrate that a combination of BET inhibitor with Top1 inhibitor is safe and exerts positive chemotherapeutic effects in colorectal cancer.

[An Improved Acute Pancreatitis Apoptosis/Necrosis In vitro Model of Acinar Cells].

OBJECTIVE: To improve the apoptosis/necrosis In vitromodel of ascites-induced acute pancreatitic (AP) acinar cells by using the co-culture system and mixed ascites technology for the first time. Furthermore, we compared this improved model with cerulein and cerulein+LPS models, and observed the effects of three models on acinar apoptotic/necrotic-related indicators. METHODS: The In vitrocultured rat acinar cells AR42J were divided in four groups: control group (medium+PBS), cerulein group (medium+10 nmol/L cerulein), cerulein+LPS group (medium+10 nmol/L cerulein+10 mg/L LPS) and improved ascites group. In the improved ascites group, the ascites of sodium taurocholate-induced rat model was mixed and added into the co-culture system to stimulated In vitrocultured homogenous acinar cells. The co-culture system was set as follows: the chambers with the pore size of 1 µm were placed in the cultue plate, and the culture medium and mixed ascites were respectively added to the culture plate and the chamber at a ratio of 1:1. The acinar cells in each group were collected after 24 h stimulation. The apoptotic/necrotic rates, the expressions of apoptosis/necrosis related proteins [B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X protein (Bax) and receptor interacting protein 1 (RIP1)], and the ultra-structure of acinar cells were detected by flow cytometry, Western blot and transmission electron microscopy (TEM). RESULTS: The acinar cells in the improved ascites model were mainly characterized by necrosis; compared with the other 3 groups, the apoptosis rate and necrosis rate were both up-regulated, RIP1 and BAX protein expression levels were up-regulated, and Bcl-2 protein was down-regulated. TEM results showed the organelle structure of acinar cells was destroyed, and the cell membrane was degraded in the improved ascites model. Compared with the control group, apoptosis rate of acinar cells in the cerulein and cerulein+LPS models were increased and necrosis rate were not changed. The expression of pro-apoptotic protein Bax was increased, while the expression level of RIP1 was not significantly increased. TEM results showed that in cerulein group and cerulein+LPS group, the chromatin of the cells was condensed into a mass, the cytoplasm was degraded and the cell membrane was intact, showing typical apoptosis characteristics. CONCLUSION: Compared with cerulein and cerulein +LPS models, which mainly focus on apoptosis of acinar cells and applied to mild acute pancreatitis study, the improved ascites model mainly focuses on the necrosis of acinar cells and is a good model for studying acinar cell necrosis and severe acute pancreatitis.

Comparative transcriptome and proteome profiling of two Citrus sinensis cultivars during fruit development and ripening.

BACKGROUND: Transcriptome and proteome analyses on fruit pulp from the blood orange 'Zaohong' and the navel orange 'twenty-first century' were performed to study Citrus sinensis quality-related molecular changes during consecutive developmental periods, including young fruit, fruit-coloring onset and fruit delayed-harvest for two months, during which fruit remained on the trees. RESULTS: The time-course analysis for the fruit developmental periods indicated a complex, dynamic gene expression pattern, with the numbers of differentially expressed genes (DEGs) between the two cultivars being 119, 426 and 904 at the three continuous stages tested during fruit development and ripening. The continuous increase in total soluble solids over the course of fruit development was correlated with up-regulated sucrose phosphate synthase (SPS) transcription levels in both cultivars. Eleven differentially expressed genes between the two cultivars involved in the flavonoid pathway were significantly enriched at the onset of the fruit-coloring stage when anthocyanins were detected in blood orange alone. Among 5185 proteins, 65 up-regulated and 29 down-regulated proteins were co-expressed with their cognate mRNAs with significant transcription and protein expression levels when the fruits from the two cultivars were compared at the fruit delayed-harvest stage. Additionally, important genes participating in the γ-aminobutyric acid (GABA) shunt were activated in blood orange at two significant expression levels in the fruit delayed-harvest stage. Thus, organic acids in fruit continuously decreased during this stage. CONCLUSIONS: This research was the first to provide a more comprehensive understanding of the differentially expressed genes involved in anthocyanin, sucrose and citrate metabolism at the transcriptome and proteome levels in C. sinensis, especially during the fruit delayed-harvest stage.

Resolvin D1 protects against inflammation in experimental acute pancreatitis and associated lung injury.

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure with considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1 in acute pancreatitis and associated lung injury. Acute pancreatitis varying from mild to severe was induced by cerulein or cerulein combined with LPS, respectively. Mice were pretreated with RvD1 at a dose of 300 ng/mouse 30 min before the first injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by Western blotting. The injection of cerulein or cerulein combined with LPS resulted in local injury in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the cerulein and LPS group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α, and IL-6 serum levels; the MPO activities in the pancreas and the lungs; the pancreatic NF-κB activation; and the severity of pancreatic injury and associated lung injury, especially in the severe acute pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas and lung and exerting anti-inflammatory effects through the inhibition of NF-κB activation in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis. These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of severe acute pancreatitis.

Effects of Tocilizumab on Experimental Severe Acute Pancreatitis and Associated Acute Lung Injury.

OBJECTIVE: To examine the therapeutic effects of tocilizumab, an antibody against interleukin-6 receptor, on experimental severe acute pancreatitis and associated acute lung injury. The optimal dose of tocilizumab and the activation of interleukin-6 inflammatory signaling were also investigated. DESIGN: Randomized experiment. SETTING: Research laboratory at a university hospital. SUBJECT: Experimental severe acute pancreatitis in rats. INTERVENTIONS: Severe acute pancreatitis was induced by retrograde injection of sodium taurocholate (50 mg/kg) into the biliopancreatic duct. In dose-study, rats were administered with different doses of tocilizumab (1, 2, 4, 8, and 16 mg/kg) through the tail vein after severe acute pancreatitis induction. In safety-study, rats without severe acute pancreatitis induction were treated with high doses of tocilizumab (8, 16, 32, and 64 mg/kg). Serum and tissue samples of rats in time-study were collected for biomolecular and histologic evaluations at different time points (2, 6, 12, 18, and 24 hr). MEASUREMENTS AND MAIN RESULTS: 1) Under the administration of tocilizumab, histopathological scores of pancreas and lung were decreased, and severity parameters related to severe acute pancreatitis and associated lung injury, including serum amylase, C-reactive protein, lung surfactant protein level, and myeloperoxidase activity, were all significant alleviated in rat models. 2) Dose-study demonstrated that 2 mg/kg tocilizumab was the optimal treatment dose. 3) Basing on multi-organ pathologic evaluation, physiological and biochemical data, no adverse effect and toxicity of tocilizumab were observed in safety-study. 4) Pancreatic nuclear factor-κB and signal transducer and activator of transcription 3 were deactivated, and the serum chemokine (C-X-C motif) ligand 1 was down-regulated after tocilizumab administration. CONCLUSIONS: Our study demonstrated tocilizumab, as a marketed drug commonly used for immune-mediated diseases, was safe and effective for the treatment of experimental severe acute pancreatitis and associated acute lung injury. Our findings provide experimental evidences for potential clinical application of tocilizumab in severe acute pancreatitis and associated complications.

PKA RIα/A-kinase anchoring proteins 10 signaling pathway and the prognosis of colorectal cancer.

BACKGROUND AND AIM: Previously study showed that the loss of the control of cAMP-dependent protein kinase A RIα (PKA RIα)/ A-kinase anchoring proteins 10 (AKAP10) signaling pathway initiate dysregulation of cellular healthy physiology leading to tumorigenesis. The aim of this study was to investigate the role of PKA RIα/AKAP10 signaling pathway in colorectal cancer (CRC). METHODS: The AKAP10 expression at the mRNA and protein level have been analyzed in colon cancer cell lines, primary CRCs and matched normal mucosa samples, and compared in accordance with specific clinicopathological features of CRC. The correlation between expression of AKAP10 and PKA RIα were also analyzed. RESULTS: Compared with HCT116 and SW480 cells, the AKAP10 was significantly upregulated in the colon cell line KM12C and its metastatic counterparts, KM12SM and KM12L4A. Moreover, the KM12SM and KM12L4A having high metastatic potentials displayed the elevated levels of AKAP10 compared with KM12C having poor metastatic potential. A notably higher level of AKAP10 expression was found in CRC tissues at both mRNA and protein levels. Increased expression of AKAP10 in CRC patients was positively associated with the depth of invasion and the grade of differentiation. Univariate survival analysis showed that the increased expression of AKAP10 was related to poorer survival. Cox multivariate regression analysis confirmed that AKAP10 was an independent predictor of the overall survival of CRC patients. PKA RIα mRNA was also expressed at high levels in CRC. The correlation coefficient between mRNA expression of AKAP10 and PKA RIα in CRC was 0.417. AKAP10 mRNA overexpression was correlated significantly with PKA RIα. CONCLUSIONS: Our data indicated that PKA RIα/AKAP10 signaling pathway is associated with the progression and prognosis of CRC.

[Potential therapeutic effect of paracrine factors from bone marrow-derived mesenchyme stem cells in the treatment of severe acute pancreatitis].

Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is one ot the most popular therapeutic measures in severe acute pancreatitis (SAP). However, technical challenges and ethical concern have hindered its clinical application. Paracrine factor, as a new safe and easy handing therapeutic measure, can work comparably effective as BMSC transplantation in SAP therapy, but bio-safe risks could be greatly reduced. In this paper, we reviewed the therapeutic effect and potential mechanism of paracrine factors in the treatment of SAP. The injection of paracrine factors yielded from cultured cell suspension will be a new cell therapeutic measure for SAP.

Critical role of myeloid differentiation factor 88 in necrotizing enterocolitis.

BACKGROUND: The importance of toll-like receptor 4 in necrotizing enterocolitis (NEC) has been intensively studied, but its downstream signaling and the potential regulatory mechanisms remain unidentified. Our study focused on the role of myeloid differentiation factor 88 (MyD88), the first downstream adaptor of toll-like receptor 4 inflammatory and apoptotic signaling in the pathogenesis of NEC. METHODS: MyD88 knockout (MyD88(-/-)-Ko) mice and lentivirus-mediated stable MyD88-knockdown cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide (LPS) in vivo, or LPS stimulation in vitro. NEC was evaluated by histology and multiple inflammatory cytokines. Enterocyte apoptosis was evaluated by terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) or Annexin analysis. Inflammatory or apoptotic molecules including NF-κB, Toll/IL-1R domain-containing adaptor-inducing IFN-ß, interferon regulatory factor 3, Bax, Bcl-2, and caspases were examined by quantitative real-time PCR (qRT-PCR). RESULTS: In the MyD88-Ko group, NEC severity and intestinal enterocyte apoptosis rate were reduced, the expression of NF-κB, caspases, and Bax, were all downregulated, while Toll/IL-1R domain-containing adaptor-inducing IFN-ß and were upregulated, and antiapoptotic gene Bcl-2 remained stable. Cytokine levels of interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α) were also all decreased. CONCLUSION: MyD88-dependent signaling is the prevailing inflammatory and apoptotic signaling in toll-like receptor 4 downstream signaling; MyD88-Ko resulted in reduced inflammatory severity and apoptosis, though MyD88-independent signaling can also be activated, but is of less dominant for the development of NEC.

Buyang Huanwu decoction ameliorates bleomycin-induced pulmonary fibrosis in rats by attenuating the apoptosis of alveolar type II epithelial cells mediated by endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the pathogenesis of idiopathic pulmonary fibrosis (IPF) can be attributed to qi deficiency and blood stasis. Buyang Huanwu decoction (BHD), a representative Chinese herbal prescription for qi deficiency and blood stasis syndrome, is widely used to treat IPF in clinical practice. However, its potential mechanisms against IPF remain unclear. AIMS OF THE STUDY: This study was carried out to explore the therapeutic effects and underlying mechanisms of BHD on bleomycin (BLM)-induced pulmonary fibrosis in rats. MATERIALS AND METHODS: UPLC-MS/MS method was performed to identify the quality of BHD used in this study. Concurrently, a IPF rat model was established by single intratracheal injection of BLM. Pulmonary function test, H&E staining, Masson staining, hydroxyproline assay were conducted to evaluate the therapeutic effects of BHD on BLM-induced pulmonary fibrosis in rats, and the regulatory effect of BHD on endoplasmic reticulum stress (ERS)-mediated alveolar type II epithelial cells (AEC2s) apoptosis in rats was further investigated by TUNEL staining, Western blot, real-time fluorescence quantitative PCR and immunofluorescence co-staining to reveal the potential mechanisms of BHD against IPF. RESULTS: The UPLC-MS/MS analysis showed that the BHD we used complied with the relevant quality control standards. The data from animal experiments confirmed that BHD administration ameliorated BLM-induced pulmonary function decline, lung fibrotic pathological changes and collagen deposition in rats. Further mechanism study revealed that BHD increased the Bcl-2 protein expression, decreased the Bax protein expression and inhibited the cleavage of CASP3 via suppressing the activation of PERK-ATF4-CHOP pathway under continuous ERS, thereby alleviating BLM-induced AEC2s apoptosis of rats. CONCLUSION: This study demonstrated that BHD ameliorated BLM-induced pulmonary fibrosis in rats by suppressing ERS-mediated AEC2s apoptosis. Our findings can provide some fundamental research basis for the clinical application of BHD in the treatment of IPF.

PD-L1 blockade in mitigating severe acute pancreatitis induced pancreatic damage through modulation of immune cell apoptosis.

Acute pancreatitis (AP) is a prevalent gastrointestinal disorder. The immune response plays a crucial role in AP progression. However, the impact of immune regulatory checkpoint PD-L1 on severe acute pancreatitis (SAP) remains uncertain. Hence, this study aimed to examine the influence of PD-L1 on SAP. We assessed PD-L1 expression in neutrophils and monocytes obtained from SAP patients. We induced SAP in C57BL/6J mice, PD-L1 gene-deficient mice, and PD-L1 humanized mice using intraperitoneal injections of cerulein plus lipopolysaccharide. Prior to the initial cerulein injection, a PD-L1 inhibitor was administered. Pancreatic tissues were collected for morphological and immunohistochemical evaluation, and serum levels of amylase, lipase, and cytokines were measured. Flow cytometry analysis was performed using peripheral blood cells. The expression of PD-L1 in neutrophils and monocytes was significantly higher in SAP patients compared to healthy individuals. Likewise, the expression of PD-L1 in inflammatory cells in the peripheral blood of SAP-induced C57BL/6J mice was notably higher than in the control group. In mice with PD-L1 deficiency, SAP model exhibited lower pancreatic pathology scores, amylase, lipase, and cytokine levels compared to wild-type mice. PD-L1 deletion resulted in reduced neutrophil apoptosis, leading to an earlier peak in neutrophil apoptosis. Furthermore, it decreased early monocyte apoptosis and diminished the peak of T lymphocyte apoptosis. Within the SAP model, administration of a PD-L1 inhibitor reduced pancreatic pathology scores, amylase, lipase, and cytokine levels in both C57BL/6J mice and PD-L1 humanized mice. These findings suggest that inhibiting PD-L1 expression can alleviate the severity of SAP.

Enhancing the emulsion properties and bioavailability of loaded astaxanthin by selecting the reaction sequence of ternary conjugate emulsifiers in nanoemulsions.

This study investigated the effects of the conjugate reaction sequences of whey protein concentrate (WPC), epigallocatechin gallate (EGCG) and dextran (DEX) on the structure and emulsion properties of conjugates and the bioaccessibility of astaxanthin (AST). Two types of ternary covalent complexes were synthesised using WPC, EGCG and DEX, which were regarded as emulsifiers of AST nanoemulsions. Results indicated that the WPC-DEX-EGCG conjugate (referred to as 'con') exhibits a darker SDS-PAGE dispersion band and higher contents of α-helix (6%), ß-angle (24%) and random coil (32%), resulting in a greater degree of unfolding structure and fluorescence quenching. These findings suggested WPC-DEX-EGCG con had the potential to exhibit better emulsification properties than WPC-EGCG-DEX con. AST encapsulation efficiency (76.22%) and bioavailability (31.89%) also demonstrated the superior performance of the WPC-DEX-EGCG con emulsifier in nanoemulsion delivery systems. These findings indicate that altering reaction sequences changes protein conformation, enhancing the emulsification properties and bioavailability of AST.

Tiao-Bu-Fei-Shen Formula Improves Glucocorticoid Resistance of Chronic Obstructive Pulmonary Disease via Downregulating the PI3K-Akt Signaling Pathway and Promoting GRα Expression.

Objective: To predict and determine the mechanism through which Tiao-Bu-Fei-Shen (TBFS) formula improves glucocorticoid resistance in chronic obstructive pulmonary disease (COPD), using network pharmacology, molecular docking technology, and in vitro studies. Methods: The main active components and associated targets of TBFS were screened using the systems pharmacology database of traditional Chinese medicine database (TCMSP). The main COPD targets were retrieved from the Human Gene (GeneCards) and DrugBank databases. A protein-protein interaction (PPI) network was constructed using the protein interaction platform STRING and Cytoscape 3.6.1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genome Pathway (KEGG) analyses were performed using the biological information annotation database Metascape. Molecular docking was performed using the AutoDock Vina software. THP-1 monocytes were treated with TBFS-containing serum and cigarette smoke extract (CSE) for 48 h, and cell proliferation in each group was determined using cell counting kit-8 (CCK-8). A COPD cell model was constructed by stimulating THP-1 monocytes with CSE for 12 h. A lentivirus vector for RNA interference of histone deacetylase 2 (HDAC2) gene was constructed and transfected into the THP-1 monocytes, and the transfection efficiency was verified using quantitative polymerase chain reaction (qPCR) and western blotting (WB). The expression of HDAC2 in each group of cells was detected using qPCR, and the expression of HDAC2, phosphoinositide-3 kinase (PI3K) p85α, glucocorticoid receptor α (GRα), and P-AKT1 in each group of cells was detected through WB. Results: A total of 344 TBFS active components, 249 related drug targets, 1,171 COPD target proteins, and 138 drug and disease intersection targets were obtained. Visual analysis of the PPI network map revealed that the core COPD targets of TBFS were AKT1, IL-6, TNF, TP53, and IL1-ß. KEGG pathway enrichment analysis resulted in the identification of 20 signaling pathways as the main pathways involved in the action of TBFS against COPD, including the PI3K-Akt, TNF, and IL-17 signaling pathways. Molecular docking experiments revealed a strong binding capacity of kaempferol, luteolin, and quercetin to the ATK1 protein in TBFS, with quercetin performing the best. PCR results showed that treatment with TBFS significantly increased the expression levels of HDAC2 in the COPD model. WB results showed that TBFS treatment significantly increased the expression levels of GRα and HDAC2 in the COPD model, while reducing the expression levels of P-AKT1. Conclusion: TBFS treatment improves glucocorticoid resistance observed in COPD through downregulation of the PI3K-Akt signaling pathway and promotion of GRα expression.

A Preliminary Study on Warm Acupuncture and Moxibustion for Treating Chronic Obstructive Pulmonary Disease with Abdominal Distension.

Most patients with COPD have a combination of abdominal distension, which has been shown to adversely affect pulmonary symptoms, frequency of acute exacerbations, and quality of life in patients with COPD. Warm acupuncture and moxibustion have been shown to be effective in relieving symptoms in patients with COPD combined with abdominal distention. Warm acupuncture and moxibustion are highly effective, easy to perform, and inexpensive forms of traditional Chinese medicine treatments. The standardized practice of warm acupuncture and moxibustion is very important for the treatment of COPD combined with abdominal distension. The specific steps include selecting the appropriate acupoints for needling through syndrome differentiation treatment and selecting moxa sticks of appropriate length for moxibustion for about 30 min after the De-qi. The course of treatment lasts for one week. The following indicators are specifically assessed: the score of the COPD Assessment Test (CAT) and the abdominal distension visual analog scale (VAS). This article will clearly illustrate how to standardize the manipulation of warm acupuncture and moxibustion to relieve COPD combined with abdominal distention.

Transcriptome and anatomical studies reveal alterations in leaf thickness under long-term drought stress in tobacco.

Drought is one of the foremost environmental factors that limit the growth of plants. Leaf thickness (LT) is an important quantitative trait in plant physiology. The experiment was carried out in a growth room and the plants were divided into two groups such as well-watered and drought-stressed. This work investigated leaf growth in terms of leaf surface growth and expansion rate, leaf stomata traits, LT, anticlinal growth, and leaf cell layers. The results showed that the leaf area and leaf surface expansion rate were decreased by drought stress (DS). Similarly, LT, anticlinal expansion rate, palisade and spongy tissue thickness, and their related expansion rates were also decreased at different days' time points (DTP) of DS. However, a steady increase was observed in the aforementioned parameters after 12 DTP of DS. The stomatal density increased while stomata size decreased at 3 DTP and 12 DTP (low leaf water potential and relative leaf water content at these time points) and vice versa at 24 DTP compared with the well-watered plants indicating adaptations in these traits in response to DS, and thus the leaf water status played a role in the regulation of leaf stomata traits. The cell length decreased in the upper epidermis, palisade and spongy tissues by DS up to 12 DTP led to lower LT while an increase was observed after 12 DTP that resulted in higher LT. The increase in the LT was supported by the upregulation of starch and sucrose metabolism, glycerolipid metabolism, protein processing in endoplasmic reticulum pathways at 18 DTP along with the differentially expressed genes induced that were related to cell wall remodeling (cellulose, expansin, xyloglucans) and cell expansion (auxin response factors and aquaporin). The results explain the response of leaf thickness to drought stress and show alterations in LT and leaf stomatal traits. This study might serve as a valuable source of gene information for functional studies and provide a theoretical basis to understand leaf growth in terms of leaf anatomy and leaf stomatal traits under drought stress.

Research progress on the mechanism of astragaloside IV in the treatment of asthma.

Asthma is a common chronic respiratory disease, and its treatment is a core problem and challenge in clinical practice. Glucocorticoids (GCs) are the first-line therapy for the treatment of asthma. Local and systemic adverse reactions caused by GCs create obstacles to the treatment of asthma. Therefore, the research target is to find a new, safe, and effective therapeutic medicine at present. Natural products are an important source for treating asthma with low cost and low toxicity. Astragaloside IV (AS-IV) is an active ingredient of traditional Chinese medicine Astragalus mongholicus Bunge. Previous studies have indicated that AS-IV plays a therapeutic role in the treatment of asthma by inhibiting airway inflammation and remodeling the airway, and by regulating immunity and neuroendocrine function (Fig. 1) . It has a variety of biological characteristics such as multi-target intervention, high safety, and good curative effect. This article reviews the specific mechanism of AS-IV for the treatment of asthma to provide references for subsequent research.

PNAS-4 expression and its relationship to p53 in colorectal cancer.

PNAS-4 is a novel pro-apoptotic protein activated during the early response to DNA damage; however, the molecular mechanisms and pathways regulating PNAS-4 expression in tumors are not well understood. We hypothesized that PNAS-4 is a p53 down-stream target gene and designed this study. We searched online for putative p53-binding sites in the entire PNAS-4 gene and did not find any corresponding information. In HCT116 colon cancer cells, after being transfected with small interfering RNA to silence p53, the expressions of PNAS-4 and other known p53 target gene (Apaf1, Bax, Fas and Dr5) were determined by real-time PCR. We found that PNAS-4 was up-regulated while Apaf1, Bax, Fas and Dr5 were down-regulated. We then examined the expression of PNAS-4 and p53 mutation in colorectal cancer patients. PNAS-4 expressed both in colorectal cancers and normal tissues, but compared with paired control, PNAS-4 was up-regulated in cancers (P=0.018). PNAS-4 overexpression ratios were correlated to the p53 mutant status (P=0.001). The mean PNAS-4 expression levels of p53 mutant homozygote group and heterozygote group were higher than that of p53 wild type group (P=0.013). The expression ratios of PNAS-4 (every sample in relative to its paired normal mucosa) were different between negative lymph node metastasis (66% up-regulated, 34% down-regulated) and positive metastasis (42% up-regulated, 58% down-regulated). Taken together, these findings suggested that PNAS-4 was not a p53 target, but overexpression of PNAS-4 was correlated to p53 inactivity in colorectal cancer.