Relationship between biofilm formation and antibiotic resistance of Klebsiella pneumoniae and updates on antibiofilm therapeutic strategies.

Klebsiella pneumoniae is a Gram-negative bacterium within the Enterobacteriaceae family that can cause multiple systemic infections, such as respiratory, blood, liver abscesses and urinary systems. Antibiotic resistance is a global health threat and K. pneumoniae warrants special attention due to its resistance to most modern day antibiotics. Biofilm formation is a critical obstruction that enhances the antibiotic resistance of K. pneumoniae. However, knowledge on the molecular mechanisms of biofilm formation and its relation with antibiotic resistance in K. pneumoniae is limited. Understanding the molecular mechanisms of biofilm formation and its correlation with antibiotic resistance is crucial for providing insight for the design of new drugs to control and treat biofilm-related infections. In this review, we summarize recent advances in genes contributing to the biofilm formation of K. pneumoniae, new progress on the relationship between biofilm formation and antibiotic resistance, and new therapeutic strategies targeting biofilms. Finally, we discuss future research directions that target biofilm formation and antibiotic resistance of this priority pathogen.

Critical review on the research of chemical structure, bioactivities, and mechanism of actions of Dendrobium officinale polysaccharide.

Dendrobium officinale (Tie-Pi-Shi-Hu) is a precious traditional Chinese medicine (TCM). The principal active components are polysaccharides (DOP), which have a high potency in therapeutic applications. However, limitations in structure analysis and underlying mechanism investigation impede its further research. This review systemically and critically summarises current understanding in both areas, and points out the influence of starch impurities and the role of gut microbiota in DOP research. As challenges faced in studying natural polysaccharide investigations are common, this review contributes to a broader understanding of polysaccharides beyond DOP.

Effects of inoculation with a binary mixture of Lactobacillus plantarum and Leuconostoc citreum on cell wall components of Chinese Dongbei suancai.

This study aimed to investigate the effects of inoculation with a starter culture consisting of Lactobacillus plantarum LNJ002 and Leuconostoc citreum BNCC 194779 on microbial community, cell wall polysaccharide characteristics, cell wall degrading enzymes, and microstructure during Chinese Dongbei suancai fermentation. The results showed that Lactobacillus (98.75%) was the dominant genus during fermentation of Dongbei suancai. The principal coordinates analysis (PCoA) suggested that inoculation with Lactobacillus promoted the stability of microbial community structure during Chinese Dongbei suancai fermentation. Besides, the lower content in cellulose (80.28 ± 2.61 ug/mg) and pectin (53.56 ± 2.67 ug/mg) observed in the inoculated fermented suancai. Simultaneously, the inoculated fermented suancai had the most decreases in SR 1 (70.35%) and SR 3 (72.06%) and the most increase in SR 2 (950%), which suggested that inoculation intensified the decrease of the linearity and the RG-1 branching degree of pectin. The contents of polygalacturonase (PG) and pectin methylesterase (PME) in inoculated fermented suancai were 21.06% and 21.86% higher than those in naturally fermented suancai. In addition, the surface of suancai leaves gradually changed from smooth to rough during fermentation, which was accelerated by inoculation. Moreover, Lactobacillus, Aspergillus, Wallemia and Mucor were all negatively correlated with cellulose and GalA. These results revealed that inoculation promoted the formation of dominant genus structure during suancai fermentation, changed the effects of enzymes on the degradation of cell wall components, thereby accelerated the formation of Chinese Dongbei suancai texture.

Recent advances in qualitative and quantitative analysis of polysaccharides in natural medicines: A critical review.

Polysaccharides from natural medicines, being safe and effective natural mixtures, show great potential to be developed into botanical drugs. However, there is yet one polysaccharide-based case that has fulfilled the Botanical Guidance definition of a botanical drug product. One of the reasons is the analytical methods commonly used for qualitative and quantitative analysis of polysaccharides fall far behind the quality control criteria of botanical drugs. Here we systemically reviewed the recent advances in analytical methods. A critical evaluation of the strength and weaknesses of these methods was provided, together with possible solutions to the difficulties. Mass spectrometry with or without robust chromatographic separation was increasingly employed. And scientists have made significant progress in simplifying polysaccharide quantification by depolymerizing it into oligosaccharides. This oligosaccharides-based strategy is promising for qualitative and quantitative analysis of polysaccharides. And continuous efforts are still needed to develop a standardized quality control method that is specific, accurate, repeatable, and applicable for analyzing individual components in natural medicine formulas.

A lymphatic route for a hyperbranched heteroglycan from Radix Astragali to trigger immune responses after oral dosing.

Gut barrier makes a huge research gap between in vivo and in vitro studies of orally bioactive polysaccharides: whether/how they contact the related cells in vivo. A hyperbranched heteroglycan RAP from Radix Astragali, exerting antitumor and immunomodulatory effects in vitro and in vivo, is right an example. Here, we determined first that RAP's antitumor activity is immune-dependent. Being undegraded and non-absorbing, RAP quickly entered Peyer's patches (PPs) in 1 h where it directly targeted follicle dendritic cells and initiated antitumor immune responses. RAP was further delivered to mesenteric lymph node, bone marrow, and tumor. By contrast, the control Dendrobium officinale polysaccharide did not enter PPs. These findings revealed a blood/microbiota-independent and selective lymphatic route for orally administrated RAP to directly contact immune cells and trigger antitumor immune responses. This route bridges the research gap between the in vitro and in vivo studies and might apply to many other bioactive polysaccharides.

Aqueous cinnamon extract ameliorates bowel dysfunction and enteric 5-HT synthesis in IBS rats.

Cinnamon protects against irritable bowel syndrome with diarrhea (IBS-D) in humans, but its efficacy and underlying mechanism of action remain poorly understood. Maternally separated (MS) IBS-D rat model and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced post-inflammatory IBS-D rat model are characterized by visceral hyperalgesia and diarrhea. This study used the two models to evaluate the effect of cinnamon extract (CE) on bowel symptoms. The MS rat model was also used to explore its underlying anti-IBS mechanism. cinnamon extract reduced defecation frequency and visceral hyperalgesia in MS rats in a dose-dependent manner and effectively improved visceral hyperalgesia in TNBS rats. The efficacy of cinnamon extract was comparable to the positive drug serotonin receptor 3 (5-HT3) selective antagonist, Ramosetron. Excessive 5-HT, a well-known pathogenic factor for IBS, in the colon and circulation of IBS rats was reduced after cinnamon extract intervention. Both, gene and protein levels of the colonic 5-HT synthetase, Tryptophan Hydroxylase 1 (Tph1), were also decreased in CE-treated IBS rats. In addition, a luciferase assay revealed that cinnamon extract and its major components, catechin, procyanidin B1/2, cinnamic acid, and cinnamyl alcohol, significantly inhibited Tph1 transcription activity in vitro. These findings illustrated that aqueous cinnamon extract partially attenuated bowel symptoms in IBS models by directly inhibiting Tph1 expression and controlling 5-HT synthesis. This provides a scientific viewpoint for the use of cinnamon as a folk medication to treat IBS.

Transcriptome sequencing and metabolome analysis reveal the mechanism of Shuanghua Baihe Tablet in the treatment of oral mucositis.

Oral mucositis (OM) caused by cancer therapy is the most common adverse reaction in the radiotherapy of head and neck tumors. In severe cases, it can lead to the interruption of treatment, which affects the control of the disease and the quality of life. Shuanghua Baihe Tablet (SBT) is a traditional Chinese medicine (TCM) formula, which is administerd to treat OM in China. It has been clinically effective for more than 30 years, but the underlying mechanism is not completely understood. With the development of multiple omics, it is possible to explore the mechanism of Chinese herbal compound prescriptions. Based on transcriptomics and metabolomics, we explored the underlying mechanism of SBT in the treatment of OM. An OM model of rats was established by 5-FU induction, and SBT was orally administered at dosages of 0.75 and 3 g·kg-1·d-1. In order to search for SBT targets and related metabolites, the dysregulated genes and metabolites were detected by transcriptomics and metabolomics. Immune related indicators such as interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Treg cell disorders was analyzed by flow cytometry. Our results showed that SBT significantly alleviated the symptoms of OM rats and the inflammatory infiltration of ulcer tissues. After SBT administration, inflammatory related metabolic pathways including linoleic acid metabolism, valine, leucine and isoleucine biosynthesis were significantly altered. Furthermore, the production of proinflammatory factors like IL-17 and TNF-α, were also dramatically reduced after SBT administration. Besides, the infiltration degree of Treg cells in the spleen of OM modeling rats was significantly improved by SBT administration, thus maintaining the immune balance of the body. The current study demonstrates that SBT regulates inoleic acid metabolism, glycerophospholipid metabolism and amino acid metabolism, and inhibits IL-17/TNF signal transduction to restore Treg and Th17 cell homeostasis in OM rats, thereby alleviating chemotherapy-induced OM.

Integrated Transcriptomic Analysis Revealed Hub Genes and Pathways Involved in Sorafenib Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), a high mortality malignancy, has become a worldwide public health concern. Acquired resistance to the multikinase inhibitor sorafenib challenges its clinical efficacy and the survival benefits it provides to patients with advanced HCC. This study aimed to identify critical genes and pathways associated with sorafenib resistance in HCC using integrated bioinformatics analysis. Differentially expressed genes (DEGs) were identified using four HCC gene expression profiles (including 34 sorafenib-resistant and 29 sorafenib-sensitive samples) based on the robust rank aggregation method and R software. Gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. A protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and small molecules reversing sorafenib resistance were searched for using the connectivity map (CMAP) database. Pearson correlation and survival analyses of hub genes were performed using cBioPortal and Gene Expression Profiling and Interactive Analysis (GEPIA). Finally, the expression levels of hub genes in sorafenib-resistant HCC cells were verified using quantitative polymerase chain reaction (q-PCR). A total of 165 integrated DEGs (66 upregulated and 99 downregulated in sorafenib resistant samples compared sorafenib sensitive ones) primarily enriched in negative regulation of endopeptidase activity, extracellular exosome, and protease binding were identified. Some pathways were commonly shared between the integrated DEGs. Seven promising therapeutic agents and 13 hub genes were identified. These findings provide a strategy and theoretical basis for overcoming sorafenib resistance in HCC patients.

Radix Astragali polysaccharide RAP directly protects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression.

Radix Astragali polysaccharide RAP has been reported to play a crucial role in hematopoiesis without a clear mechanism. In this study, RAP's effects to enhance the recovery of cyclophosphamide (Cy)-suppressed bone marrow and blood cells is confirmed in vivo first. Confocal micrographs demonstrated the interesting direct binding of FITC-RAP to hematopoietic stem cells (HSC) in bone marrow. RAP protects both mice and human HSC in terms of cell morphology, proliferation, and apoptosis. RNA-sequencing and shRNA approaches revealed FOS to be a key regulator in RAP's protection. These evidences provide an unreported mechanism that RAP directly protects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression.

Illumination of Molecular Pathways in Multiple Sclerosis Lesions and the Immune Mechanism of Matrine Treatment in EAE, a Mouse Model of MS.

The etiology of multiple sclerosis (MS) is not clear, and the treatment of MS presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of MS and to define target genes of matrine, a quinolizidine alkaloid component derived from the root of Sophorae flavescens that effectively suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To this end, the GSE108000 gene data set in the Gene Expression Omnibus Database, which included 7 chronic active MS lesions and 10 control samples of white matter, was analyzed for differentially expressed genes (DEGs). X cell was used to analyze the microenvironmental differences in brain tissue samples of MS patients, including 64 types of immune cells and stromal cells. The biological functions and enriched signaling pathways of DEGs were analyzed by multiple approaches, including GO, KEGG, GSEA, and GSVA. The results by X cell showed significantly increased numbers of immune cell populations in the MS lesions, with decreased erythrocytes, megakaryocytes, adipocytes, keratinocytes, endothelial cells, Th1 cells and Tregs. In GSE108000, there were 637 DEGs, including 428 up-regulated and 209 down-regulated genes. Potential target genes of matrine were then predicted by the network pharmacology method of Traditional Chinese medicine, and 12 key genes were obtained by cross analysis of the target genes of matrine and DEGs in MS lesions. Finally, we confirmed by RT-PCR the predicted expression of these genes in brain tissues of matrine-treated EAE mice. Among these genes, 2 were significantly downregulated and 6 upregulated by matrine treatment, and the significance of this gene regulation was further investigated. In conclusion, our study defined several possible matrine target genes, which can be further elucidated as mechanism(s) of matrine action, and novel targets in the treatment of MS.

Temporal variation analysis and risk assessment of neonicotinoid residues from tea in China.

The extensive use of neonicotinoids (NEOs) has caused the release of wide-ranging of residues to the environment and food, and their potential health risks are now receiving more attention. In this study, three surveys were conducted to obtain the overall profiles of NEO residue levels (seven NEOs and one metabolite) in Chinese tea over a period of seven years. A total of 726 tea samples were tested, and nearly 87% of the samples were found to have detectable NEO residues. The overall average detection frequency of acetamiprid was the highest, reaching 73%. Imidacloprid residues in 4.6% of the samples exceeded the Chinese maximum residue limits, whereas clothianidin and nitenpyram had been detected in Chinese tea samples since 2014. The applications of thiacloprid and thiamethoxam gradually increased, and some tea samples with high residue levels appeared in China. These findings signal the replacement of new and old varieties of NEOs in China. Both long- and short-term cumulative exposures to NEOs were calculated based on optimistic and pessimistic models recommended in the EFSA guidelines. In the three survey periods, the average total imidacloprid-equivalent concentrations were 484.63, 1713.36, and 1148.34 µg/kg, respectively. Combined with the refined point estimates and probabilistic models used in this study, the hazard quotients of NEO residues in tea for Chinese tea consumers were found to be low and within the bounds of safety.

A highly-efficient and cost-effective pretreatment method for selective extraction and detection of perchlorate in tea and dairy products.

In view of the high polarity and ubiquitous occurrence of perchlorate, achieving an ultra-trace analysis has become a challenging task. The present study aimed to develop a simple and generic pretreatment protocol based on cold-induced liquid-liquid extraction to efficiently extract perchlorate from tea and dairy products and remarkably decrease potential matrix interferences and laborious cleanup. By optimizing the pretreatment conditions, the enrichment factor of perchlorate increased by 7.79 times under the compromise between the matrix effect and extraction recovery. The validated method presented satisfactory selectivity, linearity, accuracy, precision, and matrix effect, providing recoveries of 78.2%-106.2% with RSDr ranging from 1.2% to 7.9% and RSDR less than 10.7% for tea and dairy products. This pretreatment protocol depended only on shaking, freezing, and centrifugation in one step, without additional equipment or tedious operations, which will be explored to a greater extent in complex biological or food matrices.

An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.

Insight into COVID-2019 for pediatricians.

Since December 2019, patients with unexplained pneumonia have been found in Wuhan City, Hubei Province, China. The pathogen in these cases is a new type of coronavirus. The World Health Organization confirmed this diagnosis and named the pathogen SARSCoV-2. The disease caused by SARSCoV-2 is called Corona Virus Disease (COVID-2019). The virus is highly infectious and pathogenic, causing human-to-human transmission. At present, SARSCoV-2 is still rampant in the world. Zhengzhou City in Henan Province serves as an example, 102 people have been confirmed to be infected with SARSCoV-2 (at 24:00 on February 5th, 2020), including three children, the youngest is 4 years old. From the perspective of clinical pediatricians as the first line fighting the epidemic, this paper will discuss the clinical characteristics, prevention and control measures, outcomes, diagnosis, and treatment of pediatric cases.

Difficulties in research of Chinese medicine polysaccharides.

Polysaccharides from Chinese medicines are attracting increasing attention to their wide range of valuable biological activities. As these polysaccharides are mostly from edible materials, their safety can be greatly ensured. Therefore, the Chinese medicine polysaccharides have been the focus of research and development of new drugs and health products. However, there are rarely successful cases. Here, based on the authors' own research experience, the difficulties and challenges in chemical analysis and mechanism study of Chinese medicine polysaccharides are discussed, in the hope of eliciting more innovative ideas and solutions.

Oligosaccharide-marker approach for qualitative and quantitative analysis of specific polysaccharide in herb formula by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry: Dendrobium officinale, a case study.

Qualitative and quantitative analysis of polysaccharides in herb formula remain challenge due to the limited choices of analytical methods concerning the intrinsic characteristics of large molecular mass. Herein, an oligosaccharide-marker approach was newly developed for quality assessment of polysaccharides in herbal materials, using Dendrobium officinale as a case study. This method involved partial acid hydrolysis of D. officinale polysaccharide (DOP) followed by p-aminobenzoic ethyl ester (ABEE) derivatization. Two ABEE-labeled oligosaccharides namely, Te-Man-ABEE and Pen-Man-ABEE, were selected as chemical markers due to their high specificity in herb formula. The linear relationship between the content of these two markers and the content of DOP was then successfully established respectively. The linear relationship was further transformed to that between peak area of chemical markers and DOP content so that chemical markers were not necessary to be isolated for analysis. This linear relationship was systemically validated in terms of precision and accuracy. The results showed that these two oligosaccharide-markers presented a good linear relationship with DOP (R2 ≥ 0.997) in the range of 0.68-16.02 µg. These markers also demonstrated satisfactory precision (RSD < 7.0%), and recovery (91.41%-118.30%) in real sample determination. Additionally, there was no significant difference between the results given by the two chemical markers as the RSD values were not more than 7.0%. While concerning the results given by the oligosaccharide-markers and the previously-published polysaccharide marker, the RSD value was not more than 6.4%. These suggest that the oligosaccharide-marker approach is a simple, quick, and reliable method to qualitatively and quantitatively determine of specific polysaccharide in herb formula.

Destiny of Dendrobium officinale Polysaccharide after Oral Administration: Indigestible and Nonabsorbing, Ends in Modulating Gut Microbiota.

Polysaccharides from functional foods have been proved to have diverse bioactivities, but little is known about what exactly happens to these polysaccharides after oral administration and even less about the underlying mechanism of action. Taking the marker polysaccharide (DOP) of Dendrobium officinale as an example, this study aims to demonstrate the dynamic distribution and degradation of orally dosed DOP in mice and in vitro using near-infrared fluorescence imaging and a kind of chromatographic analysis. The results indicate that (1) neither DOP nor fluorescence-labeled DOP (FDOP) was absorbed, (2) both DOP and FDOP were undigested and were quickly degraded to short-chain fatty acids in the large intestine, (3) DOP modulated gut microbiota, which could be associated with DOP's suppression of 4T1 tumor growth in mice. All of these findings suggest that some (maybe not all) bioactive polysaccharides share a common destiny: indigestible and nonabsorbing, ends in modulating bioactivities-associated gut microbiota.

Bushenshugan Formula Attenuates the Development of Lung Cancer by Inhibiting Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: BushenShugan Formula (BSF) is a traditional Chinese medicine that has therapeutic effects on middle- and late-stage lung adenocarcinoma in clinical application. It was reported that Bushen Chinese medicine suppressed the onset of pre-metastatic niches in a murine model of spontaneous lung metastasis. However, the mechanisms of BSF on human lung adenocarcinoma remain unknown. METHODS: Cell proliferation was determined by CCK8 and colony formation. Cell apoptosis and cell cycle were detected by flow cytometry. Cancer stem cells properties were examined by spheroid body formation. The migration and invasion abilities were analyzed by wound healing assay and transwell invasion assay. The mRNA expressions were determined by qRT-PCR. Western blotting analysis showed the protein levels. RESULTS: BSF was shown to inhibit the proliferation of A549 cells in time- and concentration-dependent manners. Colony formation assays also indicated the antiproliferative effect of BSF against A549 cells. Cellular mechanistic studies demonstrated that BSF arrested the cell cycle in G2/M phase and induced apoptosis. Importantly, BSF could inhibit the epithelial-mesenchymal transition(EMT) of A549 cells through PI3K/AKT/NF-κB pathway. CONCLUSIONS: BSF effectively inhibited tumour growth, suggesting that it is a promising anticancer treatment for further clinical development.

Dihydromyricetin relieves rheumatoid arthritis symptoms and suppresses expression of pro-inflammatory cytokines via the activation of Nrf2 pathway in rheumatoid arthritis model.

Rheumatoid arthritis (RA) is a systemic inflammatory and autoimmune disease. In this research, we estimated the protective effects of Dihydromyricetin (DMY) on RA induced by Complete Freund's Adjuvant (CFA). We found that DMY effectively relieved rheumatoid arthritis symptoms, such as body weight change, paw swelling and rheumatoid arthritis scores. In addition, we also observed that DMY significantly lowered the immune organ indexes (including thymus and spleen) and exhibited the anti-inflammatory effect in CFA-induced rheumatoid arthritis. The results demonstrated that the increased expression levels of interleukin-1ß (IL-1ß), interleukin-6(IL-6), tumor necrosis factor-α (TNF-α) were significantly inhibited by DMY. Furthermore, the key inflammatory mediator, cyclooxygenase-2 (COX-2) was markedly lowered after treatment with DMY. A mechanistic study indicated that DMY could up-regulate the down-regulation levels of the mRNA and protein of Nrf2, HO-1 and NQO1. Moreover, the Nrf2 activation of DMY was abolished by Nrf2 inhibitor brusatol. Thus, DMY inhibits the expressions of pro-inflammatory cytokines via activating Nrf2 pathway in RA model, which suggests that DMY has potential for further investigation as a candidate anti-arthritic agent in future.

Validation and application of an rapid HPLC-MS method for the determination of salvianic acid A in human plasma.

A rapid liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed and validated for the determination of salvianic acid A in plasma of Chinese healthy subjects after oral administration of Qishenyiqi dropping pills. After liquid-liquid extraction with ethyl acetate, salvianic acid A was chromatographed on a Agilent Zorbax XDB-C18 column using a gradient mobile phase consisting of water (0.1% formic acid)-acetonitrile (0.1% formic acid) at a flow rate of 0.45 mL/min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 196.9→134.8 and m/z 320.9→151.9 for salvianic acid A and chloroamphenicol, respectively. The method was linear over the range of 0.50-500 ng/mL using only 100 µL of plasma and the lower limit of quantification was 0.50 ng/mL. Intra-day and inter-day precisions (in terms of % RSD) were all <15% and the accuracies (in terms of % RE) were within the range of±15%, and recoveries were between 85.0 and 115%. The validated method was successfully applied to pharmacokinetic study of Qishenyiqi dropping pills in Chinese healthy subjects. After oral administration, Tmax and Cmax values were 1.33 ± 0.52 h and 21.1 ± 3.92 ng/mL, respectively. Plasma concentrations declined with t1/2Z of 1.76 ± 0.33 h.