Synergy of lytic phage pB23 and meropenem combination against carbapenem-resistant Acinetobacter baumannii.

Phage-antibiotic combination treatment is a novel noteworthy drug delivery method in anti-infection. In the current study, we have isolated a new phage, pB23, against carbapenem-resistant Acinetobacter baumannii 2023. Synergistic antibacterial effect between phage pB23 and meropenem combination could be more stable, using moderate doses of phage (multiplicity of infection ranging from 0.1 to 1,000) based on results of in vitro antibacterial activity. Phage pB23 and meropenem combination could effectively clear mature biofilms and prevent biofilm formation of carbapenem-resistant Acinetobacter baumannii in vitro. Phage pB23 and meropenem combination also has good synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii in different growth phases under static culture conditions. The pig skin explant model shows that phage pB23 and meropenem combination has a synergistic effect to remove bacteria from wounds ex vivo. Phage pB23 and meropenem combination also exhibited a synergistic antibacterial effect in vivo using a zebrafish infection mode. The potential promotion of phage proliferation by meropenem and the sensitivity recovery of phage-resistant bacteria to meropenem might elucidate the mechanism of the synergistic antimicrobial activity. In summary, our study illustrates that phage pB23 and meropenem combination could produce synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii under static growth conditions. This study also demonstrates that phage-antibiotic combination will become an effective strategy to enhance antibacterial activity of individual drug and provide a new idea of the drug development for the treatment of infections due to carbapenem-resistant Acinetobacter baumannii and other multidrug-resistant bacteria.

A map of the spatial distribution and tumour-associated macrophage states in glioblastoma and grade 4 IDH-mutant astrocytoma.

Tumour-associated macrophages (TAMs) abundantly infiltrate high-grade gliomas and orchestrate immune response, but their diversity in isocitrate dehydrogenase (IDH)-differential grade 4 gliomas remains largely unknown. This study aimed to dissect the transcriptional states, spatial distribution, and clinicopathological significance of distinct monocyte-derived TAM (Mo-TAM) and microglia-derived TAM (Mg-TAM) clusters across glioblastoma-IDH-wild type and astrocytoma-IDH-mutant-grade 4 (Astro-IDH-mut-G4). Single-cell RNA sequencing was performed on four cases of human glioblastoma and three cases of Astro-IDH-mut-G4. Cell clustering, single-cell regulatory network inference, and gene set enrichment analysis were performed to characterize the functional states of myeloid clusters. The spatial distribution of TAM subsets was determined in human glioma tissues using multiplex immunostaining. The prognostic value of different TAM-cluster specific gene sets was evaluated in the TCGA glioma cohort. Profiling and unbiased clustering of 24,227 myeloid cells from glioblastoma and Astro-IDH-mut-G4 identified nine myeloid cell clusters including monocytes, six Mo/Mg-TAM subsets, dendritic cells, and proliferative myeloid clusters. Different Mo/Mg-TAM clusters manifest functional and transcriptional diversity controlled by specific regulons. Multiplex immunostaining of subset-specific markers identified spatial enrichment of distinct TAM clusters at peri-vascular/necrotic areas in tumour parenchyma or at the tumour-brain interface. Glioblastoma harboured a substantially higher number of monocytes and Mo-TAM-inflammatory clusters, whereas Astro-IDH-mut-G4 had a higher proportion of TAM subsets mediating antigen presentation. Glioblastomas with a higher proportion of monocytes exhibited a mesenchymal signature, increased angiogenesis, and worse patient outcome. Our findings provide insight into myeloid cell diversity and its clinical relevance in IDH-differential grade 4 gliomas, and may serve as a resource for immunotherapy development. © 2022 The Pathological Society of Great Britain and Ireland.

High levels of TIMP1 are associated with increased extracellular matrix stiffness in isocitrate dehydrogenase 1-wild type gliomas.

Glioma progression is accompanied with increased tumor tissue stiffness, yet the underlying mechanisms are unclear. Herein, we employed atomic force microscopy analysis to show that tissue stiffness was higher in isocitrate dehydrogenase (IDH)-wild type gliomas than IDH-mutant gliomas. Bioinformatic analyses revealed that tissue inhibitor of metalloproteinase-1 (TIMP1) was one of the preferentially upregulated genes in IDH-wild type gliomas as compared to IDH-mutant gliomas, and its higher expression indicated worse prognosis of glioma patients. TIMP1 intensity determined by immunofluorescence staining on glioma tissues positively correlated with glioma tissue stiffness. Mechanistically, TIMP1 expression was positively correlated with the gene expression of two predominant extracellular matrix components, tenascin C and fibronectin, both of which were also highly expressed in IDH-wild type gliomas. By introducing IDH1-R132H-containing vectors into human IDH1-wild type glioma cells to obtain an IDH1-mutant cell line, we found that IDH1 mutation increased the TIMP1 promoter methylation through methylation-specific PCR. More importantly, IDH1-R132H mutation decreased both the expression of TIMP1, fibronectin, tenascin C, and the tumor tissue stiffness in IDH1-mutant glioma xenografts in contrast to IDH1-wild type counterparts. Moreover, TIMP1 knockdown in IDH-wild type glioma cells inhibited the expression of tenascin C and fibronectin, and decreased tissue stiffness in intracranial glioma xenografts. Conclusively, we revealed an IDH mutation status-mediated mechanism in regulating glioma tissue stiffness through modulating TIMP1 and downstream extracellular matrix components.

Overexpression of carnitine palmitoyltransferase 1A promotes mitochondrial fusion and differentiation of glioblastoma stem cells.

Glioma stem cells (GSCs) are self-renewing tumor cells with multi-lineage differentiation potential and the capacity of construct glioblastoma (GBM) heterogenicity. Mitochondrial morphology is associated with the metabolic plasticity of GBM cells. Previous studies have revealed distinct mitochondrial morphologies and metabolic phenotypes between GSCs and non-stem tumor cells (NSTCs), whereas the molecules regulating mitochondrial dynamics in GBM cells are largely unknown. Herein, we report that carnitine palmitoyltransferase 1A (CPT1A) is preferentially expressed in NSTCs, and governs mitochondrial dynamics and GSC differentiation. Expressions of CPT1A and GSC marker CD133 were mutually exclusive in human GBMs. Overexpression of CPT1A inhibited GSC self-renewal but promoted mitochondrial fusion. In contrast, disruption of CPT1A in NSTCs promoted mitochondrial fission and reprogrammed NSTCs toward GSC feature. Mechanistically, CPT1A overexpression increased the phosphorylation of dynamin-related protein 1 at Ser-637 to promote mitochondrial fusion. In vivo, CPT1A overexpression decreased the percentage of GSCs, impaired GSC-derived xenograft growth and prolonged tumor-bearing mice survival. Our work identified CPT1A as a critical regulator of mitochondrial dynamics and GSC differentiation, indicating that CPT1A could be developed as a molecular target for GBM cell-differentiation strategy.

Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii.

BACKGROUND: Rapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria. METHODS: In the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage P53 and rapid ultrasensitive identification of Acinetobacter baumannii (A. baumannii) was evaluated. RESULTS: The assay was capable of quantifying P53 phage DNA without DNA extraction and the detection limit of the assay was 550 PFU/mL. The agreement bias between the quantitative results of three different phage concentrations in this assay and double agar overlay plaque assay were under 3.38%. Through the built detection system, down to 1 log CFU/mL of viable A. baumannii can be detected within 4 h in A. baumannii spiked swab and bronchoalveolar lavage fluid samples. Compared with the Taqman qPCR that targets the conserved sequence of A. baumannii, the sensitivity of the assay built in this study could increase four orders of magnitude. CONCLUSIONS: The methodology offers a valid alternative for enumeration of freshly prepared phage solution and diagnosis of bacterial infection caused by A. baumannii or other bacterial infection in complicated samples through switching to phages against other bacteria. Furthermore, the assay could offer drug adjustment strategy timely owing to the detection of bacteria vitality.

Evaluation of (sdLDLc*HCYc)/HDLc ratio in clinical auxiliary diagnosis of primary cerebral infarction.

BACKGROUND: Timely detection of cerebral infarction is of vital importance in planning intervention effect of rapid rehabilitation. The clinical auxiliary diagnosis value of single biomarker, including small dense low-density lipoprotein concentration (sdLDLc), homocysteine concentration (HCYc) and high-density lipoprotein cholesterol concentration (HDLc) for cerebral infarction has been confirmed by many studies. Whether the use of three biomarkers in combination by calculating (sdLDLc*HCYc)/HDLc ratio could improve the diagnosis ability for primary cerebral infarction remains to be unclear. In the present study, we conducted a cross-sectional study to evaluate the value of (sdLDLc*HCYc)/HDLc ratio in clinical auxiliary diagnosis of primary cerebral infarction. METHODS: A total of 583 participants, including 299 healthy participants as control group and 284 participants diagnosed with first cerebral infarction as experiment group, were included in this respective study. The serum sdLDLc, HDLc and HCYc were measured by peroxidase method, enzyme-linked immunosorbent assay and an enzymatic method, respectively. RESULTS: The average concentration of sdLDL and HCY (0.69 ± 0.29 mmol/L and 18.14 ± 6.62 µmol/L) in experiment group was significantly higher than those in the control group (0.55 ± 0.22 mmol/L and 10.77 ± 2.67 µmol/L, P < 0.05). However, the average concentration of HDL (1.47 ± 0.25 mmol/L) in the control group was higher than that in the experiment group (1.33 ± 0.28 mmol/L, P < 0.05). Spearman correlation coefficient showed the three indicators are independent of each other. The positive predictive value of (sdLDLc*HCYc)/HDLc ratio (61.27%, 95% CI: 55.31-66.92) is higher than that in single biomarker (sdLDLc: 6.69 95% CI: 4.19-10.42, HCYc: 38.38%, 95% CI: 32.75-44.33, HDLc: 3.87%, 95% CI: 2.05-7.02). Receiver-operating characteristic curve (ROC) analysis illustrated that predictive power of (sdLDLc*HCYc)/HDLc was higher than single biomarker, including sdLDLc, HCYc and HDLc, in primary cerebral infarction. CONCLUSIONS: Therefore, (sdLDLc*HCYc)/HDLc ratio might be a better new indicator in clinical auxiliary diagnosis of primary cerebral infarction, which could be contributed to predicting cerebral infarction occurrence and provide a scientific basis for early prevention.

Electrochemical Enzyme Sensor Based on the Two-Dimensional Metal-Organic Layers Supported Horseradish Peroxidase.

Metal-organic frames (MOFs) have recently been used to support redox enzymes for highly sensitive and selective chemical sensors for small biomolecules such as oxygen (O2), hydrogen peroxide (H2O2), etc. However, most MOFs are insulative and their three-dimensional (3D) porous structures hinder the electron transfer pathway between the current collector and the redox enzyme molecules. In order to facilitate electron transfer, here we adopt two-dimensional (2D) metal-organic layers (MOLs) to support the HRP molecules in the detection of H2O2. The correlation between the current response and the H2O2 concentration presents a linear range from 7.5 µM to 1500 µM with a detection limit of 0.87 µM (S/N = 3). The sensitivity, reproducibility, and stability of the enzyme sensor are promoted due to the facilitated electron transfer.

Zyxin (ZYX) promotes invasion and acts as a biomarker for aggressive phenotypes of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is characterized by highly invasive growth, which leads to extensive infiltration and makes complete tumor excision difficult. Since cytoskeleton proteins are related to leading processes and cell motility, and through analysis of public GBM databases, we determined that an actin-interacting protein, zyxin (ZYX), may involved in GBM invasion. Our own glioma cohort as well as the cancer genome atlas (TCGA), Rembrandt, and Gravendeel databases consistently showed that increased ZYX expression was related to tumor progression and poor prognosis of glioma patients. In vitro and in vivo experiments further confirmed the oncogenic roles of ZYX and demonstrated the role of ZYX in GBM invasive growth. Moreover, RNA-seq and mass-spectrum data from GBM cells with or without ZYX revealed that stathmin 1 (STMN1) was a potential target of ZYX. Subsequently, we found that both mRNA and protein levels of STMN1 were positively regulated by ZYX. Functionally, STMN1 not only promoted invasion of GBM cells but also rescued the invasion repression caused by ZYX loss. Taken together, our results indicate that high ZYX expression was associated with worse prognosis and highlighted that the ZYX-STMN1 axis might be a potential therapeutic target for GBM.

Microgravity-induced hepatogenic differentiation of rBMSCs on board the SJ-10 satellite.

Bone marrow-derived mesenchymal stem cells (BMSCs) are able to differentiate into functional hepatocytelike cells, which are expected to serve as a potential cell source in regenerative medicine, tissue engineering, and clinical treatment of liver injury. Little is known about whether and how space microgravity is able to direct the hepatogenic differentiation of BMSCs in the actual space microenvironment. In this study, we examined the effects of space microgravity on BMSC hepatogenic differentiation on board the SJ-10 Recoverable Scientific Satellite. Rat BMSCs were cultured and induced in hepatogenic induction medium for 3 and 10 d in custom-made space cell culture hardware. Cell growth was monitored periodically in orbit, and the fixed cells and collected supernatants were retrieved back to the Earth for further analyses. Data indicated that space microgravity improves the differentiating capability of the cells by up-regulating hepatocyte-specific albumin and cytokeratin 18. The resulting cells tended to be maturated, with an in-orbit period of up to 10 d. In space, mechanosensitive molecules of ß1-integrin, ß-actin, α-tubulin, and Ras homolog gene family member A presented enhanced expression, whereas those of cell-surface glycoprotein CD44, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, vinculin, cell division control protein 42 homolog, and Rho-associated coiled-coil kinase yielded reduced expression. Also observed in space were the depolymerization of actin filaments and the accumulation of microtubules and vimentin through the altered expression and location of focal adhesion complexes, Rho guanosine 5'-triphosphatases, as well as the enhanced exosome-mediated mRNA transfer. This work furthers the understanding of the underlying mechanisms of space microgravity in directing hepatogenic differentiation of BMSCs.-Lü, D., Sun, S., Zhang, F., Luo, C., Zheng, L., Wu, Y., Li, N., Zhang, C., Wang, C., Chen, Q., Long, M. Microgravity-induced hepatogenic differentiation of rBMSCs on board the SJ-10 satellite.

A novel "six stitches" procedures for pediatric and adult buried penis.

INTRODUCTION: The buried penis, if not treated before adolescence, will lead to psychological and physical disorders in adulthood. Therefore, early surgical intervention is necessary. At present, the common surgical methods include the penile corpus fixation, the Johnson's operation, the Devine's method, the modified Devine's method, Shiraki's method, etc. However, we found that these traditional surgeries showed various postoperative complications, such as long-term prepuce edema, avascular necrosis of skin flaps, stenotic prepuce, scarring, and poor appearance. This video shows the main technical steps of our innovative surgical procedure "Six Stitch" (SS) method for the buried penis. MATERIALS AND METHODS: The designation of the so-called SS method was based on the total knots made (six knots were made for the SS procedure). After the crura penis was fully exposed via a longitudinal incision at the penoscrotal junction, at the 2 o'clock position (around the penis), the superficial layer of albuginea of the crura penis was sutured to the prepubic ligament with 2-0 non-absorbable sutures to prevent the retraction of the penis (the 1st knot). The same procedure was used for the 10 o'clock position (the 2nd knot); At the 2 o'clock position, the skin and subcutaneous tissue at the pubic mound were sutured to the prepubic ligament to reconstruct the appearance of dorsum penis (the 3rd knot). The same procedures were used for the 10 o'clock position (the 4th knot). At the 5 o'clock position, the ventral albuginea was sutured to the tunica dartos and subcutaneous tissue at the penoscrotal junction to reconstruct the penoscrotal angle (the 5th knot). The same procedures were used for the 4 o'clock position (the 6th knot). Finally, the gloved prepuce was reset and circumcision was conducted if the redundant prepuce existed. RESULTS: We have done a total of 64 cases of SS procedures for concealed penis; mean length improvement was 3.8 ± 0.5 cm, with a satisfying 95 percent (61 / 64), which was much longer than the outcome of the above-mentioned methods. Mean operative time was 62.3 ± 12.1 minutes, and there was no serious intraoperative or postoperative complication (only 2 presented scar hyperplasia at the incision site). CONCLUSIONS: In conclusion, after the SS procedure, patients with buried penis can acquire an almost 4 cm improvement of penile length and covert incision at the midline of the scrotum, with an acceptable and low incidence of adverse events. This safe and effective procedure may be a viable option for the surgical management of pediatric and adult buried penis.

Longer length improvement and more covert incision: a single-center, prospective study of two innovative surgical methods "one stitch" and "four stitch" for pediatric buried penis.

BACKGROUND: To introduce the detailed procedures of two innovative surgical options for pediatric buried penis and prospectively compare their efficacy and safety. METHODS: A single-center, non-randomized, prospective study was conducted at the Zhongnan Hospital of Wuhan University, where patients were operated on using the so-called "one stitch" (OS) or "four stitch" (FS) methods. The operation time, adverse events, and satisfaction were recorded for both groups. RESULTS: Finally, 156 patients underwent the so-called OS (n = 65) or FS (n = 91) method, with a follow-up rate of 86.5% (135/156). During the perioperative period, the FS group spent much longer in surgery (P < 0.001), had more blood loss (P < 0.001), and took longer to recover from edema (P < 0.001) than the OS group. In contrast to the satisfaction after 12 months' follow up, both the objective length improvement (2.5 ± 0.6 vs 3.8 ± 0.5 cm, P < 0.001) and subjective satisfaction percent (86 vs 95%, P = 0.678) in the FS group were superior to those in the OS group. No significant differences were detected in postoperative infection, stenosis circle, scar hyperplasia, and relapse. CONCLUSIONS: In conclusion, the two surgical options for pediatric buried penis are both safe and effective. The OS method has a simple procedure, so with shorter operation time and faster postoperative recovery; though the FS method with more complex procedure, patients can acquire a satisfactory improvement of penile length almost 4 cm and more covert incision at the midline of the scrotum. We primarily recommend the FS method for patients with moderate or severe buried cases; but for mild cases, we preferred the OS method.

ß1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model.

BACKGROUND: Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. But the mechanisms of how mechanical stimuli regulate the migration of keratinocytes have been poorly understood. METHODS: Human immortalized keratinocyte HaCaT cells were co-cultured with skin fibroblasts on PDMS membranes and transferred to the static stretch device developed in-house for additional 6 day culture under mechanical stretch to mimic surface tension in skin. To detect the expression of proteins on different position at different time points and the effect of ß1 integrin mechanotransduction on HaCaT migration, Immunofluorescence, Reverse transcription-polymerase chain reaction, Flow cytometry, Western blotting assays were applied. RESULTS: Mechanical receptor of ß1 integrin that recognizes its ligand of collagen I was found to be strongly associated with migration of HaCaT cells since the knockdown of ß1 integrin via RNA silence eliminated the key protein expression dynamically. Here the expression of vinculin was lower but that of Cdc42 was higher for the cells at outward edge than those at inward edge, respectively, supporting that the migration capability of keratinocytes is inversely correlated with the formation of focal adhesion complexes but positively related to the lamellipodia formation. This asymmetric expression feature was further confirmed by high or low expression of PI3K for outward- or inward-migrating cells. And ERK1/2 phosphorylation was up-regulated by mechanical stretch. CONCLUSION: We reported here, a novel mechanotransduction signaling pathways were ß1 integrin-dependent pattern of keratinocytes migration under static stretch in an in vitro co-culture model. These results provided an insight into underlying molecular mechanisms of keratinocyte migration under mechanical stimuli.

Clinical significance of S100A2 expression in gastric cancer.

Gastric carcinoma (GC) is one of the most common malignancies worldwide. To identify the candidate carcinoma-related biomarker in GC, comparative proteome technique was performed in resected GC tissues and matched adjacent non-cancerous gastric tissues (ANGT). As a result, S100A2 was successfully identified to be down-regulated significantly in GC compared with ANGT. Western blot analysis validated decreased expression of S100A2, and its expression level was related with the degree of tumor differentiation and status of lymph node metastasis in GC. Furthermore, immunohistochemistry analysis showed S100A2 down-expression was significantly associated with poor differentiation (P < 0.05), advanced depth of invasion (P < 0.05) and lymph node metastasis (P < 0.05) in GC. Kaplan-Meier curves showed that the relapse-free probability and the overall survival rate were significantly decreased with S100A2 expression decreasing (P < 0.05). Cox regression analysis indicated S100A2 down-expression was a negative independent prognostic biomarker for GC. A supplement of S100A2 protein by S100A2 expression vector significantly decreased the number of invaded cancer cells MGC-803. However, knockdown of S100A2 expression by siRNA interference compromised the invasion ability of MGC-803 cells. Moreover, S100A2 negatively regulated MEK/ERK signaling pathway, and activation of this signaling pathway by S100A2 down-regulation increased in vitro invasion of MGC-803 cells. In conclusion, this study demonstrated the clinical significance of S100A2 expression in GC, and loss of S100A2 expression contributes to GC development and progression. Therefore, the determination of S100A2 expression levels contributes to predict the outcome of GC patients.

Inserted rest period resensitizes MC3T3-E1 cells to fluid shear stress in a time-dependent manner via F-actin-regulated mechanosensitive channel(s).

The underlying cellular mechanism of anabolic effect recovered by inserting rest is not fully understood. In this work, we studied the role of F-actin regulated mechanosensitive channel(s) re-activation in mechanosensitivity modulation in vitro. Results showed that steady fluid shear stress (sFSS) stimulation with 30-min rest period was more potential in increasing alkalinephosphatase (ALP) activity than 10 and 0-min rest periods, and insertion of 30 min, but not 0 or 10 min, recovered the [Ca(2+)]i transient and contribution of the mechanosensitive channel(s). During the rest period, F-actin experienced polymerization (0-10 min), followed by depolymerization (10-30 min); inhibition of F-actin polymerization/depolymerization significantly increased/decreased the [Ca(2+)]i transient, as well as the contribution of the mechanosensitive channel(s) in subsequent sFSS stimulation. Our results demonstrated that the long rest period between sFSS loadings recruited [Ca(2+)]i transient via F-actin depolymerization-induced reactivation of mechanosensitive channel(s), suggesting that F-actin-regulated cellular stiffness could account for the decreased anabolic response during continuous mechanical loading in bone cells.

Integrating rapid pathogen identification and antimicrobial susceptibility testing through multiplex TaqMan qPCR assay.

Timely bacterial identification (ID) and antimicrobial susceptibility testing (AST) are of significance for therapy of bacteria-infected patients. In the present study, we developed a multiplex TaqMan qPCR assay for rapid and accurate ID and AST of three common hospital acquired pneumonia species, namely Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus. In this assay, DNA extraction and bacterial co-incubation with antibiotics are accomplished based on a common PCR instrument. ID of three bacteria is based on specific conserved DNA sequence fragment (gltA for A. baumannii, phoE for K. pneumoniae and nuc for S. aureus) detection through multiplex TaqMan qPCR assay within 80 min. AST of three bacteria could be acquired within 200 min based on genomic DNA fold change detection after 2 h of antibiotic exposure. Testing of 23 bronchoalveolar lavage fluid samples spiked by different A. baumannii isolates, 20 bronchoalveolar lavage fluid samples spiked by different K. pneumoniae isolates, and 14 bronchoalveolar lavage fluid samples spiked by different S. aureus isolates showed that the multiplex TaqMan qPCR assay had 100% (95% CI: 85.69-100), 100% (95% CI: 83.89-100) and 100% (95% CI:78.47-100) identification agreement with the initial spiked bacteria. Subsequent AST results compared with the standard broth microdilution method showed an overall agreement of 91.30% (95% CI: 73.20 to 97.58) for A. baumannii, 90% (95% CI: 69.90 to 97.21) for K. pneumoniae and 92.86% (95% CI: 68.53 to 98.73) for S. aureus based on the current multiplex TaqMan assay. Due to the high rapidity, good agreement, simplicity, and high throughput, this multiplex TaqMan assay could be helpful for ID and broad-spectrum AST in A. baumannii, K. pneumoniae and S. aureus, as well as potentially applicable for other clinical bacteria by changing the primers and probes.

Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization.

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

Poly(3,4-ethylenedioxythiophene)-modified Ni/silicon microchannel plate electrode for the simultaneous determination of ascorbic acid, dopamine and uric acid.

A Ni/silicon microchannel plate (Ni/Si MCP) electrode modified with poly(3,4-ethylenedioxythiophene) (PEDOT) was successfully fabricated. The analytical performance of prepared electrode for the simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) was investigated by using cyclic voltammetry and differential pulse voltammetry (DPV). The sensitivities for AA, DA and UA obtained by DPV are 5.39 A m(-2) mM(-1), 0.054 A m(-2) µM(-1) and 0.022 A m(-2) µM(-1), respectively. The calculated detection limits were 10 µM (AA), 1.5 µM (DA) and 2.7 µM (UA). The prepared electrode was successfully applied to the detection of AA, DA and UA in urine samples. The experiments illustrate that Ni/Si MCP is a good electrode material which provides a large surface-to-volume ratio and enhances the selectivity and sensitivity.

Yeast α-glucosidase inhibitory phenolic compounds isolated from Gynura medica leaf.

Gynura medica leaf extract contains significant amounts of flavonols and phenolic acids and exhibits powerful hypoglycemic activity against diabetic rats in vivo. However, the hypoglycemic active constituents that exist in the plant have not been fully elaborated. The purpose of this study is to isolate and elaborate the hypoglycemic activity compounds against inhibition the yeast α-glucosidase in vitro. Seven phenolic compounds including five flavonols and two phenolic acids were isolated from the leaf of G. medica. Their structures were identified by the extensive NMR and mass spectral analyses as: kaempferol (1), quercetin (2), kaempferol-3-O-ß-D-glucopyranoside (3), kaempferol-3-O-rutinoside (4), rutin (5), chlorogenic acid (6) and 3,5-dicaffeoylquinic acid methyl ester (7). All of the compounds except 1 and 3 were isolated for the first time from G. medica. Compounds 1-7 were also assayed for their hypoglycemic activity against yeast α-glucosidase in vitro. All of the compounds except 1 and 6 showed good yeast α-glucosidase inhibitory activity with the IC(50) values of 1.67 mg/mL, 1.46 mg/mL, 0.38 mg/mL, 0.10 mg/mL and 0.53 mg/mL, respectively.

Iridoid glycosides extracted from zhizi (fructus gardeniae) decrease collagen-induced platelet aggregation and reduce carotid artery thrombosis in an in vivo rat model.

OBJECTIVE: To investigate the anti-platelet aggregation and antithrombotic effects in rats of iridoid glycosides extracted from Zhizi (Fructus Gardeniae). METHODS: The present study evaluated the antithrombotic activity of iridoid glycosides (IGs) in a rat model of carotid artery thrombosis. The effects on coagulation, such as thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT), and the effect on collagen-induced platelet aggregation in vivo were investigated. Rats were intragastrically administered IGs (50, 100 or 200 mg/ kg) twice daily for 3 days. RESULTS: IGs were shown for the first time to have an antithrombotic action through the inhibition of platelet aggregation, with little effect on the coagulation time of peripheral blood. Our results also showed that IGs may significantly and dose-dependently reduce arterial thrombus load in a model of carotid artery thrombosis and inhibit collagen-induced platelet aggregation in rats. IGs (100 or 200 mg/kg) had no significant effect on APTT and PT, but did lengthen TT at a higher dose. CONCLUSION: These data, together with the previously reported neuroprotective effects of IGs in rats with cerebral ischemia, suggest that the antithrombotic action of IGs may potentially contribute to the treatment of cerebral ischemic diseases, including cerebral apoplexy.

Antithrombotic effect of ligustrazine hydrochloride injection on the model of induced arteriovenous shunt thrombosis.

BACKGROUND: The objective of this study is to optimize the effective dose of heparin and ligustrazine hydrochloride injection (LHI) for drug combination. MATERIALS AND METHODS: The animal clinical study of LHI was performed by the rat's model of induced arteriovenous shunt thrombosis. Experimental animals were grouped into several groups and separately treated with both LHI (20, 40, 80 mg/kg, i.p.) and heparin (60, 55, and 50 U/kg; 5000 U/ml; Sigma, i.v). The study had used thrombus weight, protein concentration in thrombus homogenate, inhibition rate of thrombosis, and plasma anti-thrombin activity as indications. RESULTS: The group combination (50, 80) got the result of 100% antithrombotic activity with 0 ± 0 mg of thrombus weight, 14 ± 3 µg/ml of protein concentration in thrombus homogenate and 1.5 ± 0.04 U/ml of plasma anti-thrombin activity. Its anti-thrombotic effect was much better than individual groups treated with LHI in a dose of 0 mg/kg and group of combination (0, 80) (P < 0.05) while antithrombotic effect of 55 and 60 U/kg heparin alone was only 37-58%. Therefore, the group of combination (50, 80) was optimal for 100% antithrombotic activity. CONCLUSION: Optimal combined doses of LHI and heparin preventing blood coagulation were determined and the results were available. It may give some hint for the further clinical application on human.