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Current antimicrobial susceptibility testing (AST) requires 16-24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for β-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours.

Structured illumination microscopy (SIM) with axially optical sectioning capability has found widespread applications in three-dimensional live cell imaging in recent years, since it combines high sensitivity, short image acquisition time, and high spatial resolution. To obtain one sectioned slice, three raw images with a fixed phase-shift, normally 2π/3, are generally required. In this paper, we report a data processing algorithm based on the one-dimensional Hilbert transform, which needs only two raw images with arbitrary phase-shift for each single slice. The proposed algorithm is different from the previous two-dimensional Hilbert spiral transform algorithm in theory. The presented algorithm has the advantages of simpler data processing procedure, faster computation speed and better reconstructed image quality. The validity of the scheme is verified by imaging biological samples in our developed DMD-based LED-illumination SIM system.

Spinal cord injury (SCI) is a devastating neurological disease without effective treatment. To generate a comprehensive view of the mechanisms involved in SCI pathology, we applied RNA-Sequencing (RNA-Seq) technology to characterize the temporal changes in global gene expression after contusive SCI in mice. We sequenced tissue samples from acute and subacute phases (2 days and 7 days after injury) and systematically characterized the transcriptomes with the goal of identifying pathways and genes critical in SCI pathology. The top enriched functional categories include "inflammation response," "neurological disease," "cell death and survival" and "nervous system development." The top enriched pathways include LXR/RXR Activation and Atherosclerosis Signaling, etc. Furthermore, we developed a systems-based analysis framework in order to identify key determinants in the global gene networks of the acute and sub-acute phases. Some candidate genes that we identified have been shown to play important roles in SCI, which demonstrates the validity of our approach. There are also many genes whose functions in SCI have not been well studied and can be further investigated by future experiments. We have also incorporated pharmacogenomic information into our analyses. Among the genes identified, the ones with existing drug information can be readily tested in SCI animal models. Therefore, in this study we have described an example of how global gene profiling can be translated to identifying genes of interest for functional tests in the future and generating new hypotheses. Additionally, the RNA-Seq enables splicing isoform identification and the estimation of expression levels, thus providing useful information for increasing the specificity of drug design and reducing potential side effect. In summary, these results provide a valuable reference data resource for a better understanding of the SCI process in the acute and sub-acute phases.

Focusing an annular laser beam can improve the axial trapping efficiency due to the reduction of the scattering force, which enables the use of a lower numerical aperture (NA) objective lens with a long working distance to trap particles in deeper aqueous medium. In this paper, we present an axicon-to-axicon scheme for producing parallel annular beams with the advantages of higher efficiency compared with the obstructed beam approach. The validity of the scheme is verified by the observation of a stable trapping of silica microspheres with relatively low NA microscope objective lenses (NA = 0.6 and 0.45), and the axial trapping depth of 5 mm is demonstrated in experiment.

Our previous study suggested that increased cytoplasmic calcium (Ca) signals may mediate aluminum (Al) toxicity in yeast (Saccharomyces cerevisiae). In this report, we found that a yeast mutant, pmc1, lacking the vacuolar calcium ion (Ca(2+)) pump Ca(2+)-ATPase (Pmc1p), was more sensitive to Al treatment than the wild-type strain. Overexpression of either PMC1 or an anti-apoptotic factor, such as Bcl-2, Ced-9 or PpBI-1, decreased cytoplasmic Ca(2+) levels and rescued yeast from Al sensitivity in both the wild-type and pmc1 mutant. Moreover, pretreatment with the Ca(2+) chelator BAPTA-AM sustained cytoplasmic Ca(2+) at low levels in the presence of Al, effectively making the cells more tolerant to Al exposure. Quantitative RT-PCR revealed that the expression of calmodulin (CaM) and phospholipase C (PLC), which are in the Ca(2+) signaling pathway, was down-regulated under Al stress. This effect was largely counteracted when cells overexpressed anti-apoptotic Ced-9 or were pretreated with BAPTA-AM. Taken together, our results suggest that the negative regulation of Al-induced cytoplasmic Ca signaling is a novel mechanism underlying internal resistance to Al toxicity.

Current antimicrobial susceptibility testing (AST) requires 16–24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for β-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours. [ABSTRACT FROM AUTHOR]

Tendinopathy is a complex multifaceted tendinopathy often associated with overuse and with its high prevalence resulting in significant health care costs. At present, the pathogenesis and effective treatment of tendinopathy are still not sufficiently elucidated. The purpose of this research is to intensely explore the genes, functional pathways, and immune infiltration characteristics of the occurrence and development of tendinopathy. The gene expression profile of GSE106292, GSE26051 and GSE167226 are downloaded from GEO (NCBI comprehensive gene expression database) and analyzed by WGCNA software bag using R software, GSE26051, GSE167226 data set is combined to screen the differential gene analysis. We subsequently performed gene enrichment analysis of Gene Ontology (GO) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG), and immune cell infiltration analysis. By constructing the LASSO regression model, Support vector machine (SVM-REF) and Gaussian mixture model (GMMs) algorithms are used to screen, to identify early diagnostic genes. We have obtained a total of 171 DEGs through WGCNA analysis and differentially expressed genes (DEGs) screening. By GO and KEGG enrichment analysis, it is found that these dysregulated genes were related to mTOR, HIF-1, MAPK, NF-κB and VEGF signaling pathways. Immune infiltration analysis showed that M1 macrophages, activated mast cells and activated NK cells had infiltration significance. After analysis of THE LASSO SVM-REF and GMMs algorithms, we found that the gene MACROD1 may be a gene for early diagnosis. We identified the potential of tendon disease early diagnosis way and immune gene regulation MACROD1 key infiltration characteristics based on comprehensive bioinformatics analysis. These hub genes and functional pathways may as early biomarkers of tendon injuries and molecular therapy level target is used to guide drug and basic research.

BACKGROUND: Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. METHODS/PRINCIPAL FINDINGS: We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. CONCLUSIONS/SIGNIFICANCE: Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.

Objective:Hyperglycemia is related to stroke. Glycated hemoglobin (HbA1c) can reflect pre-stroke glycaemia status. However, the information on the direct association between HbA1c and recurrence after non-cardioembolic acute ischemic strokes is rare and there is no consistent conclusion. Methods:The ACROSS-China database comprised of 2186 consecutive first-ever acute ischemic stroke patients with baseline HbA1c values. After excluding patients who died from non-stroke recurrence and patients lost to follow up, 1817 and 1540 were eligible for 3-month and 1-year analyses, respectively. Multivariate Cox regression was performed to evaluate the associations between HbA1c and 3-month and 1-year stroke recurrence. Results:The HbA1c values at admission were divided into 4 levels by quartiles: Q1 (<5.5%); Q2 (5.5 to <6.1%); Q3 (6.1% to <7.2%); and Q4 (≥7.2%). The cumulative recurrence rates were 8.3% and 11.0% for 3 months and 1 year, respectively. In multivariate analyses, when compared with Q1, the adjusted hazard ratios (AHRs) were 2.83 (95% confidence interval (CI) 1.28-6.26) in Q3 and 3.71(95% CI 1.68-8.21) in Q4 for 3-month stroke recurrence; 3.30 (95% CI 1.31-8.34) in Q3 and 3.35 (95% CI 1.36-8.21) in Q4 for 1-year stroke recurrence. Adding fasting plasma glucose in the multivariate analyses did not modify the association: AHRs were 2.75 (95% CI 1.24-6.11) in Q3 and 3.67 (95% CI 1.59-8.53) in Q4 for 3-month analysis; AHRs were 3.08 (95% CI 1.10-8.64) in Q3 and 3.31(95% CI 1.35-8.14) in Q4 for 1-year analysis. Conclusions:A higher “normal” HbA1c level reflecting pre-stroke glycaemia status independently predicts stroke recurrence within one year after non-cardioembolic acute ischemic stroke onset. HbA1c is recommended as a routine test in acute ischemic stroke patients. [ABSTRACT FROM AUTHOR]

OBJECTIVES: To explore the anatomy of the plantar aponeurosis (PA) and its biomechanical effects on the first metatarsophalangeal (MTP) joint and foot arch. METHODS: Anatomic parameters (length, width and thickness of each central PA bundle and the main body of the central part) were measured in 8 cadaveric specimens. The ratios of the length and width of each bundle to the length and width of the central part were used to describe these bundles. Six cadaveric specimens were used to measure the range of motion of the first MTP joint before and after releasing the first bundle of the PA. Another 6 specimens were used to evaluate simulated static weight-bearing. Changes in foot arch height and plantar pressure were measured before and after dividing the first bundle. RESULTS: The average width and thickness of the origin of the central part at the calcaneal tubercle were 15.45 mm and 2.79 mm respectively. The ratio of the length of each bundle to the length of the central part was (from medial to lateral) 0.29, 0.30, 0.28, 0.25, and 0.27, respectively. Similarly, the ratio of the widths was 0.26, 0.25, 0.23, 0.19 and 0.17. The thickness of each bundle at the bifurcation of the PA into bundles was (from medial to lateral) 1.26 mm, 1.04 mm, 0.91 mm, 0.84 mm and 0.72 mm. The average dorsiflexion of the first MTP joint increased 10.16° after the first bundle was divided. Marked acute changes in the foot arch height and the plantar pressure were not observed after division. CONCLUSIONS: The first PA bundle was not the longest, widest, or the thickest bundle. Releasing the first bundle increased the range of motion of the first MTP joint, but did not acutely change foot arch height or plantar pressure during static load testing.

BACKGROUND: Pharmaceuticals and their metabolites are inevitably emitted into the waters. The adverse environmental and human health effects of pharmaceutical residues in water could take place under a very low concentration range; from several µg/L to ng/L. These are challenges to the global water industries as there is no unit process specifically designed to remove these pollutants. An efficient technology is thus sought to treat these pollutants in water and waste water. METHODOLOGY/MAJOR RESULTS: A novel chemical, ferrate, was assessed using a standard jar test procedure for the removal of pharmaceuticals. The analytical protocols of pharmaceuticals were standard solid phase extraction together with various instrumentation methods including LC-MS, HPLC-UV and UV/Vis spectroscopy. Ferrate can remove more than 80% of ciprofloxacin (CIP) at ferrate dose of 1 mg Fe/L and 30% of ibuprofen (IBU) at ferrate dose of 2 mg Fe/L. Removal of pharmaceuticals by ferrate was pH dependant and this was in coordinate to the chemical/physical properties of pharmaceuticals. Ferrate has shown higher capability in the degradation of CIP than IBU; this is because CIP has electron-rich organic moieties (EOM) which can be readily degraded by ferrate oxidation and IBU has electron-withdrawing groups which has slow reaction rate with ferrate. Promising performance of ferrate in the treatment of real waste water effluent at both pH 6 and 8 and dose range of 1-5 mg Fe/L was observed. Removal efficiency of ciprofloxacin was the highest among the target compounds (63%), followed by naproxen (43%). On the other hand, n-acetyl sulphamethoxazole was the hardest to be removed by ferrate (8% only). CONCLUSIONS: Ferrate is a promising chemical to be used to treat pharmaceuticals in waste water. Adjusting operating conditions in terms of the properties of target pharmaceuticals can maximise the pharmaceutical removal efficiency.

Background: Pharmaceuticals and their metabolites are inevitably emitted into the waters. The adverse environmental and human health effects of pharmaceutical residues in water could take place under a very low concentration range; from several µg/L to ng/L. These are challenges to the global water industries as there is no unit process specifically designed to remove these pollutants. An efficient technology is thus sought to treat these pollutants in water and waste water., Methodology/major Results: A novel chemical, ferrate, was assessed using a standard jar test procedure for the removal of pharmaceuticals. The analytical protocols of pharmaceuticals were standard solid phase extraction together with various instrumentation methods including LC-MS, HPLC-UV and UV/Vis spectroscopy. Ferrate can remove more than 80% of ciprofloxacin (CIP) at ferrate dose of 1 mg Fe/L and 30% of ibuprofen (IBU) at ferrate dose of 2 mg Fe/L. Removal of pharmaceuticals by ferrate was pH dependant and this was in coordinate to the chemical/physical properties of pharmaceuticals. Ferrate has shown higher capability in the degradation of CIP than IBU; this is because CIP has electron-rich organic moieties (EOM) which can be readily degraded by ferrate oxidation and IBU has electron-withdrawing groups which has slow reaction rate with ferrate. Promising performance of ferrate in the treatment of real waste water effluent at both pH 6 and 8 and dose range of 1-5 mg Fe/L was observed. Removal efficiency of ciprofloxacin was the highest among the target compounds (63%), followed by naproxen (43%). On the other hand, n-acetyl sulphamethoxazole was the hardest to be removed by ferrate (8% only)., Conclusions: Ferrate is a promising chemical to be used to treat pharmaceuticals in waste water. Adjusting operating conditions in terms of the properties of target pharmaceuticals can maximise the pharmaceutical removal efficiency.

Objective: Metabolic syndrome has emerged as a novel risk factor in cardiovascular disease due to its potential for predicting stroke in population-based studies. We investigated the relationship of metabolic syndrome with stroke recurrence., Methods: This was a retrospective analysis of Chinese patients enrolled in the prospective Abnormal gluCose Regulation in patients with acute strOke acroSS China (ACROSS-China) study after their first ischemic stroke. Metabolic syndrome was defined using the International Diabetes Federation (IDF) criteria. Vascular risk factors were assessed. Outcome was defined as recurrence of stroke within one year after the index ischemic stroke. Cox proportional hazards regression was performed to identify potential predictors of stroke recurrence., Results: The prevalence of metabolic syndrome among 2639 ischemic stroke patients was 51.35%. During the one-year follow-up, 195 strokes (7.4%) recurred. The multivariate hazard ratio (95% CI) of stroke recurrence was 1.94 (1.39-2.73) for metabolic syndrome. After adjustment for components, metabolic syndrome lost its association with stroke recurrence; in this model, high fasting plasma glucose (IDF definition) was a predictor for stroke recurrence., Conclusion: Metabolic syndrome may not be predictive for stroke recurrence beyond its component individual factors for Chinese ischemic stroke patients.