Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.

Single-particle imaging of nanomedicine entering the brain.

Nanomedicine has emerged as a revolutionary strategy of drug delivery. However, fundamentals of the nano-neuro interaction are elusive. In particular, whether nanocarriers can cross the blood-brain barrier (BBB) and release the drug cargo inside the brain, a basic process depicted in numerous books and reviews, remains controversial. Here, we develop an optical method, based on stimulated Raman scattering, for imaging nanocarriers in tissues. Our method achieves a suite of capabilities-single-particle sensitivity, chemical specificity, and particle counting capability. With this method, we visualize individual intact nanocarriers crossing the BBB of mouse brains and quantify the absolute number by particle counting. The fate of nanocarriers after crossing the BBB shows remarkable heterogeneity across multiple scales. With a mouse model of aging, we find that blood-brain transport of nanocarriers decreases with age substantially. This technology would facilitate development of effective therapeutics for brain diseases and clinical translation of nanocarrier-based treatment in general.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Quantitative Label-Free Chemical Imaging of PLGA Nanoparticles in Cells and Tissues with Single-Particle Sensitivity.

Nanomedicine has brought significant advancements to healthcare by utilizing nanotechnology in medicine. Despite much promise, the further development of nanocarriers for clinical use has been hindered by a lack of understanding and visualization of nano-bio interactions. Conventional imaging methods have limitations in resolution, sensitivity, and specificity. This study introduces a label-free optical approach using stimulated Raman scattering (SRS) microscopy to image poly(lactic-co-glycolic acid) (PLGA) nanocarriers, the most widely used polymeric nanocarrier for delivery therapeutic agents, with single-particle sensitivity and quantification capabilities. A unique Raman peak was identified for PLGA ester, enabling generalized bio-orthogonal bond imaging. We demonstrated quantitative SRS imaging of PLGA nanocarriers across different biological systems from cells to animal tissues. This label-free imaging method provides a powerful tool for studying this prevalent nanocarrier and quantitatively visualizing their distribution, interaction, and clearance in vivo.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Transcriptome and Quasi-Targeted Metabolome Analyze Overexpression of 4-Hydroxyphenylpyruvate Dioxygenase Alleviates Fungal Toxicity of 9-Phenanthrol in Magnaporthe oryzae.

Magnaporthe oryzae, the causal agent of rice blast disease, produces devastating damage to global rice production. It is urgent to explore novel strategies to overcome the losses caused by this disease. 9-phenanthrol is often used as a transient receptor potential melastatin 4 (TRPM4) channel inhibitor for animals, but we found its fungal toxicity to M. oryzae. Thus, we explored the antimicrobial mechanism through transcriptome and metabolome analyses. Moreover, we found that overexpression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase involved in the tyrosine degradative pathway enhanced the tolerance of 9-phenanthrol in M. oryzae. Thus, our results highlight the potential fungal toxicity mechanism of 9-phenanthrol at metabolic and transcriptomic levels and identify a gene involving 9-phenanthrol alleviation. Importantly, our results demonstrate the novel mechanism of 9-phenanthrol on fungal toxicity that will provide new insights of 9-phenanthrol for application on other organisms.

c-Fos separation from Lamin A/C by GDF15 promotes colon cancer invasion and metastasis in inflammatory microenvironment.

Inflammatory microenvironment is an important factor for promoting cancer invasion and metastasis, but the underlying molecular mechanisms remain unclear. Here, we mimicked an inflammatory microenvironment both in vitro and in vivo and investigated its effects on the invasion and metastasis of colon cancer. Moreover, colon cancer patient samples were also analyzed statistically. Conditioned medium from the differentiated macrophages induced invasion and migration of colon cancer cells in vitro, which could be reversed by the treatment of a neutralizing anti-growth differentiation factor 15 (GDF15) antibody, indicating GDF15 involvement in inflammation-induced invasiveness. Also, we observed similar effects of human recombinant GDF15 on colon cancer cells. Mechanistically, GDF15 activated c-Fos by separating it from Lamin A/C, increasing transcriptional activity of c-Fos and regulating EMT gene expressions. However, c-Fos knockdown using lentivirus shRNA plasmid inhibited GDF15-triggered invasion and migration in vitro. In vivo, inflammation caused by lipopolysaccharides obviously increased GDF15 secretion, and c-Fos knockdown reduced the lung metastasis of colon cancer cells in mice model. In addition, c-Fos expressions in patient samples were found to be associated with colon cancer metastasis and TNM stages. Taken together, GDF15 in inflammatory microenvironment induces colon cancer invasion and metastasis by regulating EMT genes by activating c-Fos, which might be a potential therapeutic target for metastatic colon cancer.

Bimodal Molecule as NIR-CT Contrast Agent for Hepatoma Specific Imaging.

With currently available molecular imaging techniques, hepatocellular carcinoma (HCC), a liver cancer with high mortality rates and poor treatment responses, is mostly diagnosed at its late stage. This is largely due to the lack of highly sensitive contrast agents with high liver specificity. Herein, we report a novel bimodal contrast agent molecule CNCI-1 for the effective detection of HCC at its early stage both in vitro and in vivo. The agent has high liver specificity with effective X-ray computed tomography (CT)/near-infrared (NIR) imaging functions. It has been successfully applied to in vivo NIR imaging with high sensitivity and high selectivity to the HCC region of the HepG2 tumor-xenografted mice model and LM3 orthotopic hepatoma mice model. Moreover, the agent was found to be noninvasive and hepatocarcinoma cells preferential. Furthermore, it also enhanced the tumor imaging by revealing the blood vessels nearby for the CT image acquisition in the VX2 orthotopic hepatoma rabbit model. Our design strategy provides a new avenue to develop the medical relevant bimodal contrast agents for diagnosis of HCC at its early stage.

Oroxylin A increases the sensitivity of temozolomide on glioma cells by hypoxia-inducible factor 1α/hedgehog pathway under hypoxia.

Microenvironmental hypoxia-mediated drug resistance is responsible for the failure of cancer therapy. To date, the role of the hedgehog pathway in resistance to temozolomide (TMZ) under hypoxia has not been investigated. In this study, we discovered that the increasing hypoxia-inducible factor 1α (HIF-1α) activated the hedgehog pathway in hypoxic microenvironment by promoting autocrine secretion of sonic hedgehog protein (Shh), and then upregulating transfer of Gli1 to the nucleus, finally contributed to TMZ resistance in glioma cells. Oroxylin A (C16H12O5), a bioactive flavonoid, could induce HIF-1α degradation via prolyl-hydroxylases-VHL signaling pathway, resulting in the inactivation of the hedgehog. Besides, oroxylin A increased the expression of Sufu, which is a negative regulator of Gli1. By this mechanism, oroxylin A sensitized TMZ on glioma cells. U251 intracranial transplantation model and GL261 xenograft model were used to confirm the reversal effects of oroxylin A in vivo. In conclusion, our results demonstrated that HIF-1α/hedgehog pathway conferred TMZ resistance under hypoxia, and oroxylin A was capable of increasing the sensitivity of TMZ on glioma cells in vitro and in vivo by inhibiting HIF-1α/hedgehog pathway and depressing the activation of Gli1 directly.

Thermopower Modulation Clarification of the Operating Mechanism in Wide Bandgap BaSnO3 -SrSnO3 Solid-Solution Based Thin Film Transistors.

The transparent oxide semiconductor (TOS) with large bandgap (Eg ≈ 4 eV) based thin-film transistors (TFTs) showing both high carrier mobility and UV-visible transparency has attracted increasing attention as a promising component for next generation optoelectronics. Among TOSs, BaSnO3 -SrSnO3 solid-solutions (Eg = 3.5-4.2 eV) are good candidates because the single crystal shows very high mobility. However, the TFT performance has not been optimized due to the lack of fundamental knowledge especially the effective thickness (teff ) and the carrier effective mass (m*). Here, it is demonstrated that the electric field thermopower (S) modulation method addresses this problem by combining with the standard volume carrier concentration (n3D ) dependence of S measurements. By comparing the electric field accumulated sheet carrier concentration (n2D ) and n3D at same S, it is clarified that the teff (n2D /n3D ) of the conducting channel becomes thicker with increasing Sr concentration, whereas the m* becomes lighter. The former would be due to the increase of Eg and latter would be due to the enhancement of overlap population of neighboring Sn 5s orbitals. The present analyses technique is useful to experimentally clarify the teff and m*, and essentially important to realize advanced TOS-based TFTs showing both high optical transparency and high mobility.

Pharmacometabolic response to pirfenidone in pulmonary fibrosis detected by MALDI-FTICR-MSI.

Idiopathic pulmonary fibrosis (IPF) is a fatal condition that reduces life expectancy and shows a limited response to available therapies. Pirfenidone has been approved for treatment of IPF, but little is known about the distinct metabolic changes that occur in the lung upon pirfenidone administration.Here, we performed a proof-of-concept study using high-resolution quantitative matrix-assisted laser desorption/ionisation Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI) to simultaneously detect, visualise and quantify in situ endogenous and exogenous metabolites in lungs of mice subjected to experimental fibrosis and human patients with IPF, and to assess the effect of pirfenidone treatment on metabolite levels.Metabolic pathway analysis and endogenous metabolite quantification revealed that pirfenidone treatment restores redox imbalance and glycolysis in IPF tissues, and downregulates ascorbate and aldarate metabolism, thereby likely contributing to in situ modulation of collagen processing. As such, we detected specific alterations in metabolite pathways in fibrosis and, importantly, metabolic recalibration following pirfenidone treatment.Together, these results highlight the suitability of high-resolution MALDI-FTICR-MSI for deciphering the therapeutic effects of pirfenidone and provide a preliminary analysis of the metabolic changes that occur during pirfenidone treatment in vivo These data may therefore contribute to improvement of currently available therapies for IPF.

Pharmacokinetic and pharmacometabolomic study of pirfenidone in normal mouse tissues using high mass resolution MALDI-FTICR-mass spectrometry imaging.

Given the importance of pirfenidone as the first worldwide-approved drug for idiopathic pulmonary fibrosis treatment, its pharmacodynamic properties and the metabolic response to pirfenidone treatment have not been fully elucidated. The aim of the present study was to get molecular insights of pirfenidone-related pharmacometabolomic response using MALDI-FTICR-MSI. Quantitative MALDI-FTICR-MSI was carried out for determining the pharmacokinetic properties of pirfenidone and its related metabolites 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone in lung, liver and kidney. To monitor the effect of pirfenidone administration on endogenous cell metabolism, additional in situ endogenous metabolite imaging was performed in lung tissue sections. While pirfenidone is highly abundant and delocalized across the whole micro-regions of lung, kidney and liver, 5-hydroxymethyl pirfenidone and 5-carboxy pirfenidone demonstrate heterogeneous distribution patterns in lung and kidney. In situ endogenous metabolite imaging study of lung tissue indicates no significant effects of pirfenidone on metabolic pathways. Remarkably, we found 129 discriminative m/z values which represent clear differences between control and treated lungs, the majority of which are currently unknown. PCA analysis and heatmap view can accurately distinguish control and treated groups. This is the first pharmacokinetic study to investigate the tissue distribution of orally administered pirfenidone and its related metabolites simultaneously in organs without labeling. The combination of pharmametabolome with histological features provides detailed mapping of drug effects on metabolism as response of healthy lung tissue to pirfenidone treatment.

Microchip dual-frequency laser with well-balanced intensity utilizing temperature control.

A continuous-wave microchip dual-frequency laser (DFL) with well balanced intensity was presented. In order to obtain such a balanced intensity distribution of the two frequency components, the DFL wavelengths were precisely tuned and spectrally matched with the emission cross section (ECS) spectrum of the gain medium by employing a temperature controller. Finally, when the heat sink temperature was controlled at -5.6°C, a 264 mW DFL signal was achieved with frequency separation at 67.52 GHz and intensity balance ratio (IBR) at 0.991.

The Effectiveness and Safety of Nafamostat Mesylate in the Treatment of COVID-19: a Meta-Analysis.

Nafamostat mesylate, a synthetic serine protease inhibitor, has been shown to have antiviral activity against SARS-CoV-2 and anticoagulant properties that may be beneficial in the treatment of COVID-19. We conducted a meta-analysis to evaluate the effectiveness and safety of nafamostat mesylate for the treatment of COVID-19. PubMed, Embase, Cochrane Library, Scopus, Web of Science, medRxiv, and bioRxiv were searched up to July 2023 for studies comparing the outcomes of nafamostat mesylate treatment and no nafamostat mesylate treatment in patients with COVID-19. Mortality, disease progression, and adverse events were analyzed. Six studies involving 16,195 patients were included in the analysis. Meta-analysis revealed no significant difference in mortality (odds ratio [OR]: 0.88, 95% CI: 0.20-3.75, P = 0.86) or disease progression (OR: 2.76, 95% CI: 0.31-24.68, P = 0.36) between groups. However, nafamostat mesylate was associated with an increased risk of hyperkalemia (OR: 7.15, 95% CI: 2.66-19.24, P < 0.0001). Nafamostat mesylate did not improve mortality or morbidity in hospitalized patients with COVID-19. The risk of hyperkalemia is a serious concern that requires monitoring and preventive measures. Further research in different COVID-19 populations is required.

Development of porous materials via protein/polysaccharides/polyphenols nanoparticles stabilized Pickering high internal phase emulsions for adsorption of Pb2+ and Cu2+ ions.

The porous materials (PM) were prepared by the Pickering high internal phase emulsion (PHIPE) template. Firstly, the nanoparticles named as ZHMNPs or MZHMNPs were fabricated based on zein, Hohenbuehelia serotina polysaccharides and Malus baccata (Linn.) Borkh polyphenols without or with Maillard reaction, the average particle sizes and zeta potentials of which were distributed in a range of 718.1-979.4 nm and -21.6-25.2 mV. ZHMNPs possessed the relatively uniform spherical morphology, while MZHMNPs were irregular in shape. With ZHMNPs or MZHMNPs serving as the stabilizers, the PHIPEs were prepared, and exhibited the good viscoelasticity and excellent storage and freeze-thaw stabilities. Based on above PHIPEs template, the constructed PM possessed the large specific surface area and uniform pore structure. Through the investigations of adsorption performances, PM showed the outstanding adsorption capacities on Pb2+ and Cu2+ ions regardless of dissolving in deionized water or simulated gastrointestinal digestive fluid. Furthermore, the results also showed that the pH, temperature and adsorbent dosage had certain impacts on the adsorption performances of PM on Pb2+ and Cu2+ ions.

Systematic notes on three new Luthela (Mesothelae, Heptathelidae) spiders from China, with their descriptions.

Three new segmented trapdoor spider species belonging to the family Heptathelidae Kishida, 1923, i.e., Luthelaasukasp. nov. (♂♀, Sichuan), L.beijingsp. nov. (♂♀, Beijing), and L.kagamisp. nov. (♂♀, Sichuan), are described from China. Their phylogenetic position and relationships within Heptathelidae are tested and assessed using a combination available COI data downloaded from GenBank with new DNA sequences obtained in this study. The results show that the new species form a clade with eight known and one undescribed species of Luthela. High-definition illustrations of the male palps and female genitalia, diagnoses, and DNA barcodes are provided for these three new species, and their distributions are mapped.

Lateral Extensional Mode Piezoelectric ZnO-on-Nickel RF MEMS Resonators for Back-End-of-Line Integration.

High motional resistance and incompatibility with post-CMOS fabrication due to thermal budget constraints are imperative issues associated with the back-end-of-line integration of lateral extensional vibrating micromechanical resonators. This paper presents piezoelectric ZnO-on-nickel resonators as a viable means for mitigating both of the issues. Lateral extensional mode resonators equipped with thin-film piezoelectric transducers can exhibit much lower motional impedances than their capacitive counterparts due to piezo-transducers' higher electromechanical coupling coefficients. Meanwhile, the employment of electroplated nickel as the structural material allows the process temperature to be kept lower than 300 °C, which is low enough for the post-CMOS resonator fabrication. In this work, various geometrical rectangular and square plates resonators are investigated. Moreover, parallel combination of several resonators into a mechanically coupled array was explored as a systematic approach to lower motional resistance from ~1 kΩs to 0.562 kΩs. Higher order modes were investigated for achieving higher resonance frequencies up to 1.57 GHz. Local annealing by Joule heating was also exploited for quality factor improvement after device fabrication by ~2× enhancement and breaking the record of MEMS electroplated nickel resonators in lowering insertion loss to ~10 dB.

Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation.

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Multiomics Analyses Reveal the Complexity of Interaction between Two Strains of Magnaporthe oryzae.

Plants growing in open environments are frequently coinfected by multiple strains of the same pathogen. However, few investigations have been carried out to reveal the outcomes and underlying mechanisms of such infections. This study aimed to observe the behaviors of two different strains under coinfection and cocultivation. We constructed an experimental system to study such interactions directly by labeling Magnaporthe oryzae strains with the green fluorescent proteins and mushroom cherry fluorescent protein to observe mixed strain behavior in vivo and in vitro. Moreover, multiomics analyses were conducted to explore the underlying mechanisms at the genomic, transcriptomic, and metabolomic levels. Our results revealed that coinfection with two strains can affect disease severity and that the more weakly virulent strain benefits from the coinfection system. We also found that amino acid variation might negatively influence such interactions at transcriptomic and metabolomic levels. In addition, we showed that the overexpression of a glutamine-related gene improved strain competitiveness during mixture cultivation. Collectively, our results provided experimental methods to analyze the interaction between two strains of M. oryzae and preliminarily explored the interacted mechanism of two strains under cocultivation through multiomics analyses.

Cell arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.