Single Cell Killing Kinetics Differentiate Phenotypic Bacterial Responses to Different Antibacterial Classes

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability. We have made available a step-by-step experimental protocol for the fabrication and handling of microfluidic devices for investigating the interactions between the antimicrobial agents and individual cells (74). Data supporting the conclusions of this article will be made available by the authors to any qualified researcher upon request.With the spread of multidrug-resistant bacteria, there has been an increasing focus on molecular classes that have not yet yielded an antibiotic. A key capability for assessing and prescribing new antibacterial treatments is to compare the effects antibacterial agents have on bacterial growth at a phenotypic, single-cell level. Here, we combined time-lapse microscopy with microfluidics to investigate the concentration-dependent killing kinetics of stationary-phase Escherichia coli cells. We used antibacterial agents from three different molecular classes, β-lactams and fluoroquinolones, with the known antibiotics ampicillin and ciprofloxacin, respectively, and a new experimental class, protein Ψ-capsids. We found that bacterial cells elongated when treated with ampicillin and ciprofloxacin used at their minimum inhibitory concentration (MIC). This was in contrast to Ψ-capsids, which arrested bacterial elongation within the first two hours of treatment. At concentrations exceeding the MIC, all the antibacterial agents tested arrested bacterial growth within the first 2 h of treatment. Further, our single-cell experiments revealed differences in the modes of action of three different agents. At the MIC, ampicillin and ciprofloxacin caused the lysis of bacterial cells, whereas at higher concentrations, the mode of action shifted toward membrane disruption. The Ψ-capsids killed cells by disrupting their membranes at all concentrations tested. Finally, at increasing concentrations, ampicillin and Ψ-capsids reduced the fraction of the population that survived treatment in a viable but nonculturable state, whereas ciprofloxacin increased this fraction. This study introduces an effective capability to differentiate the killing kinetics of antibacterial agents from different molecular classes and offers a high content analysis of antibacterial mechanisms at the single-cell level. IMPORTANCE Antibiotics act against bacterial pathogens by inhibiting their growth or killing them directly. Different modes of action determine different antibacterial responses, whereas phenotypic differences in bacteria can challenge the efficacy of antibiotics. Therefore, it is important to be able to differentiate the concentration-dependent killing kinetics of antibacterial agents at a single-cell level, in particular for molecular classes which have not yielded an antibiotic before. Here, we measured single-cell responses using microfluidics-enabled imaging, revealing that a novel class of antibacterial agents, protein Ψ-capsids, arrests bacterial elongation at the onset of treatment, whereas elongation continues for cells treated with β-lactam and fluoroquinolone antibiotics. The study advances our current understanding of antibacterial function and offers an effective strategy for the comparative design of new antibacterial therapies, as well as clinical antibiotic susceptibility testing.Biotechnology and Biological Sciences Research Council (BBSRC)Medical Research Council (MRC)UK Department for Business, Energy and Industrial Strategy (BEIS

Segmentation of Retinal Ganglion Cells From Fluorescent Microscopy Imaging

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Unsupervised Spike Sorting for Large-Scale, High-Density Multielectrode Arrays

We present a method for automated spike sorting for recordings with high-density, large-scale multielectrode arrays. Exploiting the dense sampling of single neurons by multiple electrodes, an efficient, low-dimensional representation of detected spikes consisting of estimated spatial spike locations and dominant spike shape features is exploited for fast and reliable clustering into single units. Millions of events can be sorted in minutes, and the method is parallelized and scales better than quadratically with the number of detected spikes. Performance is demonstrated using recordings with a 4,096-channel array and validated using anatomical imaging, optogenetic stimulation, and model-based quality control. A comparison with semi-automated, shape-based spike sorting exposes significant limitations of conventional methods. Our approach demonstrates that it is feasible to reliably isolate the activity of up to thousands of neurons and that dense, multi-channel probes substantially aid reliable spike sorting

Phytochemical Analysis, Antioxidant and Antimicrobial Properties of Hexane, Ethyl acetate, and Methanol Leaf Extracts of Ipomoea hildebrandtii Vatke Shrub Plant

Ipomoea hildebrandtii Vatke have been effectively used to relieve general body pains, treat wounds and cuts in the semi-arid region of Kenya. Hence the objective of this paper was to evaluate the phytochemical analysis, antioxidant and antimicrobial properties of hexane, ethyl acetate and methanol leaf extracts of Ipomoea hildebrandtii Vatke shrub plant. The profiling of the phytochemicals was carried out using the Fourier-transform infrared (FTIR) and Gas Chromatography-Mass spectroscopy (GC-MS) analyses. Antimicrobial and antioxidant activities of the methanol, hexane and ethyl acetate extracts of I. hildebrandtii were determined using disk diffusion and 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging assays respectively. Preliminary phytochemical screening revealed that the leaf extracts are enriched with alkaloids, tannins, terpenoids, steroids, glycosides, flavonoids, phenols, quinones, and saponins. With GC-MS, the major compound tetratetracontane (73.14%) was identified in hexane and 9-octadecanamide, (Z)- in ethyl acetate and methanol extracts (30.99%) and (43.09%) respectively. The IR spectroscopy revealed various functional groups such as C=O, C=C, –OH and –NH. The methanol extract showed good antimicrobial activity at 1000 mg/mL against all the microorganisms tested except Escherichia coli while the hexane extract was the best antimicrobial activity against E. coli at 1000 mg/mL. The ethyl acetate extract showed the best antioxidant activity against DPPH (IC50 = 48.70 ± 1.54 µg/mL) compared to the standard ascorbic acid (IC50 = 21.24 ± 0.12 µg/mL). The experimental findings showed that all extracts from I. hildebrandtii leaves possess significant antimicrobial and antioxidant activities justifying its use in traditional medicine. These biological activities might be due to the presence of the presence of the identified phytochemicals in them

Unsupervised spike sorting for large-scale, high-density multielectrode arrays

electrophysiology; high-density multielectrode array; neural cultures; retina; spike sortin

Deep sequencing reveals the complex and coordinated transcriptional regulation of genes related to grain quality in rice cultivars

<p>Abstract</p> <p>Background</p> <p>Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control).</p> <p>Results</p> <p>The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved <it>cis </it>regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development.</p> <p>Conclusion</p> <p>This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate family amino acids, and storage proteins. Some of the differentially expressed genes could be useful for the development of molecular markers if they are located in a known QTL region for milling yield or eating quality in the rice genome. Therefore, our comprehensive and deep survey of the developing seed transcriptome in five rice cultivars has provided a rich genomic resource for further elucidating the molecular basis of grain quality in rice.</p

AP1S3 Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production

Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting AP1S3, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that AP1S3 expression is distinctively elevated in keratinocytes. Because AP1S3 encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that AP1S3 knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-kappa B activation. We showed that as a consequence, AP1S3-deficient cells up-regulate IL-1 signaling and overexpress IL-36 alpha, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.Peer reviewe

High-resolution retinal imaging with a compact adaptive optics ophthalmoscope

This thesis presents work on the development of a compact adaptive optics ophthalmoscope to visualize microscopic details of the human fovea. Conventional ophthalmoscopes currently employed in retinal imaging for diagnostic purposes help to detect disorders in real-time; however, their resolution is limited by the optical quality of the last focusing lens, the human eye. In recent years there has been a significant increase in studying retinal alterations, including the complication of non-ophthalmic diseases. In a number of cases, especially for visually impaired and elderly people, when the ocular media become less transparent, fixation is hard for the patients. It is often difficult to repeat the measurements during the usual clinical diagnostic routine; the dynamic changes and imperfection in the optics of the eye also significantly degrade the retinal image quality. In order to resolve cellular level details and hence detect ocular diseases in their infancy, dynamic correction of ocular aberrations is required. Developments in ophthalmoscopy have extended its application to high-resolution imaging using adaptive optics. This technology enables the in-vivo study of finer microscopic structures by dynamically correcting higher-order ocular aberrations. To date, such systems have been large and confined to research laboratory conditions. This thesis investigates the performance of a compact adaptive optics ophthalmoscope built in a cost effective way to provide a diagnostic tool that is more affordable and usable in a general clinical environment. It also highlights some of the problems associated with retinal imaging and discusses the limitations of retinal imaging systems. The results obtained with this system suggest that it is possible to non-invasively detect structural and functional changes of the retina in their early phases of development and enable precise monitoring of the effect of therapies in later clinical research