Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

Insertional mutagenesis in a haploid background can lead to complete disruption of gene function1. Here we generate a population of human cells that contain insertions in >98% of their expressed genes. We established Phenotypic Interrogation via Tag Sequencing (PhITSeq) as a method to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of selected cells rather than individual clones provides a rapid assessment of the spectrum of genes involved in phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic gram-negative bacteria that cause tissue damage at distinct anatomical sites2. We identified 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. While related CDTs may share host factors, they also exploit unique host factors yielding a characteristic profile for each CDT.

BackgroundPrevious studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics.Methodology/Principal FindingsWe used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes.Conclusions/SignificanceThis study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted.

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

About 100 years ago, the first antibiotic drug was introduced into health care. Since then, antibiotics have made an outstanding impact on human medicine. However, our society increasingly suffers from collateral damage exerted by these highly effective drugs. The rise of resistant pathogen strains, combined with a reduction of microbiota diversity upon antibiotic treatment, has become a significant obstacle in the fight against invasive infections worldwide.Alternative and complementary strategies to classical "Fleming antibiotics" comprise microbiotabased treatments such as fecal microbiota transfer and administration of probiotics, livebiotherapeutics, prebiotics, and postbiotics. Other promising interventions, whose efficacy may also be influenced by the human microbiota, are phages and vaccines. They will facilitate antimicrobial stewardship, to date the only globally applied antibiotic resistance mitigation strategy.In this review, we present the available evidence on these nontraditional interventions, highlight their interaction with the human microbiota, and discuss their clinical applicability.

The association of gut microbiota dysbiosis with various human diseases is being substantiated with increasing evidence. Metabolites derived from both, microbiota and the human host play a central role in disease susceptibility and disease progression by extensively modulating host physiology and metabolism. Several of these metabolites have the potential to serve as diagnostic biomarkers for monitoring disease states in conjunction with intestinal microbiota dysbiosis. In this narrative review we evaluate the potential of trimethylamine-N-oxide, short-chain fatty acids, 3-indoxyl sulfate, p-cresyl sulfate, secondary bile acids, hippurate, human β-defensin-2, chromogranin A, secreted immunoglobulins and zonulin to serve as biomarkers for metabolite profiling and diagnostic suitability for dysbiosis and disease.

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Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.