Dynamic microfluidic single-cell screening identifies pheno-tuning compounds to potentiate tuberculosis therapy.

Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat.

Microfluidic dose-response platform to track the dynamics of drug response in single mycobacterial cells.

Preclinical analysis of drug efficacy is critical for drug development. However, conventional bulk-cell assays statically assess the mean population behavior, lacking resolution on drug-escaping cells. Inaccurate estimation of efficacy can lead to overestimation of compounds, whose efficacy will not be confirmed in the clinic, or lead to rejection of valuable candidates. Time-lapse microfluidic microscopy is a powerful approach to characterize drugs at high spatiotemporal resolution, but hard to apply on a large scale. Here we report the development of a microfluidic platform based on a pneumatic operating principle, which is scalable and compatible with long-term live-cell imaging and with simultaneous analysis of different drug concentrations. We tested the platform with mycobacterial cells, including the tubercular pathogen, providing the first proof of concept of a single-cell dose-response assay. This dynamic in-vitro model will prove useful to probe the fate of drug-stressed cells, providing improved predictions of drug efficacy in the clinic.

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

Species-specific impact of the autophagy machinery on Chikungunya virus infection.

Chikungunya virus (CHIKV) is a recently re-emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52--but not its mouse orthologue--interacts with the non-structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.

Chikungunya virus infection of corneal grafts.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus with a high potential to spread globally. We investigated whether CHIKV is transmittable via corneal grafts. METHODS: Serum specimens from 69 potential corneal donors living in La Réunion during the 2005­2006 outbreak of CHIKV infection were screened for anti-CHIKV antibodies. Serum specimens and corneoscleral rims were subjected to quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) for detection of CHIKV. CHIKV isolation and immunolabeling were performed on eye tissue specimens. Viral transmission via the ocular route was assessed in an animal model of human CHIKV infection. RESULTS: Twelve apparently uninfected donors were viremic and/or positive for immunoglobulin M (IgM) and/or immunoglobulin G. Eye tissue specimens from 12 donors who were or were not viremic and were or were not seropositive were investigated. qRT-PCR detected CHIKV RNA in corneoscleral rims from 4 patients: 1 patient was viremic, 2 were viremic and IgM positive, and 1 was IgM positive. Infectious CHIKV was isolated from all qRT-PCR­positive samples, and antigens were detected in corneal and scleral specimens, the iris, the ciliary body, and oculomotor muscles. CONCLUSIONS: One-third of eligible corneas (4 of 12) from donors apparently uninfected with CHIKV were infected with CHIKV during the study period. CHIKV infects the human cornea and can be transmitted via the ocular route. In the absence of systematic CHIKV screening in donors, cornea donation should be banned in areas where CHIKV circulates.

Type I IFN controls chikungunya virus via its action on nonhematopoietic cells.

Chikungunya virus (CHIKV) is the causative agent of an outbreak that began in La Réunion in 2005 and remains a major public health concern in India, Southeast Asia, and southern Europe. CHIKV is transmitted to humans by mosquitoes and the associated disease is characterized by fever, myalgia, arthralgia, and rash. As viral load in infected patients declines before the appearance of neutralizing antibodies, we studied the role of type I interferon (IFN) in CHIKV pathogenesis. Based on human studies and mouse experimentation, we show that CHIKV does not directly stimulate type I IFN production in immune cells. Instead, infected nonhematopoietic cells sense viral RNA in a Cardif-dependent manner and participate in the control of infection through their production of type I IFNs. Although the Cardif signaling pathway contributes to the immune response, we also find evidence for a MyD88-dependent sensor that is critical for preventing viral dissemination. Moreover, we demonstrate that IFN-alpha/beta receptor (IFNAR) expression is required in the periphery but not on immune cells, as IFNAR(-/-)-->WT bone marrow chimeras are capable of clearing the infection, whereas WT-->IFNAR(-/-) chimeras succumb. This study defines an essential role for type I IFN, produced via cooperation between multiple host sensors and acting directly on nonhematopoietic cells, in the control of CHIKV.

Prophylaxis and therapy for Chikungunya virus infection.

BACKGROUND: Chikungunya virus (CHIKV) is a recently reemerged arbovirus responsible for a massive outbreak of infection in the Indian Ocean region and India that has a very significant potential to spread globally because of the worldwide distribution of its mosquito vectors. CHIKV induces a usually self-limited disease in humans that is characterized by fever, arthralgia, myalgia, and rash; however, cases of severe CHIKV infection have recently been described, particularly in adults with underlying condition and neonates born to viremic mothers. METHODS: Human polyvalent immunoglobulins were purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection, and the preventive and curative effects of these immunoglobulins were investigated in 2 mouse models of CHIKV infection that we developed. RESULTS: CHIKV immunoglobulins contain anti-CHIKV antibodies and exhibit a high in vitro neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection in vivo, including in the neonate. CONCLUSIONS: Administration of CHIKV immunoglobulins may constitute a safe and efficacious prevention strategy and treatment for individuals exposed to CHIKV who are at risk of severe infection, such as neonates born to viremic mothers and adults with underlying conditions. These results provide a proof-of-concept for purifying human immunoglobulins from plasma samples from patients in the convalescent phase of an emerging infectious disease for which neither prevention nor treatment is available.

O-GlcNAc modification of FoxO1 increases its transcriptional activity: a role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.

O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene.

Mono-O-glycosylations post-translationally regulate the activity of nucleocytoplasmic proteins. We showed that glucosamine and an inhibitor of deglycosylation (PUGNAc) induced O-glycosylation of FoxO1, resulting in increased expression of a glucose-6-phosphatase reporter gene. This effect was independent of FoxO1 re-localisation, since it was also observed with constitutively nuclear FoxO1-AAA mutant. Moreover, in HepG2 cells, glucosamine and PUGNAc have a synergistic effect on the glucose-6-phosphatase reporter gene, and this effect was inhibited by FoxO1 siRNAs. Since glucose-6-phosphatase plays a key role in hepatic glucose production, our observation may be of importance with regard to glucotoxicity associated with chronic hyperglycaemia in diabetes.

The expression of BIRC5 is correlated with loss of specific chromosomal regions in breast carcinomas.

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosis protein (IAP) family, is elevated in fetal tissues and in various human cancers. Mechanisms up-regulating BIRC5 in cancer are poorly understood. Here, we show that overexpression of BIRC5 induces a high proliferation level in MCF-7 breast tumor cells. In a population of 191 breast carcinomas, BIRC5 expression is not affected by BIRC5 promoter polymorphism at -31, or BIRC5 gene copy number. However, a significant correlation was found between expression of demethylase (dMTase) and expression of BIRC5. In addition, among 13 chromosomal regions tested for allelic loss [loss of heterozygosity (LOH)], two regions close to D3S1478 and D6S264 were related to BIRC5 expression. In tumors with LOH at D3S1478 and/or D6S264, BIRC5 expression was significantly increased. These regions have been suggested to harbor tumor suppressor genes and/or common fragile sites that may play a role in increasing genetic instability. These results suggest that genes located near D3S1478 and D6S264 might work by inhibiting, directly or indirectly, BIRC5 expression and thus their loss leads to its up-regulation. In addition, BIRC5 expression may induce breast tumor proliferation by promoting genetic instability. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.