Background:The role of endoplasmic reticulum (ER) stress in influenza A viral infection is unknown. Results: Influenza A virus induces the IRE1 pathway of the ER stress response. Inhibition of IRE1 activity leads to decreased viral replication. Conclusion: IRE1 is a potential therapeutic target for influenza A virus. Significance: Targeting a host molecular mechanism is a novel therapeutic strategy that is less likely to be invalidated by viral mutagenesis.

Neisseria gonorrhoeae is a strict human pathogen that is, primarily, transmitted by close sexual contact with an infected individual. Gonococcal infection of the male urogenital tract has been well studied in experimental human models and in urethral cell culture systems. Recent studies, using tissue culture cell systems, have suggested a role for the cervical epithelium in gonococcal infection of females; however, the nature of gonococcal infection of the normal uterine cervix remains controversial. To address this enigma, we have developed two primary human cervical epithelial cell systems from surgical biopsies. Gonococcal infection studies and electron microscopy show that N. gonorrhoeae is capable of infecting and invading both the endo-and the ectocervix. Invasion was found to occur primarily in an actin-dependent manner, but it does not appear to require de novo protein synthesis by either the bacterium or the host cervical cell. Membrane ruffles appear to be induced in response to gonococci. Consistent with membrane ruffling, gonococci were found residing within macropinosomes, and a concentrated accumulation of actin-associated proteins was observed to occur in response to gonococcal infection. Electron microscopy of clinically derived cervical biopsies show that lamellipodia formation and cytoskeletal changes, suggestive of membrane ruffles, also occur in the cervical epithelium of women with naturally acquired gonococcal cervicitis. These studies demonstrate the ability of N. gonorrhoeae to infect and invade both the endo-and the ectocervix of the normal uterine cervix. Gonococcal induced ruffling is a novel finding and may be unique to the cervical epithelium.

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate ␣-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with ␣2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were twoto fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing ␣2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

For a comprehensive understanding of cells or tissues, it is important to enable multiple studies under the controllable microenvironment of a chip. In this report, we present an integrated microfluidic cell culture platform in which endothelial cells (ECs) are under static conditions or exposed to a pulsatile and oscillatory shear stress. Through the integration of a microgap, self-contained flow loop, pneumatic pumps, and valves, the novel microfluidic chip achieved multiple functions: pulsatile and oscillatory fluid circulation, cell trapping, cell culture, the formation of ECs barrier, and adding shear stress on cells. After being introduced into the chip by gravity, the ECs arranged along the microgap with the help of hydrodynamic forces and grew in the microchannel for more than 7 days. The cells proliferated and migrated to form a barrier at the microgap to mimic the vessel wall, which separated the microenvironment into two compartments, microchannel and microchamber. An optimized pneumatic micropump was embedded to actuate flow circulation in a self-contained loop that induced a pulsatile and oscillatory shear stress at physiological levels on the ECs in the microchannel. All the analyses were performed under either static or dynamic conditions. The performance of the barrier was evaluated by the diffusion and distribution behaviors of fluorescently labeled albumin. The permeability of the barrier was comparable to that in traditional in vitro assays. The concentration gradients of the tracer formed in the microchamber can potentially be used to study cell polarization, migration and communications in the future. Additionally, the morphology and cytoskeleton of the ECs response to the pulsatile and oscillatory shear stress were analyzed. The microfluidic chip provided a multifunctional platform to enable comprehensive studies of blood vessels at the cell or tissue level.

Neisseria gonorrhoeae forms a biofilm in flow cells on glass coverslips as well as on primary cervical epithelial cells. Electron microscopic studies of cervical biopsy specimens from 10 patients with culture-proven N. gonorrhoeae infection revealed evidence of biofilm formation in 3 of the biopsy specimens. These biofilms showed gonococci in networks of bacterial membrane within the biofilm structure. This finding was also observed in biofilms formed over glass cover slips and after infection of primary cervical tissue in vitro. The importance of membranous networks in Neisseria biofilm formation was demonstrated with N. gonorrhoeae strain 1291-msbB, which shows a markedly decreased ability to bleb. This mutant formed significantly less biofilm over glass surfaces and cervical epithelial cells, and complementation showed reversion to wild-type biofilms. Gonoccal biofilms, as part of the cervical infection, may be involved in the mechanisms by which asymptomatic infections, persistence, and increased antibiotic resistance occur.

Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 M sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.Neisseria gonorrhoeae is a human-adapted, gram-negative diplococcus that infects the human male and female reproductive tracts. N. gonorrhoeae infections in women frequently go unnoticed. This can eventually lead to serious upper genital tract infections which ultimately can lead to infertility (13). Currently, no studies have discussed the ability of N. gonorrhoeae to produce biofilms. Bacterial biofilms have been defined as communities of bacteria intimately associated with each other and included within an exopolymer matrix. These biological units exhibit their own properties, which are quite different from those shown by the single species in planktonic form (15). Numerous bacterial species are capable of producing biofilms. Biofilms confer a number of survival advantages to the bacteria, including increased resistance to antimicrobial agents (7,18).Our interest in the capability of N. gonorrhoeae to form a biofilm came about by observations made in our laboratory during 4-and 8-day infections of primary human urethral and cervical epithelial cells (8,12). Those studies showed that the gonococcus was forming microcolonies on these surfaces, and eventually these transitioned into structures that resembled bacterial biofilms.The purpose of this study was twofold. The first objective was to verify that N. gonorrhoeae can produce a biofilm both in biofilm chambers and over primary human genital tract epithelial cells in culture. The second objective was to gain information ...

SUMMARYDeÿnitions and equations are provided for the quantitative assessment of numerical (veriÿcation) and modelling (validation) errors and uncertainties for CFD simulations and of intervals of certiÿcation for CFD codes. Veriÿcation, validation, and certiÿcation methodology and procedures are described. Examples of application of quantitative certiÿcation of RANS codes are presented for ship hydrodynamics. Opportunities and challenges for achieving consensus and standard V&V and certiÿcation methodology and procedures are discussed.

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.