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Aims We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnF2) toothpaste against a simulated human plaque biofilm (SPB). Methods and results First, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnF2 toothpaste solution, or a sham rise was administered twice daily for 3 days to mimic routine oral hygiene. SPB biomass was assessed by total bacterial DNA and methylene blue (MB) staining. Early colonizer aerobes and late colonizer anaerobes species were detected in the HA and DTBM, and the trends in changing abundance were consistent with those seen clinically. Conclusions Treatment with the SnF2 solution showed significant reductions of 53.05% and 54.4% in the SPB by MB staining and DNA, respectively. Significance and impact of study: The model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.

We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnFFirst, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnFTreatment with the SnFThe model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.

Neuraminidase (NA) is an integral membrane protein of influenza A virus (IAV) and primarily aids in the release of progeny virions, following the intracellular viral replication cycle. In an attempt to discover new functions of NA, we conducted a classical yeast two-hybrid screen and found acute myeloid leukaemia marker 1 (AML1) as a novel interacting partner of IAV-NA. The interaction was further validated by co-immunoprecipitation in IAV-infected cells and in an in vitro coupled transcription/translation system. Interestingly, we found an increase in the expression of AML1 upon IAV infection in a dose-dependent manner. As expected, we also observed an increase in the IFN-β levels, the first line of defence against viral infections. Subsequently, when AML1 was downregulated using siRNA, the IFN-β levels were found to be remarkably reduced. Our study also shows that AML1 is induced upon IAV infection and results in the induction of IFN-β. Thus, AML1 is proposed to be an important player in IFN induction and has a role in an antiviral response against IAV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Influenza epidemics and pandemics are constant threats to human health. Development of antiviral therapeutics has focused on important and major IAV proteins as targets. However, the rate at which this virus mutates makes the task challenging. Thus, next-generation approaches aim at host cellular proteins that aid the virus in its replication. This study reports a new host-virus interaction, of acute myeloid leukaemia marker 1 (AML1) with influenza A neuraminidase (IAV-NA). We have found that this interaction has a direct effect on the upregulation of host IFN-β response. Further studies may lead to a greater understanding of this new innate defence pathway in infected cells.

Opportunistic bacterial infections amongst HIV–infected individuals contribute significantly to HIV-associated mortality. The role of HIV-mediated modulation of innate mechanisms like autophagy in promoting opportunistic infections, however, remains obscure. Here we show, HIV reactivation in or infection of macrophages inhibits autophagy and helps the survival of pathogenic Mycobacterium tuberculosis (Mtb) and nonpathogenic non-tuberculous mycobacterial strains (NTMs). The HIV-mediated impairment of xenophagy flux facilitated bacterial survival. Activation of autophagy by trehalose could induce xenophagy flux and kill intracellular Mtb or NTMs either during single or co-infections. Trehalose, we delineate, activates PIKFYVE leading to TFEB nuclear translocation in MCOLN1-dependent manner to induce autophagy. Remarkably, trehalose significantly reduced HIV-p24 levels in ex–vivo-infected PBMCs or PBMCs from treatment-naive HIV patients and also controlled mycobacterial survival within Mtb-infected animals. To conclude, we report leveraging of HIV-mediated perturbed host innate-immunity by opportunistic bacterial pathogens and show an attractive therapeutic strategy for HIV and associated co-morbidities. Abbreviations: AIDS: acquired immune deficiency syndrome; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; BafA1: bafilomycin A1; CFU: colony forming unit; CTSD: cathepsin D; CD63: CD63 molecule; EGFP: enhanced green fluorescent protein; FRET: Förster resonance energy transfer; GABARAP: gamma-aminobutyric acid receptor-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GLUT: glucose transporter; HIV: human immunodeficiency virus; hMDMs: human monocyte derived macrophages; IL2: interleukin 2; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: lipidated microtubule-associated proteins 1A/1B light chain 3B; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin; mRFP: monomeric red fluorescent protein; M6PR: mannose-6-phosphate receptor; NAC: N- acetyl- L –cysteine; NTM’s: non-tuberculous mycobacteria; PBMC: Peripheral Blood Mononuclear cells; PIKFYVE: phosphoinositide kinase; FYVE-Type Zinc Finger; PHA: phytohemagglutinin; PMA: phorbol 12-myristate 13-acetate; PtdIns(3,5)P2: Phosphatidylinositol 3,5-bisphosphate; ptfLC3: pEGFP-mRFP-LC3; ROS: reactive oxygen species; SQSTM1: sequestosome1; TFEB: transcription factor EB; MCOLN1/TRPML1: mucolipin 1; PIP4P1/TMEM55B: Human trans-membrane Protein 55B; UVRAG: UV Radiation Resistance Associate; VPS35: vacuolar protein sorting associated protein 35; WDR45: WD repeat domain 45; YCAM: Yellow Chameleon.

Given the oral health implications of smoking, vaping, and using smokeless tobacco products (STPs), the dental office can be an effective setting for clinicians to offer smoking cessation counseling and treatment (SCCT).A survey tool was developed to assess dentists' approach and experience with SCCT. This survey was deployed electronically on May 21, 2021, to the American Dental Association Clinical Evaluators (ACE) Panel (N = 982), and 1 week later, nonresponders were sent reminders. Results were summarized in SAS Version 9.4.Of the 283 survey responders, one-half offered SCCT to their patients. Of those who offered SCCT, 69% would be willing to prescribe pharmacologic agents to help with cessation. Nine of 10 times, SCCT conversations included the dentist. Of the 49% of respondents who did not offer SCCT, one-quarter were current or past smokers, vapers, or STP users. Out of all of the survey respondents, 40% were aware that SCCT is part of most medical benefits. Most respondents (82%) perform surgeries on patients who smoke, vape, or use STPs.Implementation of SCCT in the dental office was split among ACE panelists, but when it was offered, the dentist was almost always involved. Awareness about SCCT medical benefits and surgical protocols for current smokers, vapers, and STP users differed among this sample.Dentists can benefit from more formal training and resources around SCCT and access to reimbursement for having these crucial conversations with their patients.

Dentists may choose to integrate intraoral scanners (IOSs) into their practices, but there are many different IOS technologies and system generations to choose from, posing a challenge for dentists who want to invest in them.A survey of IOS use rates, advantages, satisfaction, and training was developed and deployed through Qualtrics to the 968 members of the American Dental Association Clinical Evaluators (ACE) Panel on February 19, 2021. Nonrespondents were sent reminders, and data were analyzed in SAS Version 9.4 (SAS).A total of 369 panelists responded to the survey. IOS use was split among the ACE Panel; 53% indicated they use one in their practice. The top reason respondents began using IOSs was to improve clinical efficiency (70%). Ninety percent of respondents use IOSs for single tooth-supported crowns, and 58% began using IOSs less than 4 years ago. Most users are at least mostly satisfied (91%) with the results. Among nonusers, the top reason for not using an IOS was the high level of financial investment (66%); 34% and 40% of nonusers are thinking of buying or training with IOSs in 2021, respectively.Uptake of IOSs is split; most users are satisfied with their results, and nonusers cited financial barriers as the most common reason for not investing in one.As IOSs continue to penetrate the market and dentists are faced with a decision whether to invest in one, they will need guidance on how to choose the most appropriate device for their patients.

Clinicians often encounter defective restorations and are faced with the difficult decision of whether to repair the existing restoration or replace it.An electronic survey on repairing or replacing defective restorations was developed to assess how clinicians are making these decisions and the technical aspects considered when making a repair. E-mails containing the survey link were sent to the American Dental Association Clinical Evaluators (ACE) Panel on August 14, 2019, and the survey remained open for 2 weeks. Nonrespondents were sent reminders 1 week after deployment.Approximately 4 of every 5 respondents repair defective restorations. The top 3 conditions for making these repairs were noncarious marginal defects (87%), partial loss or fracture of the restoration (79%), and crown margin repair due to carious lesions (73%). Among respondents who repair defective restorations, almost all repair direct resin composite (98%), whereas approximately one-third do not repair the other restorative materials (that is, amalgam, glass ionomer, and fractured indirect all-ceramic crowns). Resin composite is used most often to repair resin direct composite restorations, and likewise, glass ionomer is used most often to repair glass ionomer restorations. Only 54% of respondents use amalgam to repair amalgam restorations. Surface treatments varied among the 3 available restorations types.Many dentists are actively making restoration repairs, but choosing clinical scenarios to make these repairs is material dependent.The repair of defective restorations is an acceptable and more conservative alternative than restoration replacement, and its success depends on proper case selection, material, and technique.

Background With many states in the United States permitting dentists to administer the COVID-19 vaccine, there is much discussion about their scope of practice in relation to delivering other vaccines. Methods Survey questions were developed to assess dentists’ awareness about their vaccine administration scope of practice and attitudes and barriers if choosing to incorporate vaccine delivery into their practice scope. The survey was deployed electronically to members of the American Dental Association Clinical Evaluators (ACE) Panel (N = 989) on September 2, 2021, and remained open for 2 weeks. Data were summarized descriptively in Qualtrics and SAS Version 9.4. Results Of the 330 ACE Panel members who responded to the survey, 42% were not aware of which vaccines their state permits them to deliver. More than one-half (55%) would be willing to administer influenza or COVID-19 vaccines in their practice setting, but at present only 2% of respondents administer vaccines. To overcome vaccine administration barriers, the top 3 resources respondents want access to are the following: training or education, financial support, and access to protocols. Of all the respondents, 91% indicated the dental hygienist should be involved in certain capacities. Conclusions Few dentists are administering vaccines, possibly owing to a number of challenges. Dental hygienists may play an integral role in the administration of vaccines in the dental clinic, but few dentists are educating their patients about vaccines. Practical Implications Although dentists wishing to administer vaccines in their practice may encounter barriers, support at the state, federal, and organizational levels could help them overcome these challenges.

Background Dental plaque biofilms are the causative agents of caries, gingivitis and periodontitis. Both mechanical and chemical strategies are used in routine oral hygiene strategies to reduce plaque build-up. If allowed to mature biofilms can create anoxic microenvironments leading to communities which harbor pathogenic Gram-negative anaerobes. When subjected to high velocity fluid jets and sprays biofilms can be fluidized which disrupts the biofilm structure and allows the more efficient delivery of antimicrobial agents. Methods To investigate how such jets may disrupt anoxic niches in the biofilm, we used planar optodes to measure the dissolved oxygen (DO) concentration at the base of in-vitro biofilms grown from human dental saliva and plaque. These biofilms were subject to “shooting” treatments with a commercial high velocity microspray (HVM) device. Results HVM treatment resulted in removal of much of the biofilm and a concurrent rapid shift from anoxic to oxic conditions at the base of the surrounding biofilm. We also assessed the impact of HVM treatment on the microbial community by tracking 7 target species by qRT-PCR. There was a general reduction in copy numbers of the universal 16S RNA by approximately 95%, and changes of individual species in the target region ranged from approximately 1 to 4 log reductions. Conclusion We concluded that high velocity microsprays removed a sufficient amount of biofilm to disrupt the anoxic region at the biofilm-surface interface.

Numerous studies have suggested a correlation between oral health, the oral microbiome, and various dermatologic conditions, particularly psoriasis. In this study, we utilize a specially designed questionnaire administered to 265 patients at The Ohio State University's dermatology clinics to explore the relationship between psoriasis and a combination of factors that included dietary habits, oral health, and oral hygiene practices. Age, family history of psoriasis, previous diagnosis of strep throat or rheumatoid arthritis, and oral pain or discomfort experienced within the last 12 months were all found to be significant predictors of psoriasis. Additionally, higher body mass index scores, poor gum health, and speech difficulties related to dental problems were all correlated with more severe psoriasis symptoms. Conversely, patients who reported consuming fresh fruit at least once a day experienced milder symptoms. Our goal is to develop a better understanding of how and why psoriasis incidence is correlated with some of the oral health factors under review.

PURPOSE Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. EXPERIMENTAL DESIGN In this case control study, small EVs are isolated from serum of 58 subjects (all male, 33 (15-70) in years) including drug naive active tuberculosis (ATB: n = 22), non-tuberculosis (NTB: n = 18), and healthy subjects (n = 18). Serum small EVs proteome analysis is carried out using isobaric tag for relative and absolute quantification (iTRAQ) experiments and an independent sample (n = 36) is used for validation. RESULTS A set of 132 and 68 proteins are identified in iTRAQ-I (ATB/Healthy) and iTRAQ-II (ATB/NTB) experiments, respectively. Four proteins (KYAT3, SERPINA1, HP, and APOC3) show deregulation (log2 -fold change > ±0.48, p

The objective of this paper is to study the effect of spacing ratio on the wake dynamics in an inline arrangement of four cylinders. Different spacing ratios, defined as ratio of distance between center of two cylinders and diameter of cylinder, are considered while. Reynolds number (Re) kept constant. At the chosen Re, far wake is expected to transition to turbulence, and hence κ-ω SST turbulence model is used for turbulence closure. The flow field and associated surface properties such as drag and lift force are strong function of spacing ratio. In the numerical solutions it is observed that, two counter rotating vortices are formed in the gap between the cylinders. These two vortices found to move towards downstream as the spacing ratio is increased. Also, the size of the vortices found increased. The two counter rotating vortices appears symmetric near cylinders, and hence lift coefficient observed to be very low for the first three cylinders. Lift coefficient observed on fourth cylinder found to one order of magnitude higher. The drag coefficient for all cylinders found to be positive except for the second cylinder which has negative drag for all spacing ratios.

Micro RNAs (miRNAs) are a class of small non-coding single-stranded RNA, which play an important role in modulating host-Influenza A virus (IAV) crosstalk. The interplay between influenza and miRNA interaction is defined by a plethora of complex mechanisms, which are not fully understood yet. Here, we demonstrate that in IAV infected A549 cells, a synchronous increase was observed in the expression of mTOR up to 24 hpi and significant downregulation at 48 hpi. Additionally, NP of IAV interacts with mTOR and modulates the levels of mTOR mRNA and protein, thus regulating the translation of host cell. RNA sequencing and qPCR analysis of IAV-infected A549 cells and NP transfected cells revealed that miR-101 downregulates mTOR transcripts at later stages of infection. Ectopic expression of miR-101 mimic led to a decrease in expression of NP, a reduction in IAV titer and replication. Moreover, treatment of the cells with Everolimus, a potent inhibitor of mTOR, resulted in an increase of miR-101 transcript levels, which further suppressed the viral protein synthesis. Collectively, the data suggest a novel mechanism that IAV stimulates mTOR pathway at early stages of infection, however, at a later time-point, positive regulation of miR-101 restrains the mTOR expression, and hence, the viral propagation.

Reticulocyte invasion by Plasmodium vivax requires interaction of the Duffy-binding protein (PvDBP) with host Duffy antigen receptor for chemokines (DARCs). The binding domain of PvDBP maps to a cysteine-rich region referred to as region II (PvDBPII). Blocking this interaction offers a potential path to prevent P. vivax blood-stage growth and P. vivax malaria. This forms the rationale for development of a vaccine based on PvDBPII. Here we report results of a Phase I randomized trial to evaluate the safety and immunogenicity of recombinant PvDBPII formulated with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Thirty-six malaria-naive, healthy Indian male subjects aged 18–45 years were assigned into three cohorts corresponding to doses of 10, 25 and 50 µg of PvDBPII formulated with 5 µg of GLA-SE. Each cohort included nine PvDBPII/GLA-SE vaccinees and three hepatitis B control vaccine recipients. Each subject received the assigned vaccine intramuscularly on days 0, 28 and 56, and was followed up till day 180. No serious AE was reported and PvDBPII/GLA-SE was well-tolerated and safe. Analysis by ELISA showed that all three doses of PvDBPII elicited antigen-specific binding-inhibitory antibodies. The 50 µg dose elicited antibodies against PvDBPII that had the highest binding-inhibitory titres and were most persistent. Importantly, the antibody responses were strain transcending and blocked receptor binding of diverse PvDBP alleles. These results support further clinical development of PvDBPII/GLA-SE to evaluate efficacy against sporozoite or blood-stage challenge in controlled human malaria infection (CHMI) models and against natural P. vivax challenge in malaria endemic areas.

Supplemental Figure S3: Functional shifts in the core subgingival microbiome during various dentition states. Figure 3a shows a Bland-Altman plot of changes in the relative abundances of core functional genes in the subgingival microbiome between the different dentitions. Each point is a functional gene. Points above and below the red median line represent genes demonstrating higher abundances in the specified dentition. Red points indicate genes with significantly different abundances (p < 0.05, FDR adjusted Wald test). Figure 3B shows the number of functional genes that were different between two subsequent dentitions. For example, the bar “Primary” represents genes that were different between predentate and primary dentitions, while “Mixed” represents number of genes differing between primary and mixed dentitions. Figures 3c and d represent the functions encoded by these genes in the primary and permanent dentitions respectively. (PDF 511 kb)

Supplemental Figure S2: Functional shifts in the core supragingival microbiome during various dentition states. Figure 2a shows a Bland-Altman plot of changes in the relative abundances of core functional genes in the supragingival microbiome between the different dentitions. Each point is a functional gene. Points above and below the red median line represent genes demonstrating higher abundances in the specified dentition. Red points indicate genes with significantly different abundances (p < 0.05, FDR adjusted Wald test). Figure 2b shows the number of functional genes that were different between two subsequent dentitions. For example, the bar “Primary” represents genes that were different between predentate and primary dentitions, while “Mixed” represents number of genes differing between primary and mixed dentitions. Figures 2c and d represent the functions encoded by these genes in the primary and permanent dentitions respectively. (PDF 428 kb)

Methods used in the study. (DOCX 172Â kb)

Supplemental Figure S1: Functional shifts in the core salivary microbiome during various dentition states. Figure 1a shows a Bland-Altman plot of changes in the relative abundance of core functional genes in the salivary microbiome between the different dentitions. Each point is a functional gene. Points above and below the red median line represent genes demonstrating higher abundances in the specified dentition. Red points indicate genes with significantly different abundances (p < 0.05, FDR adjusted Wald test). Figure 1b shows the number of functional genes that were different between two subsequent dentitions. For example, the bar “Primary” represents genes that were different between predentate and primary dentitions, while “Mixed” represents number of genes differing between primary and mixed dentitions. Figures 1c and d represent the functions encoded by these genes in the primary and permanent dentitions respectively. (PDF 541 kb)

Opportunistic bacterial infections amongst HIV-infected individuals pose serious health challenge. While immediate control of bacterial pathogens is typically attributed to innate defense mechanisms, whether HIV-mediated modulation of innate mechanisms like autophagy promote opportunistic infections, remains obscure. Using U1.1 and U937 macrophages, we show, HIV activation or infection inhibits autophagy and helps survival of pathogenic Mycobacterium tuberculosis and non-pathogenic non-tuberculous mycobacterial strains (NTMs) like Mycobacterium avium complex and Mycobacterium fortuitum. HIV achieves this by blocking xenophagy flux, which could be reversed by the autophagy inducer trehalose that kills intracellular Mtb and NTMs. We found trehalose acts as a PI (3,5) P2 agonist and activates TRPML1 to induce autophagy. Remarkably, trehalose treatment significantly reduced p24 levels in PBMCs infected with clinical HIV strains and in PBMCs derived from treatment-naive HIV patients. Taken together, our study highlights the immense potential of autophagy modulators in the therapeutic intervention of HIV and associated opportunistic infection.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

Controlling inflammatory response is important to avoid chronic inflammation in many diseases including atopic dermatitis (AD). In this research, we tried using a phosphatidylserine (PS)-coated microparticles in the AD mouse model for achieving the modulation of the macrophage phenotype to an anti-inflammatory state. Here, we prepared poly (D,L-lactic acid) microparticle coated with PS on the outside shell. We confirmed the cellular uptake of the PS-coated microparticle, which leads to the significant downregulation of the inflammatory cytokine production. In the mouse model of AD, the PS-coated microparticle was injected subcutaneously for a period of 12 days. The mice showed significant reduction in the development of AD symptoms comparing with the mice treated with the PC-coated microparticle.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease (HFMD). Several recent outbreaks of HFMD in East Asia were associated with neurological complications and numerous deaths. In 2000, an outbreak in Singapore afflicted thousands of children, resulting in four fatal cases from whom EV71 was isolated. The virus possesses four structural proteins VP1, VP2, VP3, and VP4, each of which is involved in forming the pentameric icosahedral structure of the virus. Here we report that the full-length VP1 structural protein of EV71 is capable of self-association. Dimerization of VP1 was tested using the yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis, in vitro coupled transcription-translation binding assays, and mammalian cell transcription-translation experiments. Dimerization of various truncated versions of the VP1 protein was also studied by mutational analysis. Systematic deletions of parts of VP1 revealed that the region spanning amino acids 66-132 of VP1 contains the major dimerization domain. However, the region between amino acids 132 and 297 was indispensable, and contributed largely to increasing the strength of the interaction. This ability of EV71 VP1 to self-associate and to participate significantly in forming the characteristic icosahedral capsid strongly enhances the pathogenicity and stability of the virus to withstand the environment of the gastrointestinal tract.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus isolated from patients exhibiting atypical pneumonia and may have originated from wild animals such as civet cats in southern China. The genome of SARS-CoV is a positive-sense, single-stranded RNA whose sequence is distantly related to all known coronaviruses that infect humans and animals. Like other known coronaviruses, SARS-CoV is an enveloped virus containing three outer structural proteins, namely the membrane (M), envelope (E), and spike (S) proteins. The nucleocapsid (N) protein together with the viral RNA genome presumably form a helical core located within the viral envelope. The SARS-CoV nucleocapsid (N) protein is a 423 amino-acid, predicted phospho-protein of 46 kDa that shares little homology with other members of the coronavirus family. A short serine-rich stretch, and a putative bipartite nuclear localization signal are unique to it, thus suggesting its involvement in many important functions during the viral life cycle. In this report we have cloned the N gene of the SARS coronavirus, and studied its property of self-association to form dimers. We expressed the N protein as a fusion protein in the yeast two-hybrid system to demonstrate self-association and confirmed dimerization of the N protein from mammalian cell lysates by coimmunoprecipitation. Furthermore, via deletion analysis, we have shown that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of the N protein to form dimers.

Background The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection. Methodology/Principal Findings Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/3) protein complex, severely affects the pathogen’s growth. TEM studies display shedding of the mycobacterial outer-coat. Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization. Conclusions/Significance With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics.

BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection.

MTX (mitoxantrone), an anti-tumour antibiotic, is known to cause cell death by intercalating the DNA bases. But how it interferes with the cellular proliferation is not well known. Hence, in the present study, we have tried to evaluate the interaction of this drug using proliferation dynamics to gain a better understanding of MTX's antineoplastic action. Inhibition of proliferation by these drugs was detected by evaluating its effect on cell proliferation and growth curve of the cells. MTX was also found to affect the cell viability and, thereby, cell physiology. Typical apoptotic morphologies such as condensation of nuclei and membrane permeabilization were observed through CLSM (confocal laser scanning microscopy) and fluorescence spectroscopy, which implicates commitment to cell death. Cell-cycle distribution was measured by flow cytometric measurements. The analysis demonstrated significant cell-cycle arrest on MTX treatment. Inhibition of lacZ gene expression was also observed on drug treatment, which implicates its interaction with gene expression.