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Today’s viticulture regions for quality wine production are located in relatively narrow geographical and therefore climatic niches that put them at greater risk from climate change than other more broad acre crops. Our target area, the Mátra Region is close to the edge of optimal wine production concerning its climate conditions. Fifty year (1961-2010) wine and quality (natural sugar content, in weight % of must) data are analysed and compared to parallel climate variables. Two sets of station-based monthly temperature, sunshine duration and precipitation data, taken from neighbouring stations, Eger-Kőlyuktető (1961-2010) and Kompolt (1976-2006) are used altogether in 132 combinations. In addition, daily grid-point data provided by the CarpatClim Project are used (www.carpatclim-eu.org/pages/home). By now it is clear that (1) wine quality, is in significant negative correlation with the annual precipitation and in positive correlation with temperature and sunshine duration. (2) Applying a wide combination of monthly data we obtain even stronger correlations (higher significance according to t-tests) even from the station-based data, but it is difficult to select and optimum model from the many proper combinations just slightly differing in performance over the test sample. (3) The interpolated areal averages of the grid-point data promise good results, but their statistical analysis by using 5-day averages is just in its initial phase. It is clearly seen that June 5-day temperature means strongly influence the sugar content in the Mátra-region of NE Hungary.

Targeting multiple viral proteins is pivotal for sustained suppression of highly mutable viruses. In recent years, broadly neutralizing antibodies that target the influenza virus hemagglutinin and neuraminidase glycoproteins have been developed, and antibody monotherapy has been tested in preclinical and clinical studies to treat or prevent influenza virus infection. However, the impact of dual neutralization of the hemagglutinin and neuraminidase on the course of infection, as well as its therapeutic potential, has not been thoroughly tested. For this purpose, we generated a bispecific antibody that neutralizes both the hemagglutinin and the neuraminidase of influenza viruses. We demonstrated that this bispecific antibody has a dual-antiviral activity as it blocks infection and prevents the release of progeny viruses from the infected cells. We show that dual neutralization of the hemagglutinin and the neuraminidase by a bispecific antibody is advantageous over monoclonal antibody combination as it resulted an improved neutralization capacity and augmented the antibody effector functions. Notably, the bispecific antibody showed enhanced antiviral activity in influenza virus-infected mice, reduced mice mortality, and limited the virus mutation profile upon antibody administration. Thus, dual neutralization of the hemagglutinin and neuraminidase could be effective in controlling influenza virus infection., Competing Interests: Declaration of interests A provisional patent on BiAb is currently being filed, in which Y.B.-O. and R.M. are inventors., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway., Competing Interests: Competing interests statement:The authors declare no competing interest.

Declining sequencing costs coupled with the increasing availability of easy-to-use kits for the isolation of DNA and RNA transcripts from single cells have driven a rapid proliferation of studies centered around genomic and transcriptomic data. Simultaneously, a wealth of new techniques have been developed that utilize single cell technologies to interrogate a broad range of cell-biological processes. One recently developed technique, transposase-accessible chromatin with sequencing (ATAC) with select antigen profiling by sequencing (ASAPseq), provides a combination of chromatin accessibility assessments with measurements of cell-surface marker expression levels. While software exists for the characterization of these datasets, there currently exists no tool explicitly designed to reformat ASAP surface marker FASTQ data into a count matrix which can then be used for these downstream analyses. To address this, we created CountASAP, an easy-to-use Python package purposefully designed to transform FASTQ files from ASAP experiments into count matrices compatible with commonly-used downstream bioinformatic analysis packages. CountASAP takes advantage of the independence of the relevant data structures to perform fully parallelized matches of each sequenced read to user-supplied input ASAP oligos and unique cell-identifier sequences., Competing Interests: Competing Interests The authors declare no competing interests.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Background: Facilitated by the inability to vaccinate, and an immature immune system, COVID-19 remains a leading cause of death among children. Vaccinated lactating mothers produce specific SARS-CoV-2 antibodies in their milk, capable of neutralizing the virus in vitro . Our objective for this study is to assess the effect of COVID-19 booster dose on SARS-CoV-2 antibody concentration and viral neutralization in milk, plasma, and infant stool., Methods: Thirty-nine mothers and 25 infants were enrolled from December 2020 to May 2022. Milk, maternal plasma, and infants' stool were collected at various time-points up to 12 months following mRNA COVID-19 vaccination. A subgroup of 14 mothers received a booster dose. SARS-CoV-2 antibody levels and their neutralization capacities were assessed., Results: Booster vaccination led to significantly higher IgG levels within human milk and breastfed infants' stool. In vitro neutralization of VSV-gfp-SARS-CoV-2-S-gp, a laboratory safe SARS-CoV-2 like pseudovirus, improved following the booster, with a 90% increase in plasma neutralization and a 60% increase in milk neutralization. We found that post-booster neutralization by human milk was highly correlated to SARS-CoV-2 IgG level. In support of our correlation result, Protein G column depletion of IgG in milk yielded a significant reduction in viral neutralization ( p = 0.04)., Discussion: The substantial increase in neutralizing IgG levels in milk and breastfed infants' stool post-booster, coupled with the decrease in milk neutralization capabilities upon IgG depletion, underscores the efficacy of booster doses in augmenting the immune response against SARS-CoV-2 in human milk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Valcarce, Stafford, Neu, Parker, Vicuna, Cross, D'Agati, Diakite, Haley, Feigenbaum, Al Mahmoud, Visvalingam, Cacho, Kosik, Yewdell and Larkin.)

Objective: Assess presence, durability, and neutralization capacity of SARS-CoV-2-specific antibodies in breastfeeding infants' stool, mother's plasma and milk following maternal vaccination., Design: Thirty-seven mothers and 25 infants were enrolled between December 2020 and November 2021 for this prospective observational study. All mothers were vaccinated during lactation except three, which were vaccinated during pregnancy. Milk, maternal plasma, and infants' stool was collected pre-vaccination and at periods up to 6 months following COVID-19 vaccine series initiation/completion. SARS-CoV-2 antibody levels and their neutralization capacities were assessed., Results: SARS-CoV-2-specific IgA and IgG levels were higher in infant stool post-maternal vaccination amongst milk-fed compared to controls. Maternal SARS-CoV-2-specific IgA and IgG concentrations decreased over 6 months post-vaccination but remained higher than pre-vaccination levels. We observed improved neutralization capacity in milk and plasma after COVID-19 vaccination., Conclusions: The presence of SARS-CoV-2-specific antibodies in infant stool following maternal vaccination offers further evidence of the lasting transfer of these antibodies through breastfeeding., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Significance: Orthopedic surgery is frequently performed but currently lacks consensus and availability of ideal guidance methods, resulting in high variability of outcomes. Misdirected insertion of surgical instruments can lead to weak anchorage and unreliable fixation along with risk to critical structures including the spinal cord. Current methods for surgical guidance using conventional medical imaging are indirect and time-consuming with unclear advantages., Aim: The purpose of this study was to investigate the potential of intraoperative in situ near-infrared Raman spectroscopy (RS) combined with machine learning in guiding pedicular screw insertion in the spine., Approach: A portable system equipped with a hand-held RS probe was used to make fingerprint measurements on freshly excised porcine vertebrae, identifying six tissue types: bone, spinal cord, fat, cartilage, ligament, and muscle. Supervised machine learning techniques were used to train-and test on independent hold-out data subsets-a six-class model as well as two-class models engineered to distinguish bone from soft tissue. The two-class models were further tested using in vivo spectral fingerprint measurements made during intra-pedicular drilling in a porcine spine model., Results: The five-class model achieved > 96 % accuracy in distinguish all six tissue classes when applied onto a hold-out testing data subset. The binary classifier detecting bone versus soft tissue (all soft tissue or spinal cord only) yielded 100% accuracy. When applied onto in vivo measurements performed during interpedicular drilling, the soft tissue detection models correctly detected all spinal canal breaches., Conclusions: We provide a foundation for RS in the orthopedic surgical guidance field. It shows that RS combined with machine learning is a rapid and accurate modality capable of discriminating tissues that are typically encountered in orthopedic procedures, including pedicle screw placement. Future development of integrated RS probes and surgical instruments promises better guidance options for the orthopedic surgeon and better patient outcomes., (© 2023 The Authors.)

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2 nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo . By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3 rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo , the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections., Competing Interests: The authors have no competing interests to declare.

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

SARS-CoV-2 nucleocapsid protein (N) induces strong antibody (Ab) and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2-infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12β, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS-CoV. Anti-N Abs bound to the surface of N-expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc-expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.

Background: False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa., Methods: From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs., Results: Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6-89.8]; specificity, 99.4% [95% CI, 97.7-99.9])., Conclusions: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

We investigated the function of the newly discovered myosin family protein myosin 18A (Myo18A) in Ab-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Myo18A deficiency led to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia and increased splenic IgM-secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing Abs against the major Ag hemagglutinin, associated with persistent accumulation of Ag-specific germinal center B cells and more Ag-specific bone marrow plasma cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into Ab-secreting cells, associated with higher AID and Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, and humoral immunity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Avian influenza A(H7N9) epidemics have a fatality rate of approximately 40%. Previous studies reported that low pathogenic avian influenza (LPAI)-derived candidate vaccine viruses (CVVs) are poorly immunogenic. Here, we assess the immunogenicity and efficacy of a highly pathogenic avian influenza (HPAI) A/Guangdong/17SF003/2016 (GD/16)-extracted hemagglutinin (eHA) vaccine. GD/16 eHA induces robust H7-specific antibody responses in mice with a marked adjuvant antigen-sparing effect. Mice immunized with adjuvanted GD/16 eHA are protected from the lethal LPAI and HPAI H7N9 challenges, in stark contrast to low antibody titers and high mortality in mice receiving adjuvanted LPAI H7 eHAs. The protection correlates well with the magnitude of the H7-specific antibody response (IgG and microneutralization) or HA group 2 stem-specific IgG. Inclusion of adjuvanted GD/16 eHA in heterologous prime-boost improves the immunogenicity and protection of LPAI H7 HAs in mice. Our findings support the inclusion of GD/16-derived CVV in the pandemic preparedness vaccine stockpile.

The Aim of the Study: Was to compare manual, semi-automatic and automatic methods for determining the maxillary sinus volume using cone beam computed tomography (CBCT)., Material and Methods: CBCT images from 48 patients (96 maxillary sinuses) with no history of sinus and alveolar bone surgery, who were presented to Minsk medical centers, were used in this study. Neural network training was performed on CBCT scans of 42 patients (84 maxillary sinuses).The height, depth and width of the sinus were measured manually on CBCT scans of 6 patients (12 maxillary sinuses). Maxillary sinus volume (V) was calculated by the formula: V=height´depth´1/3 width. Semi-automatic segmentation was carried out by an expert radiologist. The convolutional neural network technology was applied for maxillary sinus automatic segmentation., Results: The largest values were revealed by using the automatic method for sinus volume measurement. These values were within the 95% confidence interval (±4.29 cm 3 ) of the average sinus volume obtained from semi-automatic method., Conclusion: The data obtained using the convolutional neural network technique (artificial intelligence) has a high correlation with the results of sinus morphometric analysis acquired through manual and semi-automatic methods. Automatic maxillary sinus segmentation technique does not require special user knowledge. This method is reproducible and it is implemented in a short time interval.

With a lifetime risk of 1 in 8, breast cancer continues to be a major concern for women and their physicians. The optimal treatment of the disease depends on the stage of the cancer at diagnosis, which is typically assessed using medical imaging. However, currently employed imaging systems for breast tumor measurement rarely agree perfectly. Our group developed an Intraoperative Photoacoustic Screening (iPAS) soft tissue scanner featuring high bulk tissue sensitivity, a clinically compatible scan-time of 6 min, imaging depths greater than 2 cm and the capability to visualize whole breast tumors based on their lipid, rather than hemoglobin, profile. Here, we report on the first clinical experience with breast cancer patients by comparing tumor-measurement using iPAS, preoperative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and gold-standard pathology. Tumor size was measured volumetrically for iPAS and DCE-MRI, and separately using maximum diameters for pathology, DCE-MRI and iPAS. Comparisons were performed using Pearson's correlation coefficients, and the non-parametric Wilcoxon signed-rank test. Twelve consecutive patients were included in the study, contingent on pathologically documented invasive carcinoma. iPAS volumetric tumor size was positively correlated to DCE-MRI (Pearson's r = 0.78, p = 0.003) and not significantly different (Wilcoxon, p = 0.97). In comparison to pathology, tumor diameters given by iPAS were positively correlated (Pearson's r = 0.87, p = 0.0002) and significantly different (Wilcoxon, p = 0.0015). The results indicated that volumetric-measurement of invasive breast tumors with iPAS is similar to that of DCE-MRI. On the other hand, tumor diameter measurements were less reliable. Beyond enhancing surgical specimen examination, an extension of this technology to diagnostic imaging promises a new perspective on tumor assessment, potentially improving our current understanding and treatment of breast cancer., Competing Interests: The authors have no relevant financial interests in the manuscript and no other potential conflicts of interest to disclose., (© 2020 The Author(s).)

A major obstacle to vaccination against antigenically variable viruses is skewing of antibody responses to variable immunodominant epitopes. For influenza virus hemagglutinin (HA), the immunodominance of the variable head impairs responses to the highly conserved stem. Here, we show that head immunodominance depends on the physical attachment of head to stem. Stem immunogenicity is enhanced by immunizing with stem-only constructs or by increasing local HA concentration in the draining lymph node. Surprisingly, coimmunization of full-length HA and stem alters stem-antibody class switching. Our findings delineate strategies for overcoming immunodominance, with important implications for human vaccination., Competing Interests: The authors declare no conflict of interest.

High re-excision rates in breast-conserving surgery call for a new intraoperative approach to the lumpectomy margin evaluation problem. The unique intraoperative imaging system, presented here, demonstrated the capability of photoacoustic tomography (PAT) to deliver optical sensitivity and specificity, along with over 2-cm imaging depth, in a clinical setting. The system enabled the evaluation of tumor extent, shape, morphology, and position within lumpectomy specimens measuring up to 11 cm in diameter. The investigation included all major breast cancer-related lesions, such as invasive ductal carcinoma (IDC), multifocal IDC, ductal carcinoma in situ and combinations of these variants. Coregistration with established ultrasound (US) technology, as well as comparison to specimen radiography, validated the performance of PAT, which appeared to facilitate better tumor visualization. Contrary to expected PA contrast mechanisms, PAT images of hemoglobin distribution correlated poorly with US-determined tumor location, while hypointense regions in lipid-weighted PAT images were in better agreement with US.

Influenza A virions possess two surface glycoproteins-the hemagglutinin (HA) and neuraminidase (NA)-which exert opposite functions. HA attaches virions to cells by binding to terminal sialic acid residues on glycoproteins/glycolipids to initiate the infectious cycle, while NA cleaves terminal sialic acids, releasing virions to complete the infectious cycle. Antibodies specific for HA or NA can protect experimental animals from IAV pathogenesis and drive antigenic variation in their target epitopes that impairs vaccine effectiveness in humans. Here, we review progress in understanding HA/NA co-evolution as each acquires epistatic mutations to restore viral fitness to mutants selected in the other protein by host innate or adaptive immune pressure. We also discuss recent exciting findings that antibodies to HA can function in vivo by blocking NA enzyme activity to prevent nascent virion release and enhance Fc receptor-based activation of innate immune cells.

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration-approved NA inhibitors enhance anti-stem-based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Probing the limits of CD8 + T cell immunosurveillance, we inserted the SIINFEKL peptide into influenza A virus (IAV)-negative strand gene segments. Although IAV genomic RNA is considered noncoding, there is a conserved, relatively long open reading frame present in segment 8, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated. Although we failed to detect NEG8 protein expression in IAV-infected mouse cells, cell surface K b -SIINFEKL complexes are generated when SIINFEKL is genetically appended to the predicted C terminus of NEG8, as shown by activation of OT-I T cells in vitro and in vivo. Moreover, recombinant IAV encoding of SIINFEKL embedded in the negative strand of the neuraminidase-stalk coding sequence also activates OT-I T cells in mice. Together, our findings demonstrate both the translation of sequences on the negative strand of a single-stranded RNA virus and its relevance in antiviral immunosurveillance.

[This corrects the article DOI: 10.1371/journal.ppat.1006796.].

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.

Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.

We studied the ability of monoclonal Abs (mAbs) recognizing the major hemagglutinin (HA) antigenic sites to inhibit neuraminidase (NA) cleavage of sialic acids on fetuin. We show that virion associated-NA activity in the enzyme linked lectin assay (ELLA) is largely dependent on HA-mediated attachment of virions to immobilized fetuin. For a Sb-antigenic site specific mAb, there is a nearly perfect correlation between neuraminidase inhibition and blocking virus attachment to immobilized fetuin. By contrast, Sa-, Ca-, and Cb- antigenic site specific mAbs block NA activity in ELLA or the traditional thiobarbituric acid assay by sterically interfering with NA access to substrate. We conclude first, that ELLA with intact virus can only be used to measure anti-NA Abs if sera lack HA-specific Abs, and second, that anti-HA Abs block NA activity by both limiting virion interaction with sialic acid containing surfaces and by sterically limiting NA access to sialic acids attached to macromolecules., (Published by Elsevier Inc.)

Accurate reconstruction of 3D photoacoustic (PA) images requires detection of photoacoustic signals from many angles. Several groups have adopted staring ultrasound arrays, but assessment of array performance has been limited. We previously reported on a method to calibrate a 3D PA tomography (PAT) staring array system and analyze system performance using singular value decomposition (SVD). The developed SVD metric, however, was impractical for large system matrices, which are typical of 3D PAT problems. The present study consisted of two main objectives. The first objective aimed to introduce the crosstalk matrix concept to the field of PAT for system design. Figures-of-merit utilized in this study were root mean square error, peak signal-to-noise ratio, mean absolute error, and a three dimensional structural similarity index, which were derived between the normalized spatial crosstalk matrix and the identity matrix. The applicability of this approach for 3D PAT was validated by observing the response of the figures-of-merit in relation to well-understood PAT sampling characteristics (i.e. spatial and temporal sampling rate). The second objective aimed to utilize the figures-of-merit to characterize and improve the performance of a near-spherical staring array design. Transducer arrangement, array radius, and array angular coverage were the design parameters examined. We observed that the performance of a 129-element staring transducer array for 3D PAT could be improved by selection of optimal values of the design parameters. The results suggested that this formulation could be used to objectively characterize 3D PAT system performance and would enable the development of efficient strategies for system design optimization.

Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.