The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the SAIfnar1 knock-in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. SAIfnar1 mice  display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not SAIfnar1 mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation-induced tissue damage can be purposely mimicked for therapeutic benefits.Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects Subject Categories Immunology; Digestive System Inflammation has evolved as an adaptive process that combines the counteractive elements of tissue destruction versus healing to limit and eventually eliminate the harmful effects of irritants and infectious agents. Initial stages of inflammation that exacerbate tissue damage are often followed by subsequent regeneration/repair of tissue to restore its function . Furthermore, this treatment led to a noticeable increase in the levels of amylase in blood plasma  and Ifnar1−/− mice displayed a similar severity of these alterations  and diverse inflammatory stimuli can induce ubiquitination of the intracellular domain of IFNAR1 leading to its endocytosis and degradation in cultured mammalian cells , IL1β (12.8-fold), VEGF (70.5-fold) TNFα (4.5-fold), and IL6 (585.2-fold) as well as stimulated p38 kinase activation in pancreatic tissue . Importantly, naïve Ifnar1SA mice developed normally and exhibit neither any signs of growth retardation nor gross abnormalities , nor hallmarks of constitutive pancreatic inflammation  compared with wild type animals. Consistent with the key role of Irf7 in subsequent IFN induction . Importantly, while the IFNAR1S526A mutant receptor remained sensitive to the proteolytic effects of extracellular trypsin in vitro , we did not observe its ubiquitination and downregulation in response to the caerulein-induced pancreatitis in vivo  than in Ifnar1+/+ (4.50-fold) mice. Levels of IL10 protein in blood plasma were higher in caerulein-treated Ifnar1SA animals (251 ± 2 versus 114 ± 2 pg/ml in wild type mice) indicative of systemic anti-inflammatory response and consistent with reported increase of this cytokine in caerulein-treated mice  and anti-inflammatory cytokines (e.g. IL10). Noticeably higher arts Fig A. In addmals Fig A. This rIfnar1SA mice. Upon caerulein injections, the pancreata from Ifnar1SA animals displayed a greater extent of infiltration by leukocytes as seen from histologic analysis  in Ifnar1SA mice  and levels of plasma IFNβ . In parallel, the severity of hepatic inflammation was prominently greater in Ifnar1SA mice compared to the wild type animals as judged by increased release of liver aminotransferases from hepatocytes into the bloodstream  strongly suggest that ubiquitination of IFNAR1 plays a protective role during hepatitis of either autoimmune or toxic etiology. Collectively, these data implicate ubiquitination and degradation of IFNAR1 as an important common mechanism that limits the extent of inflammatory liver injury.Furthermore, livers harvested from ions Fig D, greateIfnar1SA animals that received BMT from either mice of the same genotype or from wild type animals. The latter chimeric mice challenged with caerulein exhibited a lesser increase in the levels of serum amylase activity  approach was utilized. We initially compared the severity of acute pancreatitis in 4. Chimeric animals that received BMT from wild type mice consistently exhibited a lesser serum level of liver aminotransferases  revealed a robustly augmented liver tissue damage and inflammatory phenotype  was previously shown to act on immune cells , whose overdose causes severe liver damage and greatly contributes to acute liver failure of non-viral etiology in human patients . Collectively, these results suggest that targeting IFNAR1 via either blocking its extracellular domain (by antibody) or inducing ubiquitination of its intracellular domain (by LPA-like agents) can be used to protect liver against excessive inflammatory damage.To further determine the benefit of targeting IFNAR1 in inflammation, we used a neutralizing antibody against IFNAR1 that binds to its extracellular domain  functionally mimic the complete loss of this receptor via genetic ablation. Under specific physiologic and pathologic scenarios, this accelerated degradation may result in a phenotypic equivalency between wild type and IFNAR1 knockout animals. Such equivalency should not be over-interpreted as the lack of role of IFN in a given physiologic process or a pathologic condition.Data presented here indicate that stimulated ubiquitination and degradation of IFNAR1 in the bone marrow-derived cells recruited to the foci of inflammation plays a major protective role during inflammation. Striking systemic and local phenotypes observed in Irf7 seen in Ifnar1SA mice can contribute to further increases in ligands production , the inability to eliminate IFNAR1 may create a feed-forward signaling amplification mechanism. Importantly, the augmented IFN signaling as well as phenotypes observed in Ifnar1SA mice are evident despite numerous negative regulators of IFN pathway that function downstream of receptor and include the expression of signaling and transcriptional inhibitors including tyrosine phosphatases, suppressors of cytokine signaling, Sprouty, Ubp43, protein inhibitors of STAT, etc.  mice, Ifnar1SA (Ifnar1tm1.1Syfu) mice  were used in previous publications  of the University of Pennsylvania approved all animal procedures of disease modeling, blood collection, euthanasia and tissue harvesting . Eight to 10 weeks old female littermate mice including wild type  and were homozygous for indicated Ifnar1 alleles. For generalized inflammation model, mice were treated with LPS  and sacrificed when they became moribund and displayed the loss of righting reflex, loss of >20% of body weight and non-responsiveness to footpad compression as previously described  or vehicle (olive oil) and sacrificed after 48 h. Alternatively, mice were injected with acetaminophen  and sacrificed after 24 h. Blood was processed for ALT/AST analyses. Livers were either flash frozen for extracting mRNA or fixed in 4% formalin for hematoxylin and eosin staining. Histopathologic analyses were carried in a double blinded manner by a qualified veterinary pathologist (A.B.). Quantification of the lesions was carried out by counting the number of lesions per field; an average of ten fields per mouse and five mice per group were scored. The assessment of the severity of hepatitis was done by evaluating tissues on a scale of 0–3 where the scores were representative of grades of 0 , 1 (mild hepatitis), 2 (moderate hepatitis with limited necrotic foci) and 3 .To induce autoimmune hepatitis, mice were injected intravenously with Concanavalin A (20 mg/kg) or vehicle  and sacrificed 10 h later for blood and tissues harvesting. To induce toxic hepatitis, mice were injected intraperitoneally with CClt-test. A P-value< 0.05 was considered statistically significant.All data are expressed as the mean ± s.d. (standard deviation). Number of samples is shown in figures or/and figure legends for each experimental cohort. Statistical analyses were performed using Microsoft Excel or GraphPad Prism software, and differences were determined by an unpaired two-tailed Student's in vitro. Should this regulation occur in vivo, it would pose a medical problem of reduced efficacy of pharmaceutical IFN in inflamed tissues. Furthermore, the very existence of such mechanism is counterintuitive for the host defense as it may limit the anti-viral and anti-tumorigenic benefits of endogenously produced IFN within the regions of inflammation. Hence, we sought to investigate the manifestation and biological significance of inflammation-induced IFNAR1 ubiquitination and downregulation in vivo.Inflammation is an adaptive process that combines the counteractive elements of tissue destruction versus healing to limit and eventually eliminate the harmful effects of irritants and infectious agents. Initial stages of inflammation that exacerbate tissue damage are often followed by subsequent regeneration/repair of tissue to restore its function. While failure to progress to these later stages contributes to development of many human diseases, the mechanisms limiting tissue injury and promoting the transition to the restorative phase are poorly understood. Type 1 interferons (IFN) protect the organism from viruses and tumors yet elicit tissue toxicities often being manifested in patients with tumors or chronic viral infections treated with IFN-based drugs. All effects of IFN are mediated by its cell surface receptor, whose levels are regulated by phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain. Importantly, these processes depriving cells from ability to respond to their future encounter with IFN can be stimulated by inflammatory stimuli Induction of pancreatitis, hepatitis or generalized inflammation robustly downregulated IFNAR1 in mice. This downregulation was not seen in the knock-in mice incapable of stimulating IFNAR1 ubiquitination. Importantly, these mice were highly susceptible to acute and chronic pancreatitis, toxic and autoimmune hepatitis and bacterial endotoxin-induced septic shock. Altered balance of pro-inflammatory/anti-inflammatory cytokines and specific chemokines resulting in the recruitment of inflammatory monocytes was observed in pancreatic tissues of IFNAR1 ubiquitination-deficient mice. These mice displayed extensive damage and persistent immune infiltration of inflamed tissues, and, in the case of generalized inflammation, overwhelming mortality. Under conditions of chronic pancreatitis, these mice also exhibited a dramatically impaired ability to repair the pancreatic tissue and restore its anatomical and functional integrity. Experiments using bone marrow transplantation determined that inability to ubiquitinate and degrade IFNAR1 in hematopoietic compartment is responsible for excessive pancreatic and hepatic inflammatory phenotypes. Importantly, pharmacologic stimulation of protein kinases that stimulate IFNAR1 ubiquitination was protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not in IFNAR1 ubiquitination-deficient mice.Studies described here uncovered an endogenous mechanism by which inflammatory process confines its harmful phase and enables the transition to tissue restoration through promoting IFNAR1 ubiquitination and degradation. In addition, mimicking this natural mechanism by preemptive induction of protein kinases that leads to stimulation of IFNAR1 ubiquitination elicits a tissue protective effect. Pharmacologic stimulation of IFNAR1 ubiquitination and degradation represents a fundamentally novel strategy to limit the extent of tissue damage and accelerate healing and restoration of the function. Furthermore, a similar strategy could be potentially used to temper the toxic side effects of the IFN-based drugs. Given that many of the currently available drugs are potentially capable of stimulating IFNAR1 ubiquitination, the validation of this concept and practical applications to eliminate IFNAR1 in acute life-threatening inflammatory syndromes is likely to be imminent.


Acoustophoresis has been utilized successfully in applications including cell trapping, focusing, and purification. One current limitation of acoustophoresis for cell sorting is the reliance on the inherent physical properties of cells  instead of selecting cells based upon biologically relevant surface-presenting antigens. Introducing an acoustophoretic cell sorting approach that allows biochemical specificity may overcome this limitation, thus advancing the value of acoustophoresis approaches for both the basic research and clinical fields.The results presented herein demonstrate the ability for negative acoustic contrast particles (NACPs) to specifically capture and transport positive acoustic contrast particles (PACPs) to the antinode of an ultrasound standing wave. Emulsification and post curing of pre-polymers, either polydimethylsiloxane (PDMS) or polyvinylmethylsiloxane (PVMS), within aqueous surfactant solution results in the formation of stable NACPs that focus onto pressure antinodes. We used either photochemical reactions with biotin-tetrafluorophenyl azide (biotin-TFPA) or end-functionalization of Pluronic F108 surfactant to biofunctionalize NACPs. These biotinylated NACPs bind specifically to streptavidin polystyrene microparticles (as cell surrogates) and transport them to the pressure antinode within an acoustofluidic chip.To the best of our knowledge, this is the first demonstration of using NACPs as carriers for transport of PACPs in an ultrasound standing wave. By using different silicones  and curing chemistries, we demonstrate versatility of silicone materials for NACPs and advance the understanding of useful approaches for preparing NACPs. This bioseparation scheme holds potential for applications requiring rapid, continuous separations such as sorting and analysis of cells and biomolecules. F) exerted on a particle depends on several factors including the acoustic pressure amplitude (P0), particle volume (Vp), wavelength (λ), and the acoustic contrast factor (ϕ) . Importantly, the sign of the acoustic contrast factor, which depends on both the density (ρ) and the compressibility (β) of the particle relative to the surrounding solution, dictates the relocation (Equation 2). For example, particles with higher compressibility (βp) than the surrounding media (βw) will move to the pressure antinode, whereas particles with a lower compressibility than the surrounding media will move to the pressure node. In general, particles with a positive ϕ , such as polystyrene beads or cells, transport to acoustic pressure nodes within aqueous media, whereas particles with a negative ϕ  transport to the acoustic pressure antinodes within aqueous media.Microparticles suspended in an ultrasound standing wave field may respond to the primary acoustic radiation force by transporting to specific locations along the wave  -4. The pet al. recently developed multi-node acoustic focusing and demonstrated up to 37 parallel flow streams [et al. exploited the inherent contrast factor of constituents from whole blood to separate and sort positive contrast erythrocytes from negative contrast lipids within an acoustofluidic device [The capacity to relocate PACPs to pressure nodes has been used in various approaches for focusing and separation of mammalian cells -11. For  streams . Petersoc device . Strategc device ,10. In cet al. reported the first use of NACPs for biomolecule quantification assays by using protein adsorption to modify the surface of PDMS particles [ We seek to augment acoustophoretic particle sorting capabilities by introducing newly designed negative acoustic contrast particles (NACPs) with the capacity for specific biomolecular recognition and relocation of PACPs to antinodes of ultrasound standing waves. Since NACPs move in the direction opposite to the majority of mammalian cells, we hypothesized that biofunctional NACPs can capture and specifically transport targeted cells (or other PACPs) to the pressure antinodes, provided the total acoustic force of the NACPs is greater than the total acoustic force of the PACPs. Figure articles . While carticles . These cHerein, we report on the preparation of NACPs and demonstrate the utility of these microparticles in a new acoustophoretic separation scheme. Specifically, NACPs are prepared using two different silicone elastomers and biotinylated using two different chemical modification approaches. The newly designed NACPs are evaluated as carriers for the transport of streptavidin PACPs to pressure antinodes within acoustofluidic devices. Our results reveal the potential of this approach for cell sorting applications.Silicone elastomers offer properties suitable for NACPs such as compressibility at mild temperature  . Here, a-1 , 1,408 cm-1 (=CH2 scissors), and 1,597 cm-1 (C=C stretch). While vinyl groups are versatile for various chemical reactions , our studies revealed that relatively simple photochemical reaction with biotin-TFPA results in biofunctionalization of PVMS particles /ozone irradiation , UV grafNext, we sought to evaluate the acoustic responsiveness of these silicone microparticles. Our results show that microparticles prepared from either PVMS or PDMS function as NACPs within aqueous media Figures  and 5. FThe separation of silicone NACPs from polystyrene microparticles demonstrated in Figure As expected, in the absence of fluid flow, NACPs accumulate at the pressure antinodes along the acoustofluidic channel walls during activation of the PZT Figures  and 5. Sboth NACPs and PACPs in opposing directions. Given this promise, it is necessary to further examine several aspects of using NACPs in cellular separations. For example, the role of bioaffinity bond strength between particles that are being subjected to force in opposite directions may need to be studied in detail. Likewise, the features that enable the primary radiation force of NACPs to dominate that of PACPs requires further investigation. The transport of PACPs to the pressure antinodes will only occur when a complex of PACP bound to NACPs exhibits an overall negative acoustic contrast factor, which can be adjusted through the volume, density, and bulk modulus of the NACPs. In the current study, these properties have converged to favor the relocation of PACP-NACP complexes to the antinode. We anticipate that future experimental and computational investigations will reveal the optimal parameters that support efficient cell separation.To the best of our knowledge, this is the first report documenting the use of NACPs as carriers for active transport of PACPs in acoustofluidic systems. Although polystyrene microparticles were used as cell surrogates in this preliminary investigation to demonstrate separation, this approach should be suitable for cell sorting based on binding of NACPs to specific cell surface antigens. Because the positive acoustic contrast factor value of cells is less than polystyrene beads , we antiThis report communicates a new approach for bioseparation that employs polysiloxane-based microparticles with a negative acoustic contrast property. Emulsifying and post-curing pre-polymers within aqueous surfactant results in stable microparticles that transport to the pressure antinode of an ultrasonic standing wave field in aqueous media. By using polysiloxanes with different chemical compositions and curing chemistries , we demonstrate versatility and general utility of silicone materials as negative acoustic contrast agents. Both photochemical and physical adsorption approaches are used to biofunctionalize NACPs, ultimately enabling the specific capture and transport of PACPs to an acoustic pressure antinode. These results encourage further pursuits aimed at using NACPs for cell separation, owing to potential advantages of this system such as high sensitivity, selectivity, portability and low cost.Preparing PVMS particles: A mixture of 1.0 g of hydroxyl-terminated PVMS [Preparing PDMS particles: A mixture comprising a 1:10 weight ratio of curing agent: base of Sylgard® 184  was thoroughly mixed and 1 gram of the mixture was subsequently combined with 1 wt% of Pluronic F108. The mixture was homogenized as previously described. The emulsion was incubated at 45°C, stirring for at least 1.5 hrs and subsequently left at ambient conditions for at least 12 hrs to permit curing. Functionalization: For reactions with biotin-TFPA (Quanta Biodesign), ~5 × 107 PVMS microparticles were washed with 1× PBS by centrifuging and resupending the pellet in a final volume of 2 mL of 1× PBS. The microparticles were transferred to a cylindrical glass vial (2.5 cm diameter) and 3 mg biotin-TFPA in 100 μL of dimethylacetamide was added. Light irradiation occurred using an Omnicure S1000 equipped with a high pressure mercury lamp and an internal 320–500 nm filter. The associated light guide was placed ~5 mm above the stirring solution for 30 min at a light intensity of ~100 mW/cm2 at a wavelength of 365 nm, . The resultant yellow solution was stored at 4°C until use. Biotinylation of Pluronic F108 surfactant followed a similarly reported protocol [ted PVMS , 0.07 g 2 resolution). Scanning electron microscopy (SEM) images were obtained using model FEI XL 30 SEM under ultra-high resolution mode after sputter coating the samples with approximately 6 nm of gold. Optical microscopy images were obtained using an upright Zeiss Axio Imager A2 microscope with appropriate filter set .Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra were acquired using a Thermo Electron Nicolet 8700 spectrometer  and PDMS NACPs  occurred by combining ~1033 = 0.3 nm/V, APC International). The channel width was designed to operate at a half wavelength resonant mode  resulting in an antinode at both channel walls and a single node in the channel center line. For the experiments, an electric signal with peak-to-peak voltage of 31 V was applied to the PZT. Prior to running experiments, the acoustofluidic channels were treated with a solution of Pluronic F108.The acoustofluidic device  of PVMS NACPs (prepared with 0.3 wt% cetyltrimethylammonium bromide surfactant) after filtration with a 12 μm polycarbonate filter. The particle diameters were determined using optical microscopy with a 40× objective. Any microparticles <1 μm would not be resolved with this technique.Click here for fileAnalysis of PVMS microparticles after functionalization with biotin-TFPA and fluorescent streptavidin. Histogram showing the signal to background (S/B) fluorescent values of: (A) PVMS microparticles (~5 × 107 particles/mL) combined with biotin-TFPA and irradiated with a 320–500 nm light source (~10 mW/cm2) for 30 minutes. The particles were subsequently labeled with streptavidin AlexaFluor® 488 and washed with 1× PBS. The negative control reactions were performed identically except (B) without light irradiation or (C) without biotin-TFPA. All fluorescent values were taken from images acquired using a 40× objective and 25 ms exposure. Three separate fluorescent images from the same sample were taken and used to calculate standard deviations.Click here for fileAcoustofluidic device. Digital camera images showing (A) the glass top and (B) the silicon underside of an exemplary acoustofluidic device. To collect downstream sorted particles, a trifurcation arrangement was designed with two side outlets and a single middle outlet, where negative and positive acoustic contrast particles would exit, respectively. The PZT is attached to the silicon underside.Click here for file


Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual.gag, one region in pol, and three regions in env.HIV diversity was measured in 203 adults: 20 with acute HIV infection , 116 with recent HIV infection , and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection.The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined. Accurate methods for measuring HIV incidence using cross-sectional samples are important for monitoring the HIV epidemic and assessing the efficacy of interventions for HIV prevention env sequences are usually homogeneous early in infection env diversity usually increases over time and stabilizes or declines in advanced stages of HIV disease env and gag may be concordant or discordant over the course of infection env and gag proteins Because HIV generally diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence While sequencing-based studies of HIV diversity have been informative, the cost and effort needed to sequence viral variants  make those methods impractical for analysis of HIV incidence in larger cohorts or surveillance studies, which may require analysis of hundreds or thousands of samples. Recent reports have found that the frequency of ambiguous nucleotide calls in population sequencing data may reflect HIV diversity gag, pol, and env diversity in samples from 203 adults with different stages of HIV infection. These data suggest that the HRM diversity assay may be useful for analysis of HIV incidence.We recently developed a rapid assay for HIV diversity based on high resolution melting (HRM) technology The EXPLORE, HIVNET 001, Johns Hopkins HIV Clinical Cohort (JHHCC), and Johns Hopkins Hospital Emergency Department (JHH ED) studies were conducted according to the ethical standards set forth by the institutional review boards of the participating institutions and the Helsinki Declaration of the World Medical Association; participants provided written, informed consent Samples were collected from adults in the United States. Acute samples , and median CD4 cell count: 43 cells/mm3 .Samples from adults with non-recent HIV infection (infected >2 years) were obtained from two sources: (1) The Johns Hopkins HIV Clinical Cohort (JHHCC) study of HIV-infected patients in Baltimore  gag and pol was prepared using the ViroSeq system. HIV DNA used to analyze env was prepared using the Qiagen OneStep RT-PCR Kit ; reverse primer JH38R (5′-GGTGARTATCCCTKCCTAAC-3′) HIV RNA was extracted from plasma or serum using the ViroSeq HIV Genotyping System . HIV DNA used to analyze gag-pol and env amplicons) amplified from five recent samples (EXPLORE study) was cloned into the vector, pCR®2.1-TOPO . Plasmids were diluted to approximately 5 ng/µl for HRM analysis.DNA . Melting curves were used to determine HRM scores, as described previously The HRM diversity assay was performed as previously described The Wilcoxon rank sum test was used to compare HRM scores from different sample sets  for each region. Outlier values were defined as greater than the third quartile + (1.5 x interquartile range [IQR]) or less than the first quartile – (1.5 x IQR). Extreme values were defined as values greater than the third quartile + (3 x IQR) or less than the first quartile – (3 x IQR). Logistic regression was used to assess HRM scores for recent and non-recent infection for all six regions both separately and jointly. The Wilcoxon rank sum test was used to compare HRM scores for adults in different subgroups . Analyses were performed using SAS software version 9.2 .gag (GAG1 and GAG2), one region in HIV pol (POL), and three regions in HIV env .The HRM diversity assay was used to analyze samples from 189 adults with different stages of HIV infection . The median HRM scores for those samples were significantly higher than the median scores for control plasmids for all of the regions except GAG2 . The higIn all six regions analyzed, the median HRM scores for adults with non-recent infection (infected >2 years) were significantly higher than the median scores for adults with recent HIV infection . In 3. As shown in HRM scores for adults with non-recent infection who had CD4 cell counts above vs. below 50 cells/mmIn the analysis described above, adults with recent infection were all MSM; three (3%) of those men reported using intravenous drugs in the six months before HIV seroconversion. In contrast, the non-recent group included both men and women, most of whom likely acquired HIV infection through intravenous drug use. Some studies indicate that the number of HIV variants present very early in HIV infection varies among different risk groups We used a novel HRM diversity assay to compare HIV diversity in adults with different stages of HIV disease. Adults with acute HIV infection had uniformly low HRM scores (low levels of HIV diversity). However, the median HRM scores for those individuals were significantly higher than those obtained for plasmid controls for all regions except GAG2. This indicates that the HRM diversity assay can detect a low level of HIV diversity very early in HIV infection. In adults with acute HIV infection, the highest median HRM score was obtained for the ENV2 region, which contains IDR cluster I of gp41. HRM scores for all regions except for ENV2 were significantly higher in seropositive adults with recent HIV infection than in adults with acute HIV infection.env in adults with likely recent vs. likely non-recent infection (classified using a detuned enzyme immunoassay strategy) In all six regions analyzed, we found significantly higher levels of HIV diversity in adults who were infected for at least two years (non-recent group) than in adults near the time of HIV seroconversion (recent group). An important finding of this study was that viruses from individuals with non-recent infection often exhibited low diversity in at least one of the regions analyzed; in each region, there was some overlap in the HRM scores from adults with recent and non-recent infection. A similar finding was reported in a study that used a heteroduplex mobility assay to compare HIV diversity in the V3-V5 region of HIV 3. In the non-recent group, HRM scores were not significantly different among adults with CD4 cell counts above vs. below 50 cells/mm3, and all of the unusually high HRM scores in this group  were from adults with CD4 cell counts <50 cells/mm3. These data show that advanced HIV disease is not associated with misclassification using the HRM diversity assay, and suggest that the HRM diversity assay may be useful for identifying samples from adults with advanced HIV disease who were misclassified as recently infected using serologic incidence assays.Advanced HIV disease is associated with misclassification of individuals with non-recent infection as recently infected using serologic incidence assays Viral suppression is also associated with misclassification of individuals with non-recent infection as recently infected using serologic incidence assays For samples that do amplify, we recognize that there is a potential to underestimate diversity when fewer copies of HIV RNA are used in the analysis. However, in a previous study The HRM diversity assay provides data that is likely to be independent of data from serologic incidence tests. Therefore, use of the HRM diversity assay in combination with serologic testing is likely to improve the precision of multi-assay algorithms for HIV incidence, lowering misclassification rates. We are exploring whether inclusion of the HRM diversity assay as part of a multi-assay algorithm will eliminate the need for CD4 cell count data, allowing all of the testing to be performed using a single plasma or serum sample. In summary, this study provides proof of principle that HIV diversity can be used as a biomarker to distinguish between adults with recent vs. non-recent HIV infection. Further studies are needed to evaluate the performance of multi-assay algorithms for HIV incidence determination that include the HRM diversity assay.


Unfortunately many of the proteins whose transit through the ER constitutes a major evidence for peroxisome biogenesis from the ER do not exhibit a similar localization in plants. Consequently, at best the ER acts as a membrane source for peroxisome in plants. However, in addition to their de novo biogenesis from the ER an increase in peroxisome numbers also occurs through fission of existing peroxisomes. In recent years live-imaging has been used to visualize peroxisomes and the ER but the precise spatio-temporal relationship between the two organelles has not been well-explored. Here we present our assessment of the peroxisome-ER relationship through imaging of living Arabidopsis thaliana plants simultaneously expressing different color combinations of fluorescent proteins targeted to both organelles. Our observations on double transgenic wild type and a drp3a/apm1 mutant Arabidopsis plants suggest strong correlations between the dynamic behavior of peroxisomes and the neighboring ER. Although peroxisomes and ER are closely aligned there appears to be no luminal continuity between the two. Similarly, differentially colored elongated peroxisomes of a drp3a mutant expressing a photoconvertible peroxisomal matrix protein are unable to fuse and share luminal protein despite considerable intermingling. Substantiation of our observations is suggested through 3D iso-surface rendering of image stacks, which shows closed ended peroxisomes enmeshed among ER tubules possibly through membrane contact sites (MCS). Our observations support the idea that increase in peroxisome numbers in a plant cell occurs mainly through the fission of existing peroxisomes in an ER aided manner.Transmission electron micrographs of peroxisomes in diverse organisms, including plants, suggest their close association and even luminal connectivity with the endoplasmic reticulum (ER). After several decades of debate  Single membrane bound, spherical-ovoid 0.1–1.5μm organelles that appeared closely associated with the ER were first reported at the ultra-structural level in the proximal convoluted tubules of mouse kidney by Rhodin  and in hWhile the biochemical characterization of peroxisomes progressed rapidly . Fluorescent highlighting of the 0.4–1.5 μm diameter peroxisomes shows their hitherto unexplained erratic motility that includes stop and go motion, sudden twists and turns including U-turns, and an almost individualistic manner of movement where one peroxisome might remain almost static while others around it move at varying velocities  and its color variants, that highlight peroxisomes and the ER separately have been developed for living plant cells . As shown in Figure −1  peroxisomes within a cell might appear to be arrested in a particular sub-cellular location while other peroxisomes move past them at varying speeds. We further investigated this line of thought by observing peroxisomal motility under conditions that are known to affect ER dynamics.The cortical ER also consists of large membrane patches that constitute ER islands. When observed using only YFP-PTS1 many peroxisomes appear moribund or execute only Brownian movement  displayed short oscillations in these cells with the rest exhibiting Brownian movement. By 6 min the number of motile peroxisomes had risen to 35 ± 2.8%, while ER-tubules started exhibiting remodeling and new areas of anastomosis to form the typical polygonal ER mesh. By 12 min the ER exhibited normal cortical and sub-cortical dynamics and 67 ± 5% peroxisomes had achieved motility with normal speeds of 4 ± 1.5μms−1 in sub-cortical strands. It was concluded that while slowing down the dynamic behavior of the ER results in slowing peroxisome motility an increase in the movement rate of the ER results in a concomitant change in peroxisomal movement.Low temperature, around 4°C slows down ER motility, leads to the disappearance of long tubular ER strands and causes a distinct increase in short ER tubules and wider cisternae  peroxisomes displayed relatively localized movements. These involved multiple circumambulations . The initially green fluorescent tubular peroxisomes in these lines could be readily photo-converted to red fluorescent ones through irradiation with a violet-blue light  strongly suggested that one is observing an ER like compartment the simultaneous time-lapse observations of peroxisomes and the ER in double transgenics expressing GFP-PTS1 and RFP-ER showed several instances where peroxisomal tubules lay in ER lined channels  seeds were obtained from the Arabidopsis Biological Research Center . Stable transgenic lines of wild type Arabidopsis and the apm1-1 mutant expressing one and two targeted fluorescent proteins were created through the floral dip method . The epi-fluorescence setup consisted of a Leica DM-6000CS microscope. Photo-conversion was performed manually by controlling the diaphragm as described earlier  or the ImageJ distribution Fiji (http://pacific.mpi-cbg.de/wiki/index.php/Fiji). Adobe Photoshop was used for annotation of movies. Imaris software  was used to render iso-surface 3 D rendering of ER and peroxisomes from confocal image stacks and x-y-time series. All experiments were carried out at least five times.All images and movies were cropped and processed for brightness/contrast as complete image or stacks using either Adobe Photoshop CS3 (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.


Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in  DNA polymerases are required during both DNA replication and various types of DNA repair. DNA double-strand breaks are frequently repaired by homologous recombination, a conservative process in which DNA is copied into the break site from a similar template. The specific polymerases that operate during homologous recombination repair of DNA double-strand breaks have not been fully characterized in multicellular organisms. In this study, we created mutant strains of Drosophila lacking one or more DNA polymerases and determined their ability to synthesize large amounts of DNA during homologous recombination. We found that the error-prone translesion polymerases eta and zeta play overlapping roles during the initiation of synthesis, while the Pol32 subunit of the replicative polymerase delta complex is required for repair involving large amounts of synthesis. In addition, we showed that flies lacking the Rev1 translesion polymerase synthesize more DNA during gap repair than their normal counterparts. Our results demonstrate that both replicative and translesion polymerases are involved in homologous recombination and identify Rev1 as a protein that may regulate the access of various polymerases to double-strand break repair intermediates. DNA double-strand breaks (DSBs) pose a serious threat to cell viability and genome integrity. DSBs can be repaired either by non-homologous end joining, in which the DSB ends are processed and directly ligated, potentially leading to loss of information and mutagenesis  delta and epsilon play important roles in repair synthesis during HR in vivo findings. In addition, a non-essential subunit of pol delta, Pol32, is required for break-induced replication, a form of HR that requires extensive DNA synthesis In the budding yeast in vitro studies using purified human proteins have identified a potential function for polymerase eta in extending D-loop intermediates TLS polymerases have also been implicated in HR repair. In chicken DT40 B lymphocytes, the absence of polymerases eta and zeta results in reduced gene conversion during antibody diversification and increased chromosomal abnormalities, respectively Thus, evidence from a variety of systems suggests that both replicative and error-prone TLS polymerases may be utilized during DSB repair. However, the roles of specific polymerases used during HR and how they are coordinated remains poorly defined. In this study, we present evidence that multiple TLS polymerases can function during the initial synthesis stage of HR repair and that they compete with polymerase delta during repair of a double-strand gap in Drosophila. Furthermore, we show that Rev1 may act to coordinate the initial recruitment of TLS polymerases, thereby preventing replicative polymerases from acting during early repair synthesis.CG3975, has been previously identified. Drosophila Pol32 possesses conserved PCNA and polymerase alpha interacting motifs , nitrogen mustard, and ionizing radiation and mildly sensitive to hydroxyurea, but were not sensitive to camptothecin . Together, the conserved domain structure, mutagen sensitivity, and female sterility suggest that CG3975 is a functional ortholog of Pol32. Thus, we will hereafter refer to CG3975 as Pol32, acknowledging that additional studies are needed to confirm this assertion.We exposed spn-A mutants, which lack the Rad51 protein and are therefore unable to carry out the initial strand invasion steps of HR, are unable to survive ionizing radiation (IR) doses in excess of 750 rads spn-A and pol32 mutants show similar survival defects following IR exposure  No excision of the a}P{w element or restoration of the intact transposon results in the original apricot eye color; (2) Repair that involves extensive synthesis  and annealing at the long terminal repeats of copia allows for full expression of the white gene and results in a red eye color (hereafter referred to as “full HR”); (3) Repair in which end joining occurs immediately upon excision, or HR in which synthesis aborts prematurely before fully copying the white gene results in yellow-eyed flies (“aborted HR”). In the third case, the amount of repair synthesis that occurred prior to end joining can be estimated by PCR. Previously, we have found that end-joining repair without synthesis is an extremely rare event in wild-type flies To further characterize the role of Pol32 in HR repair, we utilized a site-specific DSB repair assay in which the mechanism of repair can be inferred using an eye color reporter construct 4 kb gap . The P{wpol32 mutants  and Rev3   . Larvae  mus205) . This li mustard . As witha}P{w assay to determine if flies lacking either pol eta or pol zeta were defective in HR repair of a site-specific DSB. Flies lacking pol eta had a 45% decrease in full HR repair relative to wildtype .Next, we utilized the wildtype , left, be by 50% , right.  mutants . This wapol eta rev3 double mutants. We initially predicted that since each single mutant showed a reduction in full HR events, the double mutant would display a further reduction in HR repair. Surprisingly, we observed no difference in the frequency of full HR repair for the pol eta rev3 mutant compared to wildtype . Thus, although repair synthesis is more processive in its absence, Rev1 does not appear to be absolutely required for the coordination of both pol eta and pol zeta during HR repair. Because rev3 and rev1 mutants are similarly sensitive to IR, we predict that the major role of Rev1 is to recruit pol zeta to early DSB repair intermediates.To test this, we obtained a g region . We werel mutant . The mutl mutant . Intereswildtype . Howeverpol32 rev3 double mutant. Full HR repair was reduced 70%, similar to pol32 single mutants, but repair synthesis tract lengths were reduced dramatically in aborted HR repair products compared to wildtype, with defects at distances as short as 250 base pairs and virtually no synthesis observed at distances ≥4.3 kilobases . This synergistic effect is consistent with the idea that pol delta (with Pol32) and pol zeta directly compete for HR intermediates. When both polymerases are impaired or eliminated, repair synthesis is greatly inhibited.Our results indicate that both TLS and replicative polymerases are acting during HR repair of a double-strand gap. These polymerases could compete for D-loop substrates, with the amount of synthesis at any given point during HR dependent upon the processivity of the polymerase synthesizing at that moment. Alternatively, a mechanism for a coordinated polymerase switch may exist, where HR synthesis initiates with a TLS polymerase and later switches to a replicative polymerase. To explore these two possibilities, we created a ilobases . The synpol32 rev3 and pol32 rev1 mutants should be identical. To test this hypothesis, we performed the a}P{w assay in a pol32 rev1 mutant background. Although full HR events were reduced by 60% in pol32 rev1 mutants, repair synthesis tract lengths were increased dramatically over wildtype  can gain access to the D-loop intermediates, resulting in longer repair tract lengths. In cases when pol delta is loaded, Pol32 appears to be important for maintaining the processivity of the delta complex . This isrev3 and pol eta mutants  multiple polymerases can initiate HR synthesis, and (2) the access of these polymerases to HR intermediates is likely regulated by Rev1. In support of the first conclusion, loss of both Pol32 and Rev3 results in extremely short synthesis tract lengths in aborted HR repair products , suggest mutants . Perhapspol32 rev1 mutants should be equal to the pol32 single mutant. Instead, repair tract lengths were increased in the double mutant  was used to excise a}P{w for rev1 and pol eta single mutants, whereas all other experiments were performed with a third chromosome transposase source . Matched wildtype controls using the appropriate transposase source were done for each experiment (the same representative control for each respective transposase source is indicated throughout). Individual males possessing both a}P{w and the transposase source were mated to females homozygous for a}P{w and repair products were recovered in female progeny. Each vial was counted as an independent sample and statistical significance was calculated using the Mann-Whitney statistical test. Genomic DNA from flies possessing independent repair events was recovered HR repair was monitored through the DSB created after excision of a Figure S1Drosophila melanogaster by Gray et al. (2004) based on its pol-alpha interacting domain (highlighted) and PCNA-interacting motif (underlined). Only the first 14 amino acids would be present in a theoretically expressed L2pol32.ClustalW alignment of Pol32 in yeast, human and fly. Pol32 was originally identified in (TIF)Click here for additional data file.Figure S2pol eta and pol zeta mutants. (A) Mutants of pol eta and rev3 (mus205) were created through imprecise excision of a P element . White box indicates open reading frame; shaded arrow, the untranslated regions; numbers indicate nucleotide position from start of transcription. (B) pol eta mutants are sensitive to ultraviolet radiation (UV) and rev3 mutants are sensitive to methyl methanesulfonate (MMS). Percent survival was calculated as homozygote eclosion relative to an untreated control. Error bars represent the standard deviations of at least three trials.Characterization of (TIF)Click here for additional data file.Figure S3rev1 mutant. (A) Minos (Mi) transposable element insertion (MB11152) in the REV1 coding region. (B) The Minos insertion is a null allele. RT-PCR was conducted using RNA isolated from MB11152 homozygous stocks. rp49  was used as a control. Primers were designed to span an intron; PCR using genomic DNA (gDNA) produces a larger PCR product. MW = molecular weight marker. (C) Flies homozygous for the MB11152 insertion (rev1 mutants) are sensitive to ionizing radiation (IR).Characterization of a (TIF)Click here for additional data file.Figure S4pol32 rev1 mutants. Aborted HR events from pol32 rev1 mutants (n = 45) have an intermediate phenotype relative to pol32 and rev1 single mutants. Each bar represents the percentage of events with at least the indicated amount of synthesis.Left end repair synthesis in (TIF)Click here for additional data file.Text S1a}P{w assay.Additional Methods. Expanded methods, including primers used, for the (DOC)Click here for additional data file.


Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events.2 and publication bias was assessed using Begg's funnel plot and Egger's regression test. Many of the 209 eligible studies included an unclear or high risk of bias. Meta-analyses showed a significant effect of preventing influenza-like illness  and laboratory confirmed influenza infection  through vaccinating immunocompromised patie nts compared to placebo or unvaccinated controls. We found no difference in the odds of influenza-like illness compared to vaccinated immunocompetent controls. The pooled odds of seroconversion were lower in vaccinated patients compared to immunocompetent controls for seasonal influenza A(H1N1), A(H3N2) and B. A similar trend was identified for seroprotection. Meta-analyses of seroconversion showed higher odds in vaccinated patients compared to placebo or unvaccinated controls, although this reached significance for influenza B only. Publication bias was not detected and narrative synthesis supported our findings. No consistent evidence of safety concerns was identified.Electronic databases and grey literature were searched and records were screened against eligibility criteria. Data extraction and risk of bias assessments were performed in duplicate. Infection prevention and control strategies should recommend vaccinating immunocompromised patients. Potential for bias and confounding and the presence of heterogeneity mean the evidence reviewed is generally weak, although the directions of effects are consistent. Areas for further research are identified. Respiratory disease is a leading cause of global mortality to which seasonal and pandemic influenza both make substantial contributions. For example, in the USA an estimated average 225,000 hospitalisations and 36,000 deaths per annum are attributable to seasonal influenza Patients with sub-optimal immune function due to disease or therapy (the immunocompromised) are recognised to be at increased risk from influenza-related complications, and are recommended for annual vaccination in many national vaccination guidelines. Concerns about influenza within immunocompromised populations include an impaired respo nse to vaccination and higher risk of complicated infection with increased mortality We conducted a systematic review and meta-analysis to assess influenza vaccination for immunocompromised patients. We report the primary analysis and its interpretation from a public health policy perspective, to assess the overall evidence. A second manuscript will be submitted for publication which reports a secondary analysis of our data, stratified by aetiology of immunocompromise.An abbreviated study protocol is available from the National Institute for Health Research international prospective register of systematic reviews (PROSPERO) The study population of interest comprised all persons immunocompromised due to primary immunodeficiency (genetic defects) or secondary immunodeficiency . Immunocompromised populations were derived from World Health Organization (WHO) and United Kingdom (UK) Department of Health immunisation policy to prevent influenza infection Single reviewers conducted searches during January 2011, based on the term construct used for MEDLINE see , which wAfter removal of duplicates a three-stage screening process applied the eligibility criteria to all records. Screening at title, abstract and full text was managed primarily within EndNote® ×4.0.2 . Records in non-compatible formats or non-English languages were manually screened. Screening was un dertaken by two reviewers in parallel, with consensus by discussion and provision for arbitration by a third reviewer. Due to insufficient resources, Spanish and Portuguese literature was screened by one reviewer.Data were extracted by two reviewers in parallel using a piloted template, with consensus by discussion and provision for arbitration by a third reviewer. No further data were sought from corresponding authors of eligible studies. Items extracted for study characteristics comprised country setting, objectives, design, sample size, methods of recruitment, inclusion and exclusion criteria, sequence generation, allocation, confounders and funding source. Population items comprised description of study groups, setting and stability of setting, age, sex, socioeconomic characteristics and risk factors for exposure to influenza. Intervention items comprised healthcare provider, setting in which health care delivered, description of intervention or exposure, vaccination type, route of administration, dosing schedule, and number of subjects allocated to and receiving the intervention or exposure. Outcome items comprised definition, measurement tool, timing and unit of measurements, blinding of assessors, duration of follow-up, number of measurements made , intervention and comparator results, detail of statistical analyses performed, and control for selection bias and confounding.Extracted outcome data on immune response were classified according to Committee for Human Medicinal Products (CHMP) seroconversion and seroprotection criteria for each influenza subtype patients were vaccinated against Risk of bias was assessed at both study and outcome level using tools produced by the Cochrane Collaboration Descriptive statistics were calculated using Microsoft® Office Excel® 2007 version 12 . Where feasible, odds ratios including 95% confidence intervals and the standard error of the natural log odds ratio were calculated for input into meta-analyses.2<40%) and abandoned where heterogeneity was high (I2>85%). Statistical significanc e of pooled odds was assumed at the 5% level and assessed using the Χ2 test. Risk of publication bias for studies subject to meta-analysis was assessed visually using Begg's funnel plots and quantified using Egger's regression test. Sub-analysis sought to utilise the UN inequality-adjusted Human Development Index 2010 (UN HDI) Primary analysis was designed to synthesize appraisal of methodological quality and extracted study data by means of tabulation, narrative and meta-analysis (where appropriate). With the exception of serological outcome measures, data pertaining to the 2009 influenza A(H1N1) pandemic were pooled together with seasonal influenza data in accordance with the research aim to assess overall evidence. Meta-analysis of pooled odds ratios estimated the effect size of vaccinating immunocompromised patients versus immunocompetent controls (VICT), and of immunocompromised patients receiving vaccination versus those receiving placebo or no vaccination (PNV). Meta-analyses were conducted using Stata® version 10  initially using a random effects model. Analyses were re-executed using a fixed effects model where heterogeneity was low (ICharacteristics of the eligible studies are summarised in A median of sixty immunocompromised patients received active influenza vaccination across the 209 studies (interquartile range [IQR] 36 to 110). Studies typically administered the vaccine by intramuscular injection (n = 138) with a minority utilising intradermal (n = 20), subcutaneous (n = 2) and intranasal routes (n = 3). Forty-nine studies did not report these data (most likely intramuscular) and three studies used multiple routes of administration. The median intervention group size for included RCTs was 55 ( IQR 26 to 103) and the median placebo or no vaccination group size was 24 (IQR 17 to 56). A median of 65 vaccinated immunocompromised subjects were recruited (IQR 40 to 116) and sources of funding were declared by 114 studies.et al (2010) scored highly within the reporting domain of the risk of bias tool whilst Vazquez-Alvarez et al (2010) scored poorly due to limited description of the study characteristics including potential confounding variables. Both studies scored poorly for external and internal validity.et al (2006) did not assess quality and validity for included studies. Anema et at (2008 ) and Atashili et al (2006) conducted meta-analyses for a pooled estimate of the effectiveness of influenza vaccination in preventing ILI or laboratory confirmed infection in HIV patients, both including three prospective studies et al (2006) additionally included a case control study Outcomes for all 209 individual studies cannot be presented due to volume of data. We pooled studies for analyses according to review questions, irrespective of aetiology of immunocompromise . Meta-analysis also pooled two studies of ILI reported in vaccinat ed immunocompromised patients compared to VICT 2 = 12.3%; p = 0.286).Meta-analysis pooled seven studies of ILI reported in vaccinated immunocompromised patients compared to PNV et al (2006) pooled one RCT, two non-randomised studies and one case-control study of ILI or laboratory confirmed influenza compared to PNV, estimating a risk difference of −0.27  but with significant heterogeneity  et al (2008) performed the same analysis excluding the case-control study, finding a risk ratio of 0.34  again with significant heterogeneity  Two earlier meta-analyses considered vaccination impact on incidence of ILI in immunocompromised patients. Atashili Of those studies unsuitable for meta-analysis, we identified 22 interventional studies and one observational design where no cases of ILI were found in vaccinated immunocompromised patients (including two RCTs with a PNV comparator and 13 non-randomised studies with VICT controls). The remaining studies we identified typically showed a low incident case number of cases, with some noteworthy exceptions. Cumulative incidence of ILI in vaccinated renal transplant recipients immunosuppressed with azathioprine is reported as 5.4% and 8.3% for those on mycophenolate mofetil, compared to 8.1% in healthy controls 2 = 50.4%; p = NS).Meta-analysis pooled two studies in vaccinated subjects with HIV compared to PNV We found limited data from non-randomised studies showing very low numbers of incident cases of laboratory confirmed influenza post vaccination in immunocompromised patients. Nine studies reported no cases during follow-up, and two studies found a single case each. A study by Tasker and colleagues Data on immune response to vaccination for each influenza subtype were pooled for meta-analyses based on CHMP definitions of seroconversion or seroprotection Of the 85 studies unsuitable for meta-analysis reporting rates of seroconversion, seroprotection or mean geometric increase in HI titre based on serology within 2–4 weeks, many were single-arm but broadly supported the above findings. Notably, statistically equivalent rates of seroconversion (SC1) were found to influenza A(H3N2) and B in patients with primary immunodeficiency post hoc selection of reported outcomes. Local and systemic adverse events were mapped to CHMP criteria Adverse event data were unsuitable for meta-analysis owing to difficulty in accurately identifying cases and denominator counts, and potential for bias due to Eighty-seven studies reported clinical or laboratory markers of vaccination impact on the underlying immunosuppressive condition. These included CD4+ count and HIV load in HIV-positive patients, relapse and complication rate in cancer patients, allograft rejection rate in transplant patients, disease activity scores in patients with autoimmune conditions and lung function tests in respiratory patients. We did not identify consistent evidence of disease progression or worsening of clinical symptoms related to underlying immunosuppressive condition following vaccination.et al (2008) report biopsy-proven allograft rejection within six months of vaccination in four paediatric liver transplant recipients Incidence of serious adverse events was reported in 21 studie s, although only 11 of these included a control group. Three hospitalisations occurred in patients with HIV Risk of publication bias was assessed using Begg's funnel plot and confirmed statistically where feasible using Egger's test. There was no evidence of biased reporting among studies subject to meta-analysis.This systematic review is the first to consider clinical and serologic outcomes following influenza vaccination in immunocompromised patients, incorporating data from the 2009 pandemic period. Our results suggest that vaccinating immunocompromised patients against influenza provides clinical protection from influenza-like illness and laboratory confirmed infection compared to placebo or no vaccination, and the rate of symptomatic disease is comparable to that observed in vaccinated healthy controls. The pooled odds of seroconversion were consistently higher in vaccinated patients compared to PNV controls, although statistical superiority was demonstrated only for influenza B. Conversely, the odds of seroconversion (SC1) and seroprotection conferred by vaccination were consistently and significantly lower in patients compared to VICT controls for seasonal influenza A(H1N1), A(H3N2) and B see . The datMany of the 209 eligible studies were at unclear or high risk of bias across most domains and the number of RCTs was relatively small (n = 23). The majority of studies (n = 137) were non-randomised trials that included a control group, but without robust randomisation selection bias between study arms cannot be excluded. Non-randomised designs may also introduce unbalanced confounding variables, and given that analyses were commonly reported unadjusted, these may reasonably influence the reported effect sizes for each pooled outcome measure. Potential confounders were anticipated and specified in the study protocol. Included cases series conducted during the 2009 influenza A(H1N1) pandemic are likewise at high risk of selection bias. Stratification of meta-analyses by risk of bias was unfeasible due to concerns with selecting a specific domain for classifying studies as ‘low’ or ‘high’. Adverse event data pr esented in Moderate to high levels of statistical heterogeneity were present in many of the reported meta-analyses, reaching significance on numerous occasions. Even where effect sizes are consistent, clinical heterogeneity may continue to challenge the validity of meta-analysis. Potential confounders related to aetiology of immunocompromise or intervention characteristics may be responsible for such heterogeneity. This includes pooling of data arising from the 2009 influenza A(H1N1) pandemic vaccine  with seasonal vaccines although <10% of studies overall involved such vaccines, only one study was included in the ILI meta-analysis (PNV comparator), and the two studies reporting data on prevention of laboratory confirmed influenza offered narrative information only. Previous exposure to influenza vaccination, timing of vaccine administration (in relation to changes in administration of immunosuppressive therapy or disease state) an d immunosenescence may also be important effect modifiers contributing to heterogeneity between the reported outcome measures. Similarly, matching between vaccine and wild type influenza strains is likely to introduce a degree of inter-seasonal variability; however, this does not affect our conclusions in terms of public health policy as these are typically designed to provide consistent advice over multiple seasons. Our analyses reported separately by aetiology of immunocompromise provide a degree of sensitivity testing for pooled results.Paucity of data limited or prevented some analyses. There were insufficient data to adequately report on seroconversion or seroprotection with a PNV comparator. The planned sub-analysis of evidence from resource-poor countries was abandoned due to insufficient data arising from this setting. In addition, it is now recognised that a large proportion of the population aged ≥55 years probably had some degree of pre-existing immunity to the 2009 influenza A(H1N1) pandemic strain, adding further difficulty to the interpretation of data from the pandemic period Our data favour a policy of routinely recommending influenza vaccination to immunocompromised patient groups, who may be at higher risk of influenza and its complications Methodological limitations affecting the current evidence base mandates new robust studies assessing the incidence of ILI and laboratory confirmed influenza in vaccinated immunocompromised patients. Similarly, robust studies are needed to inform revised CHMP seroconversion and seroprotection criteria applicable to immunocompromised patients. Further primary research is warranted to quantify factors contributing to heterogeneity, including the utility of second ‘booster’ doses, immunological adjuvants and degree of immunosuppression on rates of clinical protection and response to vaccination. Systematic reviews and meta-analyses are indicated to assess the impact of vaccinating immunocompromised patients on influenza-related morbidity and mortality. In addition, resource poor countries should be supported to conduct robust studies of influenza vaccination in their immunocompromised populations. Proportionally different comorbidities such as malnutrition or co-in fection with HIV may be encountered and response to vaccination among indigenous groups and ethnic minorities may differ in these settings compared to developed countries.Our systematic review and meta-analyses suggest immunocompromised patients do manifest an immune response to vaccination that, while not as vigorous as that of healthy controls, probably confers a similar level of clinical protection against influenza and, importantly, does so without causing excess harm. Limitations including potential for bias and confounding and the presence of statistical or clinical heterogeneity mean the evidence for these assertions is generally weak, but the direction of effects are remarkably consistent. Nevertheless, our study supports national and international public health policy recommendations for the targeting of immunocompromised patients for influenza vaccination.Figure S1Summary of risk of bias using the Cochrane Collaboration tool (n = 191). Legend: green = low risk of bias; yellow = unclear risk of bias; red = high risk of bias.(PDF)Click here for additional data file.Figure S2Forest plots for immune response to vaccination question.. Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): seasonal influenza A(H1N1), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): influenza A(H3N2), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): seasonal influenza B, vaccinated immunocompromised patients versus vaccinated immunocompetent contr ols. . Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): seasonal influenza A(H1N1), vaccinated immunocompromised patients versus placebo or no vaccination. . Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): influenza A(H3N2), vaccinated immunocompromised patients versus placebo or no vaccination. . Forest plot of studies of seroconversion (≥4 fold rise in haemagglutination inhibition titre): influenza B, vaccinated immunocompromised patients versus placebo or no vaccination. . Forest plo t of studies of seroconversion (<1∶40 pre-vaccination to ≥1∶40 haemagglutination inhibition titre post vaccination): seasonal influenza A(H1N1), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroconversion (<1∶40 pre-vaccination to ≥1∶40 haemagglutination inhibition titre post vaccination): influenza A(H3N2), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroconversion (<1∶40 pre-vaccination to ≥1∶40 haemagglutination inhibition titre post vaccination): seasonal influenza B, vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroprotection (≥1∶40 haemagglutination inhibition titre post vaccination): seasonal influenza A(H1N1), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroprotection (≥1∶40 haemagglutination inhibition titre post vaccination): influenza A(H3N2), vaccinated immunocompromised patients versus vaccinated immunocompetent controls. . Forest plot of studies of seroprotection (≥1∶40 haemagglutination inhibition titre post vaccination): seasonal influenza B, vaccinated immunocompromised patients versus vaccinated immunocompetent controls. Figure S2< /xref>.13. Forest plot of studies of seroprotection (≥1∶40 haemagglutination inhibition titre post vaccination): pandemic influenza A(H1N1), vaccinated immunocompromised patients versus vaccinated immunocompetent controls.(PDF)Click here for additional data file.Table S1MEDLINE search construct. Legend: PICO = research question in terms of population, intervention, comparators and outcomes. MeSH = Medical Subject Headings .(PDF)Click here for additional data file.Table S2Summary of risk of bias using the Downs and Black (1998) tool (n = 2). Legend: N/A = not applicable; higher score = less risk of bias.(PDF)Click here for additional data file.Table S3Summary of risk of bias using the US AHRQ tool (n = 3). Legend: Response indicates whether associated elements for reduction of bias have been met.(PDF)Click here for additional data file.Protocol S1(PDF)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Efforts to emulate the formidable information processing capabilities of the brain through neuromorphic engineering have been bolstered by recent progress in the fabrication of nonlinear, nanoscale circuit elements that exhibit synapse-like operational characteristics. However, conventional fabrication techniques are unable to efficiently generate structures with the highly complex interconnectivity found in biological neuronal networks. Here we demonstrate the physical realization of a self-assembled neuromorphic device which implements basic concepts of systems neuroscience through a hardware-based platform comprised of over a billion interconnected atomic-switch inorganic synapses embedded in a complex network of silver nanowires. Observations of network activation and passive harmonic generation demonstrate a collective response to input stimulus in agreement with recent theoretical predictions. Further, emergent behaviors unique to the complex network of atomic switches and akin to brain function are observed, namely spatially distributed memory, recurrent dynamics and the activation of feedforward subnetworks. These devices display the functional characteristics required for implementing unconventional, biologically and neurally inspired computational methodologies in a synthetic experimental system. The human brain is the most powerful information processor known to man. Although the activity of individual neurons occurs orders of magnitude slower (ms) than the clock speeds of modern microprocessors (ns), the human brain can greatly outperform CMOS computers in a variety of tasks such as image recognition, especially in extracting semantic content from limited or distorted information, when images are presented at drastically reduced resolutions In the brain, these excitatory/inhibitory connections between neurons, known as synapses, are nonlinear electroionic junctions whose conductivity changes in response to electrical and chemical signals. The relative timing of signals arriving from either side of the synaptic terminals, as well as larger-scale spatiotemporal patterns of network activity during these events, strongly influence the resultant change in synaptic strength, or plasticity We present a detailed analysis regarding the consequences of coupling many atomic switches together in a highly interconnected, recurrent structure to create an operational neuromorphic device that self-assembles into a functional state. The motivation for building complex network-based computing devices extends beyond an interest in understanding and emulating brain function. Alongside efforts to reduce the dimensions of circuit elements while increasing their integration, the wiring of interconnects has become the limiting factor in both design and performance of electronic devices Previously we reported an operational regime near the “edge of chaos” in similar network devices, as characterized by power law scaling of temporal metastability, avalanche dynamics and criticality Atomic switch network devices were characterized using a range of potentiostatic inputs, including constant and ramped DC as well as sinusoidal AC signals. These complex atomic switch networks are shown to exhibit various nonlinear behaviors, depending on the magnitude and timing of both present and prior input signals. Behaviors include both weak (continuous I–V loop hysteresis) and strong (discrete threshold switching) memristance as well as nonlinear frequency response (higher harmonic generation) and persistent fluctuations in conductivity under constant bias (recurrent connectivity); results which were found to agree with a recent theoretical study of current flow in memristor networks + migration, (2) electrochemical redox reactions involving Ag+/Ag0 to produce metallic filaments, and (3) an associated non-equilibrium α/β-Ag2S phase transition Previous reports on the synapse-like properties of single atomic switches have demonstrated features similar to short-term plasticity and long-term potentiation, where applied bias voltage produced a junction conductance dependent on the history of stimulation  + dopant cations across the insulator leads to ‘strong’ memristance characterized by abrupt switching through metallic filaments formed once the Ag+ cations reach the cathode and are reduced to metallic silver 2S, a conductive phase of silver sulfide normally unstable below 170°C 0 and α-Ag2S, the electrically insulating room temperature phase 2S to its room-T α-phase, which drives the dissolution of the Ago filaments and turns the atomic switch OFF at a rate dependent on the history of applied bias, producing the observed memory effects.These mechanisms collectively produce the memristive switching and synaptic memory functions exhibited by a single atomic switch. Specifically, ‘weak’ memristance resulting from redistribution of AgA great deal of effort has been put towards building biologically inspired computational hardware 9 junctions/cm2 according to analysis of SEM images), silver nanowire networks functionalized with interfacial Ag|Ag2S|Ag atomic switches. These nanowire networks were prepared through self-assembly without pre-patterning of the network topology using the electroless deposition of Ag from Cu inside the SU-8 reaction well of an I/O device platform 3 produces a metallic silver structures with variable morphologies depending on the concentration of Ag+ and distribution of Cu 2S|Ag interfaces were formed spontaneously within the network during gas phase sulfurization Based on the view that recurrent connectivity is essential to brain-like function, we have built, characterized and operated devices using massively interconnected (10a). This behavior represents activation of the network and is shown as an illustrative example of a network device undergoing a behavioral phase transition similar to the bias-driven forming step required to activate single resistive switches. Following network activation, devices subjected to repeated bias sweeping generally exhibit robust, strong memristive behavior, typified by hard switching (inset). Robust switching over 10,000 cycles was demonstrated at an operational threshold voltage (Vt) of reduced magnitude . Both the distributed power dissipation As recent theoretical models predict passive generation of second harmonics in both singular memristors and in random networks, the distribution of switch function throughout the network was examined through analysis of the device's frequency response Having characterized atomic switch operation in an interconnected complex network, we examined the device for emergent behaviors specific to its brain-like recurrent structure. Structurally, the atomic switch network is recurrent in the sense that there exist pathways such that electrical signals produced at one junction may lead to (delayed) feedback at the same junction. Here we present experimental evidence of spatially distributed and correlated network dynamics, which are attributed to such recurrent connectivity. These recurrent dynamics are presented as an emergent property of the atomic switch network.Applying a constant 1 V DC bias  producedOFF>10 MΩ) and fluctuations of ΔR/R∼1. In order to produce relative resistance changes of such high magnitude, switching events within the network must be correlated. While stochastic processes may be involved in the correlation of these fluctuations These fluctuations are of a magnitude significantly greater than what can be considered noise. An internal control experiment compared Fourier transformed current responses  of the dON/ROFF ratio greater than 30. In the example shown, the conductive paths between the two channels overlap spatially, yet are switched independently, indicating that local sub-regions of the network can transition to distinct operational modes despite being embedded within a highly interconnected, largely metallic structure. This is analogous to the presence of feedforward subnetworks within the recurrent architecture of the cortex. Single pulses of sufficient magnitude overwhelm the recurrent dynamics and induce feedforward activation of local sub-regions along a path connecting the involved pair of electrodes without significantly altering the conductivity of other spatially intertwined channels within the same nanowire network.Inside the generally recurrent structure of the brain's neural network, there is evidence for the existence of feedforward subnetworks utilized for the fast propagation of certain signals The degree to which pulse-mode channel creation influences overall network connectivity can be visualized in electrode resistance cross-correlation matrices . In thisUsing a simple, two-step fabrication procedure combining top-down and bottom-up fabrication techniques, we have created functional neuromorphic devices based on a self-assembled, complex network architecture. We describe these atomic switch networks as neuromorphic not only in that the massively interconnected, dendritic features observed in biological neural networks inspired the device architecture, but also due to several important network scale properties reported here. The devices demonstrate weak and strong memristive behaviors, as well as higher harmonic generation, confirming theoretical predictions on current flow through memristor networks. Previously unreported emergent behaviors specific to the complex architecture were observed as persistent bidirectional fluctuations of the current in response to constant applied voltage and the pulse-based feedforward switching ON and OFF of localized conductance channels within the highly interconnected network. Despite lacking the brain's rich assortment of neurotransmitter systems, with distinct excitatory and inhibitory neurons, the complex network of atomic switches produces multiple behaviors from a single basic unit through a capacity for localizing function in subnetworks inside a structure correlated by the nonlinear memory response of individual atomic switches. This diversity of function indicates the device's potential as a universal approximator of dynamical systems 2 followed by a post-exposure bake beginning at 65°C and ramping up to 95°C before cooling to room temperature at 1°C/min. The SU-8 was developed by immersion in PGMEA (Propylene Glycol Methyl Ether Acetate). Fully developed wafers were rinsed with isopropanol and hard baked at 130°C on a hotplate in N2 atmosphere to increase SU-8 resistance to high temperatures.Electrodes were patterned on the surface of a Si wafer  by photolithography. A Cr/Pt (15/150 nm) bilayer was deposited using e-beam evaporation. Subsequently, microfluidic reaction wells were patterned from a thick layer of SU-8 (approx. 500 µm) deposited by spin coating. The resist was UV exposed with a dose of 1200 mJ/cm3  at concentrations ranging from 0.1–100 mM into microfluidic cells containing Cu seed posts, leading to a spontaneous reaction between Ag+ and Cu. Optimal conditions were achieved with Cu posts ranging from 0.25–4 µm in diameter at pitches of 0.5–4 µm reacted with 50 mM AgNO3, sulfurized at 130°C for 10 minutes under N2 flow at atmospheric pressure. The silver networks self-assembled during this processes, and were then functionalized by reaction with sulfur  in a Pyrex tube. The sulfur was melted in an evaporation boat at 130°C and delivered to the substrate by N2 flow.Electroless deposition of Ag from Cu was performed by pipetting aqueous AgNOElectrical characterization of the devices was conducted using four Pt electrodes positioned around the edges of the Ag network. Current-voltage spectroscopy was conducted using a bipotentiostat (Pine Instruments model AFCBP1) in conjunction with a DAQ module  at a sample rate of 10 kHz. Measurements were performed in a two-electrode configuration. Multi-channel resistance measurements were obtained using a multiplexed  SMU . The entire I/O system was housed in a Faraday cage and mounted on a vibration isolation table (TMC). Devices were characterized after each stage of the fabrication cycle. Subsequent data analyses were carried out using MATLAB 2010b (MathWorks) and Origin 8.1 (OriginLab Corporation).The full dataset used in Figure S1Device Activation. (a) Initial bias sweeps (±7.5 V at 1 V/s) demonstrate weakly memristive behavior with increasing hysteresis magnitude . (b) Bias sweeps from (a) rescaled to include the hard switching  phase transition event at Va≈7.5 V.(TIF)Click here for additional data file.Figure S2Robust Switching. Operation of a device following the phase transition (activation) exhibiting typical, robust pinched hysteresis/switching. Shown device parameters: sweep rate = 103 V/s (1 kHz), RON = 1 kΩ, ROFF>20 kΩ, Vt = 0.5 V.(TIF)Click here for additional data file.


Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the  The differentiation of multipotent progenitor cells into terminal cell types must involve a number of coordinated changes. Since different cell types have distinct proliferative properties , the determination of cell fate must be coordinated with cell cycle behavior. The end of proliferation is likely to be coordinated with changes in cellular growth, since doubling of cell components is necessary for mitotic cell populations to maintain cell size, but is not necessary for non-dividing cells. It is likely that changes in growth are further correlated with other aspects of cell physiology, including metabolism and energetics. Mechanisms should therefore exist to co-regulate multiple cellular processes during terminal differentiation.Drosophila eye imaginal disc, like many other developing tissues, grows rapidly and then becomes largely post-mitotic on terminal differentiation The Drosophila, nucleoli are readily revealed by antibody labeling for the nucleolar protein fibrillarin, a rRNA 2′-O-methyltransferase that processes pre-ribosomal RNA The nucleolus is the site of ribosomal RNA transcription and initial assembly of ribosomal subunits. Accordingly, nucleolar size is one simple albeit indirect measure of ribosome biogenesis and cellular growth, and is proportional to growth rate in cell lines and in tumor cells Cell fate specification begins during the third and final larval instar. A wave of differentiation moves anteriorly across the eye imaginal disc, starting from the posterior margin, until after two days the entire retinal field is differentiating  [4]. A dThe G1 arrest of eye cells depends on the secreted signals Dpp and Hh It was of interest to determine whether nucleolar size, an indirect indicator of ribosome biogenesis, decreased with larval eye disc differentiation, when such changes would occur, and how they might be regulated. It would be useful to assess how changes in nucleolar size correlated with particular cell cycle arrest or cell proliferation events, and how any such coordination might be achieved. Accordingly, substantial changes in nucleolar size in the eye disc that accompany the transition from multipotent progenitors to differentiating retinal cells are described.Nucleoli can be visualized through antibody labeling to detect the fibrillarin component of the nucleolar snRNP particle To define a time-course, cross-sectional areas of individual nucleoli were recorded across the anterior-posterior axis of eye discs . The knoMean nucleolar cross-section was nearly 65% smaller in zone 9 near the posterior disc margin than in zone 1 in the anterior. The results of plotting median nucleolar size were similar (not shown). There often appeared to be a temporary halt or small reversal of the trend towards smaller nucleoli around zones 5–6, which could be related to the Second Mitotic Wave . Which cCip1/WAF1 posterior to the morphogenetic furrow GMR-Gal4/+) the mean size of apical and basal nuclei was significantly different in zone 6, being larger in the basal nuclei that participate in the Second Mitotic Wave, and smaller in the apical nuclei of cells that are differentiating not dividing at this stage , mouse and rabbit anti-GFP antibodies (Invitrogen #A11120 and A11122); rabbit anti-phosphoHistone3 , mouse monoclonal anti-fibrillarin , and mouse monoclonal anti-Cyclin B (F2F4). Secondary antibodies were Cy2- and Cy3-conjugates from Jackson Immunoresearch. Labeling of eye discs was performed as described Mothers against dpp (Mad) clones were obtained in hsF; 1Mad2 FRT40/M(2)24F [armLacZ] FRT40. 1Mad2 is a null allele that lacks the N-terminal sequences for phosphorylation by BMP family receptors smo clones were obtained in hsF; 1Mad2 FRT40/ubiGFP FRT40. 3smo is a missense allele that is genetically null smo Mad clones were obtained in hsF; 3 Mad1smo2 FRT40/M(2)24F [armLacZ] FRT40.Clones of cells mutant for genes were obtained by the FLP-mediated mitotic recombination technique