White matter (WM) neuroimaging studies have shown varied findings at different stages of schizophrenia (SZ). Understanding these variations may elucidate distinct markers of genetic vulnerability and conversion to psychosis. To examine the similarities and differences in WM connectivity between those at-risk for and in early stages of SZ, a cross-sectional diffusion tensor imaging study of 48 individuals diagnosed with first-episode SZ (FE-SZ), 37 nonpsychotic individuals at a high genetic risk of SZ (GHR-SZ), and 67 healthy controls (HC) was conducted. Decreased fractional anisotropy (FA) in the corpus callosum (CC), anterior cingulum (AC), and uncinate fasciculus (UF) was observed in both the GHR-SZ and FE-SZ groups, while decreased FAs in the superior longitudinal fasciculus (SLF) and the fornix were only seen in the FE-SZ participants. Additionally, both GHR-SZ and FE-SZ showed worse executive performance than HC. The left SLF III FA was significantly positively correlated with hallucinations, and right SLF II was positively correlated with thought disorder. The presence of shared WM deficits in both FE-SZ and GHR-SZ individuals may reflect the genetic liability to SZ, while the disparate FA changes in the FE-SZ group may represent symptom-generating circuitry that mediates perceptual and cognitive disturbances of SZ and ultimately culminates in the onset of psychotic episodes. Disconnectivity of different brain regions, which is putatively mediated by white matter (WM) abnormalities, is widely considered to be a key feature of schizophrenia (SZ) –3. PostmLongitudinal studies have observed deviations in cognition, emotion, and behavior in individuals with SZ years before illness onset , implicaAmong the first-degree relatives, the offsprings' prevalence is higher than normal people , meaningIn this study, we examined WM fractional anisotropy (FA) in FE-SZ, GHR-SZ, and healthy control (HC) participants using a voxel-based DTI approach. We opted to apply a voxel-based analysis because it has been shown to be more sensitive to subtle WM differences than other approaches such as tract-based and ROI-based analysis . FurtherForty-eight FE-SZ, 37 GHR-SZ, and 67 HC individuals took part in the study. FE-SZ and GHR-SZ participants were recruited at the outpatient clinic of the Department of Psychiatry, First Affiliated Hospital of China Medical University, Shenyang, China. HC participants were recruited from local community via advertisements. The study was approved by the Institutional Review Board of China Medical University. All participants provided written informed consent.Absence or presence of Axis I and Axis II disorders was confirmed by consensus between two trained psychiatrists using the Structured Clinical Interview for DSM-IV (SCID)  or the KThe FE-SZ participants met DSM-IV criteria for schizophrenia, schizophreniform disorder, or schizoaffective disorder without any other Axis I disorders. All FE-SZ participants were in their first psychotic episode with illness duration of less than one year. With regard to medication status, 24 of the FE-SZ participants were antipsychotic-naïve, while the other 24 participants were taking antipsychotic medications. The GHR-SZ participants had one parent with SZ and did not meet criteria for any DSM-IV Axis I disorders. The HC participants were selected to match in age and sex the FE-SZ and GHR-SZ participants. They had no personal or familial history of Axis I psychiatric disorders. Detailed demographic and clinical characteristics are presented in 2, 65 contiguous slices of 2 mm without gap, 25 noncollinear directions, and one no diffusion-weighting baseline image.All participants were scanned using a GE Signa HDX 3.0T magnetic resonance imaging scanner with a standard 12-channel head coil at The First Affiliated Hospital of China Medical University. Head motion was minimized with foam padding. Diffusion-weighted images were acquired using a spin-echo planar imaging sequence parallel to the anterior-posterior commissure plane with the following parameters: TR = 17000 ms, TE = 85.4 ms, image matrix = 120 × 120, field of view = 240 × 240 mmhttp://www.nitrc.org/projects/panda), a fully automated program for processing of brain diffusion images. After motion and eddy current correction were performed, individual FA images of native space were registered to the FA template in MNI  space, followed by resampling the images to a customized spatial resolution 1 × 1 × 1 mm with subsequent warping transformations. Lastly, the FA images were smoothed by a 6 mm Gaussian kernel to reduce noise and misalignment. The resulting images were then used in statistical analyses.Image preprocessing was performed using PANDA (http://www.fil.ion.ucl.ac.uk/spm) with diagnostic group as an independent factor and age and sex as covariates. Statistical significance was defined by corrected p < 0.05 with thresholds at uncorrected p < 0.01 and cluster size > 109 voxels as determined by Monte Carlo simulation in AlphaSim , and (3rrected) . Group dPost hoc analyses revealed decreased FA in the CC, AC, and UF in both the FE-SZ and GHR-SZ group when compared to the HC group, with no significant difference in FA between FE-SZ and GHR-SZ participants . SignifiFAs did not differ significantly between nonmedicated and medicated FE-SZ participants in any WM cluster identified in ANCOVA, indicating no confounding effect from medications. Additionally, when the medication was used as additional covariate, the same results were obtained except CC. Decreased FAs were shown only in GHR-SZ, but not in FE-SZ, when compared to HC.p = 0.069). Post hoc analyses showed that the BPRS scores were significantly higher in FE-SZ compared to GHR-SZ and HC, but not between GHR-SZ and HC. WCST performance in FE-SZ subjects was worse than GHR-SZ and HC, but no differences existed between GHR-SZ and HC.All the subjects except 5 FE-SZ patients finished the BPRS assessment. 26 FE-SZ, 32 GHR-SZ, and 35 HC finished the WCST . We founr = 0.309, p = 0.044), and right SLF II was positively correlated with thought disorder subscore . The Pearson's correlation analyses between WCST performance and the FA values of the CC, AC, and UF did not show significant associations.Pearson's correlation analyses were performed between the BPRS scores and FA value of left SLF III, right SLF II, and fornix in FE-SZ. The left SLF III FA was significantly positively correlated with hallucinations score of BPRS , such as the inferior parietal lobule, ventral prefrontal cortex, and superior temporal gyrus. The HASC serve as convergence points for multimodal sensory inputs and organize these inputs to guide responses to environmental stimuli and demands . The dynDecreased FAs in the CC, AC, and UF were observed in both the FE-SZ and GHR-SZ groups, relative to the HC group. Findings of abnormalities in the CC have been fairly consistent across the different stages of SZ , 40–43. Shared WM deficits were also found in the frontotemporal  and frontolimbic  pathways in the FE-SZ and GHR-SZ groups. Skewed distribution of FA in the left UF has been previously reported in studies of FE-SZ, implicating abnormalities at the center of the UF. Correlation between FA in the UF and negative symptoms, as well as an accelerated decline in UF FA with aging, has been shown in chronic SZ . In our Distinct from the GHR-SZ group, the FE-SZ group showed additional WM alterations in the right SLF II, left SLF III, and FX, when compared to the HC group. The SLF II and SLF III play overlapping roles in integrating information in the frontoparietotemporal circuitry , 49, parOur findings of shared and distinct abnormalities of WM integrity in FE-SZ and GHR-SZ raise intriguing questions about the role of genetic, neural, and other factors in conversion to psychosis among individuals at-risk for SZ. Are there differential markers for vulnerability to SZ versus definite progression to psychosis? Are certain factors more critical in conversion to psychosis? Our findings appear to suggest that disruptions in the CC, AC, and UF, shared by the FE-SZ and GHR-SZ groups, may represent genetic vulnerability to SZ, but do not result in symptomatic onset of SZ, whereas abnormalities in the right SLF II, left SLF III, and FX, found only in the FE-SZ group, may reflect key neural processes that generate definitive symptoms of SZ. In addition, our findings in the SLF and FX in FE-SZ may represent symptom-generating processes that are present in all psychotic disorders. Studies of these regions in other types of psychosis could provide insight into the level of neural differentiation among psychotic disorders. Our findings also speak to the synergistic effects of susceptibility and illness-generating factors in psychotic conversion of at-risk individuals. Longitudinal studies have shown higher rates of conversion to psychosis when genetic predisposition was coupled with environmental adversities . ConceivThe inclusion of both FE-SZ and GHR-SZ individuals is the major strength of our study. This allows us to dissociate WM abnormalities related to genetic vulnerability and those related to illness itself. Importantly, the SZ participants in our study were in their first episode of illness. Studying those in early stages of illness is substantially advantageous for identifying primary neuropathological processes proximal to initial symptom onset; findings from such samples are not confounded by illness chronicity and recurrence. Half of the FE-SZ participants (24/48) were taking antipsychotic medications at the time of scan. Post hoc analyses indicated no significant effects of medications on our primary WM findings, except CC. How medications influenced CC FA values in SZ needs further investigation in larger sample without taking medications.While our central findings are consistent with previous literature, there are some differences regarding the laterality of findings in the SLF and UF and the specific subregions of CC that are affected in SZ , 43, 45.Consistent with the previous studies , 54, FE-Our study is limited by the use of a cross-sectional design. Longitudinal studies are required to track the eventual development of schizophrenia in the high-risk group to confirm that findings are related to risk for the development of schizophrenia rather than protective factors. Second, the HR group in current study included only 37 individuals who are the children of patients with SZ. The sample size may be small considering heterogeneity in this sample, as only a portion of these individuals will later develop SZ. Future studies should distinguish between genetically high-risk relatives without symptoms, and those with existing prodromal symptoms without familial risk. Third, there are some potential problems with the voxel-based DTI analyses that were performed in the study to explore whole brain white matter abnormalities. For example, there is no satisfactory solution to aligning FA images from multiple subjects , and theIn summary, this study lends further support to the critical involvement of WM disruptions in SZ. Importantly, shared and distinct alterations in WM integrity were observed between FE-SZ and GHR-SZ. The shared abnormalities in the CC, AC, and UF may stem from genetic vulnerability to SZ that are present in both FE-SZ and GHR-SZ; whereas the distinct changes in the SLF and FX seen only in the FE-SZ group may point to separate neural mechanisms responsible for psychotic symptoms in SZ, such as hallucinations. Future studies may further elucidate differentiating features between FE-SZ and GHR-SZ, which would improve prediction of psychotic conversion in at-risk individuals. Such differentiation could significantly strengthen the effectiveness of early identification and intervention strategies in SZ, a disorder in which initiation of treatment at the time of psychotic onset is often too late for substantial impact on clinical outcome in affected individuals.


The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells  By opsonizing the tumor cell, antibodies can also mark tumor cells for attack by the complement system, NK cells and macrophages. Antibody engineering provides powerful technologies to improve antibody effector functions and to generate novel, bispecific biologics. The use of mAbs has revolutionized antitumor therapy, with impressive achievements in the treatment of both hematological malignancies and solid tumors  and antibody-derived biologics are essential tools for cancer research and therapy , 2. Antid tumors .via a hydrophobic interface—poses obstacles to the development of bispecific biologics. These aspects illustrate the need for new antibody formats that provide the same binding specificity of mAbs but with better stability and in vivo pharmacodynamics.However, certain inherent structural properties limit the applicability of mAbs and antibody-derived biologics for tumor therapy. The large size of mAbs  can hamper access to tumor cells. Moreover, the nature of the antibody recognition module—a pair of variable domains non-covalently associated The discovery of naturally occurring heavy chain antibodies  containing a highly stable and soluble single antigen-binding V-domain—designated VHH or nanobody (15 kDa)—has opened the way for a new generation of antitumor therapeutics. Other excellent reviews describe the discovery and structure of nanobodies, their potential applications in oncology, infection, immunity, and other diseases –18. HereConventional mAbs are composed of two heavy and two light chains Figure . Both chvia a linker peptide into a small (30 kD) single polypeptide binding module, designated single-chain variable fragment (scFv)  Figure . In an s) Figure .The VHH domains of camelid heavy chain antibodies have been shaped by more than 50 million years of evolution for high solubility and stability, independent of a partner VL domain. As recombinant proteins, VHH are designated single-domain antibodies or nanobodies  in referA notable difference between the camelid VHH and the human VH domain is the length and orientation of the CDR3 loop Figure . The CDRin vivo  is a drawback, as this can limit penetration into tumors in vivo , 28. It in vivo .In 1993, naturally occurring heavy chain antibodies were discovered serendipitously while analyzing the serum of a dromedary in a practical biochemistry course at the University of Brussels . It was The use of animal-derived antibodies for antitumor therapy is limited, because the immune system typically mounts an antibody response against the foreign components of a therapeutic antibody , 33. NeuSome highly successful antitumor antibodies have been generated simply by replacing the constant domains of the parental mouse antibody with the corresponding domains of human IgG heavy chain and kappa or lambda light chains Figure . RituximWith respect to immunogenicity, it is important to note that CDR3 loops likely contribute more to immunogenicity than framework residues of the V-domain . Indeed,VHH domains typically display a high sequence identity with human type 3 VH domains (VH3), likely accounting for their low immunogenicity . In addiIn order to reduce the residual immunogenicity of animal-derived V-domains in conventional antitumor antibodies, the six CDR loops have been successfully grafted from animal VH and VL domains onto human VH and VL domains . CampathAttempts have also been made to generate fully human heavy chain antibodies  Figure . Such fuvia disulfide bonds in the hinge region to a CD3-specific “half-mAb”  , 48 . Thus, unbound nanobodies are rapidly cleared from the bloodstream by renal elimination. The resulting short in vivo half-life of 1–2 h reduces the time interval to bind to their target molecule within the tumor , and ecto-enzymes. Nanobodies against “classical” receptor targets can antagonize ligand binding and activation of signaling cascades by targeted tumor cells . For exar growth , 68 and/r growth . An examr growth . Fusion r growth . A recenr growth . The resThe antitumor effects of nanobodies can be enhanced by coupling nanobodies to protein, peptide, and chemical effectors Figure . EffectoGenetic fusion and chemical conjugation provide two, partially overlapping, options for equipping nanobodies with effectors. The following sections describe the combination of nanobodies with different effectors, including genetic fusion of nanobodies to protein toxins and peptides, and chemical conjugation to radionuclides, photosensitizers, and anti-neoplastic drugs.Pseudomonas exotoxin, Diphtheria toxin, ricin, cucurmosin) induces cell death, while the targeting moiety binds to antigens preferentially expressed on the cancer cell surface, minimizing cytotoxic side effects on normal cells. Nanobody-based immunotoxins typically are generated by genetic fusion of the protein toxin to the C-terminus of the nanobody, i.e., akin to fusion of an nanobody to the hinge and Fc domains described above  effectively inhibited the proliferation of VEGFR2-expressing cells in vitro . This has been demonstrated recently in an elegant proof of principle study with an EGFR-specific nanobody fused to the E5B9 peptide tag (the UniTag) . Opsoniz18F-labeled CD11c-specific nanobodies  can be used to site-specifically biotinylate nanobodies carrying an Avi-tag . BirA-cananobody , 104. Bivia a photosensitive, NIRF.Nanobodies can readily be conjugated to radionuclides and fluorochromes, using either sortagging or classical chemical conjugation strategies Figure B. Such nConventional protein conjugation strategies use random conjugation to reactive side chains, most commonly amide conjugation to the amino group of lysine side chains or to the N-terminus of the protein . Random conjugation is difficult to control and may compromise the functionality of the nanobody, e.g., by sterically interfering with target binding, by sterically compromising tissue distribution, and by providing potentially immunogenic epitopes. Two elegant approaches have been developed to conjugate chemicals to nanobodies at a specific site. One involves the introduction of a cysteine residue at the C-terminus or at specific framework residues, providing a basis for site-specific maleimide conjugations , 102. ThTargeted radionuclide therapy is a systemic treatment that aims to deliver cytotoxic radiation to cancer cells and to cause at the same time minimal toxicity to surrounding healthy tissues. Radiopharmaceuticals consist of two components: a targeting moiety that specifically determines the accumulation of the radiopharmaceutical in the tumor and a radionuclide that delivers cytotoxic radiation through its decay . There iin vivo study demonstrated that 177Lu-labeled anti-HER2 nanobodies efficiently targeted HER2-positive xenografts and prevented tumor growth, while keeping radioactivity levels low in normal organs , as encapsulation of drugs overcomes problems, such as poor solubility, limited stability, and rapid clearance  Figure C. Nanopaet cells , 121. Inarticles , 123. InRelease of the drugs from the particles can be achieved by leakage or by mechanical destruction by ultrasound or intracellular degradation. The first liposomes that were decorated with anti-EGFR nanobodies were internalized into the target cell . Anti-EGvia the blood and lymphatics, diffusion through the endothelial cell and basement membrane into the interstitial space, hydrodynamic pressure in the blood vs. the tumor tissues, elimination of biologics from the system , binding to non-tumor cells, and binding to proteins . It may, therefore, be of interest to consider and test other options for the delivery of antitumor nanobodies and heavy chain antibodies.While size does matter, it is not the only factor determining the delivery of nanobody-based biologics to tumor cells. Tumor delivery is controlled by a complex interplay of factors, many of which are still poorly understood: the site of injection , transport via a transmembrane domain to cytosolic activation domains. In nanobody-based CARs, the relatively unstable scFv is simply replaced by a stable nanobody  have been used successfully as gene-therapy vectors, i.e., for the long-term expression of a therapeutic proteins in vivo , 131. Foin vivo . In a reloidosis . It is cIn vivo studies have underscored the favorable biodistribution of nanobodies, including deep penetration into tumors. Numerous nanobody-based biologics have shown antitumor efficacy in preclinical studies in vivo. Naked nanobodies can antagonize growth factor receptors and block ion channels and ecto-enzymes in the tumor microenvironment. Fusion of one or more nanobodies to the hinge and Fc-domains of a human immunoglobulin yields highly soluble and versatile heavy chain antibodies. Importantly, because nanobodies do not bind light chains and because they do not show any tendency to aggregate, nanobody-based bispecific hcAbs do not suffer from the VH–VL pairing problem of bispecifc conventional antibodies. Heavy chain antibodies are roughly half the size of conventional antibodies and, thus, may show better tissue penetration than conventional mAbs, while retaining the capacity to recruit the complement system, NK cells, and macrophages. Genetic fusion of nanobodies to peptide tags opens a path for marking tumor cells for attack by tag-specific T cells or NK cells transduced with a tag-specific CAR or with universal tag-specific cytotoxic antibodies. Sortagging and introduction of cysteine residues at specific framework positions allow easy conjugation to virtually any chemical moiety, including chelators for radionuclides, NIRFs, polyethylene glycol, liposomes, and nanoparticles. Preliminary studies indicate that it may well be worthwhile to further explore other modes for effective targeting of tumor cells with antitumor nanobodies, including Nb-CAR-expressing T cells and NK-cells and AAV encoding antitumor nanobodies.Nanobodies, nanobody-based heavy chain antibodies, and nanobody-drug conjugates have a huge potential as antitumor therapeutics. The US Food and Drug Administration recently granted fast track designation for caplacizumab, a bivalent nanobody targeting von Willebrand factor. Ablynx, the leading nanobody biotech company, has submitted an application for European Marketing Authorisation for caplacizumab, which thus may well become the first nanobody approved for therapy. Before nanobody-based antitumor therapeutics follow suite, additional preclinical and clinical studies are warranted. Antitumor nanobodies and antitumor Nb-hcAbs may overcome some of the obstacles that hamper therapies with antitumor mAbs. V-domains display a much lower intrinsic immunogenicity than Fc domains across species barriers. Moreover, nanobodies show higher sequence identity to human VH domains than do murine VH domains and divergent framework residues routinely are “humanized” in clinical nanobodies. While fusion to the hinge and Fc domains of human IgG does not add any additional immunogenicity, fusion to toxins, peptide tags, and chemical conjugation add potentially immunogenic epitopes. Moreover, as with conventional antibodies, the antibody paratope can induce an anti-idiotypic antibody response in a fraction of patients. As distinct nanobodies to the same target protein become available, switching to a different antitumor nanobody will become a therapeutic option. Assuming that progress will continue at the present pace, it is likely that the future repertoire of clinicians will include an increasing battery of nanobody-based antitumor therapeutics.FK-N and PB conceived the topic; PB, JH, and FK-N wrote the manuscript.via MediGate GmbH, a wholly owned subsidiary of the University Medical Center Hamburg-Eppendorf.PB and FK-N are coinventors on a patent application on CD38-specific nanobodies. FK-N receives a share of antibody and protein sales


In 2015, the WHO End TB Strategy laid out ambitious goals to dramatically reduce tuberculosis (TB) deaths, incidence, and catastrophic costs through research, bold new strategies, and patient-centered care. In this commentary, recent evidence on sputum collection strategies for smear microscopy is reviewed, and the argument is made that redesigning smear microscopy as a patient-centered service offers the only realistic and widely available strategy to advance TB diagnostic care towards the initial End TB Strategy goals laid out for 2025. Finally, the successful adoption of same-day sputum smear microscopy as a model for patient-centered TB care is suggested to be synergistic with and to form part of the scale-up of new TB diagnostic tools.https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-017-0947-9Please see related article:  The last decade has produced major advances in diagnostic testing for active tuberculosis (TB), including the approval of several novel diagnostic assays by the World Health Organization (WHO) following rigorous evidence-based reviews , 2. UnfoBMC Medicine on October 27, 2017, Murphy et al. [Two recent articles provide welcome data to advance this agenda. In an article published in y et al.  reportedLancet Global Health, Datta et al. [In another article published in the August 2017 issue of a et al.  describeThe above studies, incorporating data from a wide range of low- and middle-income countries, supplement substantial prior evidence showing that collecting an early morning specimen for acid-fast bacilli smear microscopy after an initial spot specimen adds very little diagnostic yield beyond that provided by collecting a second spot specimen on the day of presentation . NeverthTaken together, these data highlight same-day smear microscopy as an immediately scalable approach to diagnostic evaluation for TB that links patient-centeredness and quality of care through enhanced efficiency and reduced cost . Both as


E. coli inhabit the large intestines of humans and animals and are important in maintaining normal intestinal homeostasis. There are also many groups of disease-causing E. coli, including diarrheagenic and extra-intestinal pathogenic E. coli (ExPEC). E. coli strains have been identified primarily based on their O- and H-antigens, defining the E. coli serotype. There are approximately 188 somatic O-antigens, 74 capsular K-antigens, 53 flagellar H-antigens, and greater than 60 fimbrial F-antigens in E. coli identified based on antigens that produce an immune response in animals. This research topic consists of articles based on the subject of an international workshop on “Emerging approaches for typing, detection and characterization of Escherichia coli” held at the Pennsylvania State University in 2015. This workshop brought together well-known scientists from throughout the world to provide a forum for the exchange of ideas with regard to examining the current serotype classification and nomenclature for E. coli, emerging pathotypes, and new technologies and whole genome sequencing (WGS) for detection, characterization, and outbreak investigation. Scientists who presented papers at the conference were affiliated with public health laboratories, regulatory agencies, academic institutions, and industry groups from the U.S., United Kingdom, France, Italy, Canada, Denmark, Germany, and Japan, as well as industry groups working on technologies for characterization, detection, identification, and subtyping E. coli. This workshop provided a forum to discuss different concepts and practices for typing E. coli based on O- and H-antigens and for characterization of pathotypes. Furthermore, there were discussions on the progress made in the area of WGS as a tool for E. coli typing, subtyping, characterization, diagnostics, and outbreak investigation, as well as on the evolution and emergence of highly pathogenic strains . Phenotypic methods, such as serotyping for O- and H-antigen determination, biotyping based on biochemical characteristics, or bacteriophage typing have been used for differentiating and characterizing E. coli for many years; however, these methods are labor intensive, time consuming, and not always accurate. Recent advances in molecular techniques and DNA sequencing technologies have led to the development of typing and subtyping methods for E. coli that are more accurate and have better discriminatory power than phenotypic typing methods. WGS of E. coli is allowing more rapid and accurate identification of pathogenic strains and is replacing pulsed-field gel electrophoresis (PFGE), a gold standard for investigating food-borne disease outbreaks , including the use of various chromogenic agars, enzyme immunoassays, and qPCR. Their group at the Provincial Laboratory of Public Health in Canada have also utilized WGS, single nucleotide polymorphism (SNP), and k-mer analysis for epidemiological investigations, and they compared these methods to pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis. In agreement with other studies, they found that the sequence-based methods offer higher resolution . They found that most isolates for which the O-group could not be identified by serotyping could be O-typed using WGS data. Furthermore, WGS data provided more accurate stx-subtyping results compared to PCR. Thus, WGS provided more reliable results for strain identification and characterization compared to traditional serotyping and PCR, enabling a higher level of strain discrimination and the ability to predict the pathogenic potential.Shigella species and E. coli. Shigella species and enteroinvasive E. coli (EIEC) are very similar genetically, and it has been proposed that Shigella and EIEC be classified as a single pathovar of E. coli. The work of Pettengill et al. based on WGS data and phylogenetic analyses of EIEC and Shigella also did not support distinct genera designations for these organisms. They suggested that Shigella be classified as EIEC, and common O-antigen designations should be used. They also identified a panel of 404 SNP markers that could be used to discriminate among different phylogenetic clades.WGS data on enteric pathogens is shedding light into the relationship between E. coli. The sequences of the O-antigen gene clusters of 6 STEC O-groups that were found to be non-typeable using a previously reported E. coli O-genotyping PCR system were published by Iguchi et al. They then developed specific PCR assays to identify these novel O-groups that they designated as OgN1, OgN8, OgN9, OgN10, OgN12, and OgN31. Therefore, many O-groups that are not able to be identified by traditional serotyping or other methods may represent new E. coli O-groups and will require new O-group designations. For distinguishing the H1 and H12 H-types of E. coli that have 97.5% identity in their fliCH1 and fliCH12 genes, Beutin et al. analyzed the sequences of E. coli H1 and H12 strains and developed a two-step real-time PCR detection procedure where the first step PCR assay would detect both H1 and H12 flagellar types followed by second step real-time PCR that discriminated H1 or H12. These technologies will ultimately lead to further clarification E. coli O- and H-genotypes and in their phylogenetic relationships.Targeted sequencing in combination with PCR has furthered the characterization and typing of Delannoy et al. tested beef enrichments for the presence of specific STEC genetic markers and found that stx, eae, espK, and espV, in combination with CRISPRO26:H11 served as a suitable set of markers for screening for STEC of concern in beef enrichments, thus lowering the number of samples that required additional testing. A High Resolution Virulence Allelic Profiling (HReVAP) approach to determine allelic variants in genes carried on STEC pathogenicity islands  was developed by Michelacci et al. Following cluster analysis of the allelic forms of 91 virulence genes, representing allelic signatures based on HReVAP analyses, they investigated the phylogeny of STEC and identified subpopulations within groups. The approach provided evidence for co-evolution of LEE and OI-122, likely acquired through a single event, and it could be used a tool for studying the evolution of STEC mobile genetic elements. Knowledge of allelic signatures in STEC isolates and the presence of specific genes associated with strains that cause severe illness (hemorrhagic colitis and hemolytic uremic syndrome) improves the ability to test for and identify STEC strains of greater clinical significance.In an effort to develop more accurate and specific methods for STEC detection, Baranzoni et al. characterized STEC strains in pigs and found that strains that carried stx2e (81%) were the more predominant STEC, followed by strains that carried stx1a (14%), stx2d (3%), and stx1c (1%) as determined by PCR and an E. coli Identification (ECID) Array. The swine isolates also carried other virulence genes such as iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The work presented new insights into the virulence genes associated with porcine isolates that may potentially cause human illness. STEC prevalence throughout the pork production chain in Argentina was studied by Colello et al. by determining the O-groups and virulence gene content of strains collected from farms, during slaughter and processing, and at retail markets. The prevalence and characterization of STEC strains throughout the system reflected vertical transmission of the strains, emphasizing the importance of integrated STEC control systems from farm to table.Knowledge of the virulence genes of food-borne pathogens carried by animals provides information to assess their genetic diversity and zoonotic potential. E. coli in dairy cattle farms in Egypt was studied by Braun et al. using DNA microarray-based assays that detect resistance genes, and the isolated strains were also serotyped using a SeroGenoTyping array. The study showed a notable prevalence of ESBL-producing strains, and carbapenemase genes (blaOXA-48 and blaOXA-181) (encode enzymes in the beta-lactamase family) were also detected in isolates resistant to imipenem and meropenem. The high prevalence of antibiotic resistant E. coli strains in cattle farms in Egypt points to the importance of increased surveillance efforts in developing countries. Another array-based approach for STEC characterization was reviewed by Carter et al. The “suspension array” technology, which can potentially analyze up to 500 targets in one reaction, was used in both multiplex PCR and immunoassay microbead-based formats to detect E. coli serogroup-specific markers, as well as virulence markers. In addition to virulence- and serotype-specific genes, high-throughput array-based systems can potentially detect antibiotic resistance determinants, as well.Overuse and misuse of antibiotics in humans and animals has led to an increase in antibiotic resistant bacteria, which is a major public health concern worldwide. The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Luedtke and Bosilevac). Each molecular method had strengths and weaknesses related to the detection and enumeration rate and dynamic range for enumeration. ExPEC cause the majority of urinary tract infections and are associated with many other types of extraintestinal infections. Food, particularly poultry, can carry ExPEC, particularly antibiotic-resistant strains that cause human illness  in ground chicken meat, purge, and chicken meat surfaces. Using non-thermal processing technologies such as high pressure processing, gamma irradiation, and ultraviolet light (UV-C) they showed significant reduction in the levels of UPEC strains in poultry meat; therefore, these treatments may be useful for the production of safer food products, particularly for at-risk consumers.Cattle are an important reservoir of STEC, and STEC that cause serious human illness can be found in higher rates from veal calves compared to beef cattle. Methods for determining the prevalence of STEC in veal hides and carcasses, as well as for enumeration of the STEC were evaluated. The methods included a molecular-based most probable number method (MPN), a qPCR assay, and a digital PCR assay ( Manges, . SommersE. coli. WGS in particular is providing a powerful and expanding range of information to identify targets for development of improved intervention strategies and is being implemented for source tracking and as part of routine surveillance systems (Bergholz et al., E. coli and ultimately provide better resources for improving public health.In summary, many new technologies have become available to enhance the ability to detect, identify, and characterize All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Cancer gives rise to an enormous number of deaths worldwide nowadays. Therefore, it is in urgent need to develop new therapies, among which combined therapies including photothermal therapy (PTT) and chemotherapy (CHT) using polymer-based nanocarriers have attracted enormous interest due to the significantly enhanced efficacy and great progress has been made so far. The preparation of such nanocarriers is a comprehensive task involving the cooperation of nanomaterial science and biomedicine science. In this review, we try to introduce and analyze the structure, preparation and synergistic therapeutic effect of various polymer-based nanocarriers composed of anti-tumor drugs, nano-sized photothermal materials and other possible parts. Our effort may bring benefit to future exploration and potential applications of similar nanocarriers. Cancer, a torturing disease that threats human health, accounts for an enormous number of deaths worldwide nowadays, constituting a huge burden on the societies of both more and less developed countries . Worse sAlthough conventional chemotherapy (CHT) performed with small-molecule drugs plays a key role in the combat against cancer, it still shows some limitations, e.g., severe side effects, lack of selectivity , poor water-solubility (hampering distribution in bloodstream and leading to low bioavailability) and multidrug resistance of tumor cells (decreasing therapeutic efficacy) ,17,18,19With the ability to avoid the above limitations, polymer-based nanocarriers, which are nanoparticles capable of loading and delivering small-molecule agents, have gained increasing interest and became a focus of research . HoweverPhotothermal therapy (PTT), a treatment that external near-infrared (NIR) light is converted to heat by photoabsorbers  to destruct adjacent cells or tissues by increasing the temperature, has attracted increasing attention in anti-cancer research ,25. HoweIn consideration of the strengths and weaknesses of the both therapies above, PTT and CHT can be complementary to each other . Namely,The integration and co-delivery of both drugs and photoabsorbers with double-cargo nanocarriers are feasible: (1) nanocarriers have plenty space to accommodate both agents ,33,34; , docetaxel (DTX) or paclitaxel (PTX) and sometimes cisplatin . Other u3O4, metal chalcogenide or dichalcogenide, indocyanine green (ICG)  and PEG-4000. The loading of camptothecin (CPT) to PEG-MoOx3− HNSs was implemented by stirring in the dark. One other interesting example is a gold nanostars-based nanocarrier prepared by coating the frame (gold nanostars) with several layers of coats, including two mitochondria-targeting peptides and hyaluronic acid (HA) [Exceptional examples still exist, though. Not all frame-coat structured nanocarriers are prepared following the three steps above. As shown in −x HNSs) . PEG-MoOcid (HA) .3O4) to the tumor [There are two common ways to increase active accumulation of nanocarriers: (1) addition of targeting ligands and (2) application of external magnetic field. Targeting ligands may bind to specific sites on the surface of cancer cells, promoting nanocarriers entrance into tumor tissues . Occasiohe tumor . Both wa3O4 nanoparticles coated by Prussian blue (PB) in another report [It is also important to prevent premature leak of drug from nanocarriers in physiological or pathological environment before they reach target tumors . If thesr report ; but we Photothermal therapy (PTT), a treatment that external near-infrared (NIR) light is converted to heat by photothermal materials to destruct adjacent cells or tissues by increasing the temperature, has attracted increasing attention in anti-cancer research ,25. NIR In principle, the synergistic effect is achieved by simultaneously affecting multiple pathways of the same disease with various therapies . PTT andMulti-drug resistance (MDR), the leading reason to tumor relapse, is mainly related to the overexpression of ATP-binding cassette (ABC) transporters, e.g., P-glycoprotein (P-gp), on cell membrane, which facilitate the efflux of drugs from tumor cells . CorrespLight-controlled drug release is a distinctive feature of the double-cargo nanocarriers. For accurate release of drug within tumors, stimuli-responsive drug delivery systems are developed and prevalent , which aHow to clarify the synergistic effect of CHT and PTT by experiments? According to the analysis of all papers we retrieved, we found a fact: the more experiments, the more persuasive. Generally, both in vitro and in vivo experiments are necessary. A paper on nanosized drug delivery system without in vivo experiments is not intact and less persuasive. Consequently, papers including detailed and comprehensive experiments containing both in vitro and in vivo ones tend to be published in high IF journals. Among these experiments, we focused on in vitro anti-tumor effect assays  and in vivo anti-tumor effect assays . Both assays offer the most convincing data that describe the synergistic anti-tumor effect. If the combined therapy of PTT + CHT efficiently exerts more anti-tumor effect than PTT alone or CHT alone does, the conclusion is tenable that the synergistic effect does exist.All papers listed in Several examples are as follows. As shown in 1-HA, PCH) nanoparticles for loading DOX and ICG [Another example illustrated the preparation of the arylboronic ester and cholesterol modified hyaluronicacid  is calculated. The experiment directly and intuitively shows the therapeutic effects. Additionally, the biodistribution is necessary to indicate the accumulation of nanocarriers within tumors. Other experiments, including in vivo photothermal effects, bodyweight, H&E staining, are needed to describe the health status of mice from different perspective. The lower RTV, the better therapeutic effects. Several examples are listed below.2 in just 8 min. (2) in vivo tumor inhibition experiment where tumor volumes were recorded, RTV were calculated and 4T1 tumor-xenografted mice coupled with their corresponding tumors at the 20th day were photographed  The H&E staining of tumor tissues  in mice livers at the 7th day after the first injection, significantly lower than other groups [Many nanocarriers with NIR radiation did not only almost eradicate in situ solid tumors but also managed to suppress recurrence during the period of treatment and observation ,106,109.Gene therapy (GNT) has also been introduced to construct triple therapy. combinational therapy: triple therapies based on GNT + CHT + PTT have been explored. A multifunctional aptamer AS1411, which is a guanosine-rich hairpin-like single-stranded DNA, may load up DOX via intercalation and bind to nucleolin that is expressed on the surface of or within the cytoplasm of cancer cells ,123. Bra® is recorded  but not yet completed. In our opinion, there are three main reasons to the phenomenon above. First, the entire process of the clinical trial is long (about 10–15 years) and expensive (about $1 billion) [While it is true that any polymer-based therapies containing nanoparticles are available in market or progressing in clinical trials ,125, it billion) . Third, 3O4, etc., need to be further clarified [2O3 might be the desired candidates for better therapeutic effects [Although researches on combined therapy of PTT and CHT through polymer-based composite nanocarriers have much progressed, there are still many hurdles that hinder clinic applications and challenge scientists to future studies. The hurdles include but not limited to, the following ones. Firstly, the barriers to extravasation from vasculature to tumor tissue should be further overcome for better accumulation. For example, the pea-like nanocomposites designed according to hydrokinetics moves faster in blood and have easier access to tumor tissues . A liposlarified . Thirdly effects . Fourthl effects . For exa effects . Sixthly effects . Some te effects ,128. How effects . In summFor ingredients, we classified various ingredients to three-plus-two categories. We suggest that DOX and a targeting ligand  should be employed to cooperate with a new photoabsorber or a new polymer for the construction of a novel nanocarriers.For micelles and/or liposomes, we anticipate that brand-new polymers are developed to prepare novel nanocarriers. According to our analysis for papers cited in this review, multi-stimuli-responsive polymers are welcome. However, the possible disintegration in vivo has severely compromised the potential applications of micelles and/or liposomes.For frame-coat structured nanocarriers, carbon-based and silica-based frames are most popular. Serving both as the frame and the photoabsorber, PPy is also favorite to many research teams. We suggest that pure carbon-based materials  and PPy should be used to construct novel nanocarriers that delivers new anti-tumor drugs or new polymers (new coats). In our opinion, silica-based frames  are also good choices. We also regard PPy as a bioavailable material which could facilitate the modification of new non-photothermal frames for the fabrication of novel nanocarriers, because the polymerization of pyrrole is controllable and the growth of PPy may take place on the outer or inner surface of the frame ,71,72. WCompared with individual CHT or PTT per se, combinatory therapy of CHT and PTT will greatly enhance anti-tumor effects. The cooperation of both therapies may lead to synergistic effect by inhibiting (or even eradicating) solid tumors, impeding recurrence or metastasis of MDR tumors and improving prognosis. The key factor of a successful synergistic therapy is to design a well-designed, stable, bioavailable, targeting and stimuli-responsive nanocarrier with satisfactory performance. In this review, we listed more than hundred nanocarriers with various strengths and weaknesses. We classify them into two categories and analyzed their structure, preparation and effects. Furthermore, we summarized common problems that may prevent potential future in vivo studies and clinic applications.In our opinion, nanocarriers with better stability , satisfactory safety  and precise controllability  should be more popular in future. The detailed mechanism of the in vivo activities of nanocarriers and the distinctive interactions between nanocarriers and tumor tissues need to be further clarified in the future. The first FDA-approved combined nanotherapy involving PTT and CHT is also expectable.


Ocular images play an essential role in ophthalmological diagnoses. Having an imbalanced dataset is an inevitable issue in automated ocular diseases diagnosis; the scarcity of positive samples always tends to result in the misdiagnosis of severe patients during the classification task. Exploring an effective computer-aided diagnostic method to deal with imbalanced ophthalmological dataset is crucial.In this paper, we develop an effective cost-sensitive deep residual convolutional neural network (CS-ResCNN) classifier to diagnose ophthalmic diseases using retro-illumination images. First, the regions of interest  are automatically identified via twice-applied Canny detection and Hough transformation. Then, the localized zones are fed into the CS-ResCNN to extract high-level features for subsequent use in automatic diagnosis. Second, the impacts of cost factors on the CS-ResCNN are further analyzed using a grid-search procedure to verify that our proposed system is robust and efficient.Qualitative analyses and quantitative experimental results demonstrate that our proposed method outperforms other conventional approaches and offers exceptional mean accuracy (92.24%), specificity (93.19%), sensitivity (89.66%) and AUC (97.11%) results. Moreover, the sensitivity of the CS-ResCNN is enhanced by over 13.6% compared to the native CNN method.Our study provides a practical strategy for addressing imbalanced ophthalmological datasets and has the potential to be applied to other medical images. The developed and deployed CS-ResCNN could serve as computer-aided diagnosis software for ophthalmologists in clinical application.The online version of this article (10.1186/s12938-017-0420-1) contains supplementary material, which is available to authorized users. Eye diseases present great challenges and are serious threats to human health and quality of life . OphthalRetro-illumination images are an inevitable and typical imbalanced dataset in the clinical diagnosis of eye diseases , 12, 13 Generally, two types of approaches, namely, data leveling –20 and aRetro-illumination images were obtained from the Childhood Cataract Program of the Chinese Ministry of Health (CCPMOH)  of the ZTP, FP, TN and FN represent the numbers of true positives, false positives, true negatives and false negatives, respectively.Quantitative indicators were employed to assess the performance of our proposed CS-ResCNN compared with four conventional features, two classifiers and three data-level methods. The four conventional evaluation indicators  were calculated as follows.F1-measure  and false positive rate (1-specificity); the PR curve depicts the precision and recall. The larger AUC is, the better the classification performance is. We adopted fivefold cross-validation (CV)  to obtaiAs shown in Fig. PCO occurs in the lens area, accompanied by strong noise from nearby regions such as the iris and sclera. The boundary of the crystalline lens is an approximately circle in the original retro-illumination images. This characteristic provides a basis for crystalline lens detection. Canny detection and Hough transformation are very suitable for this kind circle detection. We employed two consecutive procedures, Canny detection and Hough transformation , 37, to After obtaining the lens ROI, we randomly selected four-fifths of the cropped images to form a training dataset; the remaining fifth functioned as the testing dataset. By adjusting the cost factor, we trained multiple classifiers to find a suitable cost factor  class in this study, we discriminatively consider the cost of misclassifying different classes and assign a large cost factor to misclassification of the minority class. Therefore, this technology can simultaneously address the imbalanced dataset problem and minimize the false-negative rates.m imbalanced samples to form a set of data sets m, w, n and k stand for the size of mini-batch, the trainable parameters, the number of inputs neurons of the softmax classification function and the number of classes, respectively. The I{a true statement} = 1 and I{a false statement} = 0), and Cmin is cost of misclassifying minority and severe PCO patients. By seeking the optimal Cmin, we can train the final learning model to have a higher discriminative capability for patients with severe PCO. Furthermore, a weight decay term J(w). To obtain the optimal parameters of the CS-ResCNN in this process, we compute the derivative of the cross-entropy function J(w) as shown in Eq. Specifically, we randomly selected atch-GD)  is adoptmentclasspt{minimaJ(w), we can achieve the most appropriate parameter weight Moreover, the parameters of all the previous layers can be achieved using the chain rule of the back-propagation (BP) method. By optimizing the cross-entropy function In this study, the CS-ResCNN was implemented using the Caffe toolbox  and traiTwice-applied Canny detections and Hough transformations , 37 wereBp and Bgt represent the prediction and ground truth bounding box of crystalline lens, respectively. Furthermore, the prevalent intersection-over-union (IoU)  indicatoHierarchical visualization technology , 50, 51 We further applied the t-SNE method to investigate whether the extracted high-level features were discriminative. High-level features were mapped into two-dimensional space to determine their discrimination ability. Visualized maps of the high-level features extracted using four conventional methods  and two deep learning methods (CS-ResCNN and native ResCNN) are displayed separately in Fig. In addition, we explored the effectiveness of another cost-sensitive method, threshold-moving method , 53, whiCmin, respectively; a correct classification is set to zero , we obtained a total of 18 comparative experiment results. We calculated the accuracy (ACC), sensitivity (SEN) and specificity (SPC) indicators for the results, which included 16 sets from conventional methods  and perform ophthalmic disease diagnosis. As implemented, the software can predict multiple images at a time. We hope that our work will help to provide high-quality medical care with personalized treatment recommendations for patients in less-developed areas where advanced medical devices and well-trained doctors are scarce. After a period of clinical application, we are able to upgrade the model to further enhance its accuracy and reliability with large amounts of accumulated datasets. This process takes only half an hour using four GPUs.https://github.com/Ophthalmology-CAD/retro-illumination-images.The source code of the CS-ResCNN for retro-illumination images is available from Github: http://www.cc-cruiser.com:5008/retro_illumination_images_prediction.The website of the computer-aided diagnosis software is available at In this paper, we proposed a feasible and automatic approach based on our CS-ResCNN model to effectively address the problem of misclassifications resulting from imbalanced ophthalmic images datasets. Our method demonstrates high performance and robustness within an adaptive cost factor range. Qualitative analyses of the visualized results illustrate that the features extracted from the CS-ResCNN are meaningful and discriminative, and quantitative assessments indicate that the CS-ResCNN model not only maintains an acceptable SPC range but also significantly boosts the ACC, SEN, F1-measure and G-mean indicators. The results of abundant experimental comparisons revealed that our proposed CS-ResCNN method outperforms both other conventional features and data-level methods  as well as the native CNN approach.In the future, we will explore and compare additional potential algorithms such as U-Net or Faster R-CNN for the segmentation and grading of the ophthalmic images. Then, we will investigate how to integrate multi-source images and multiple deep learning models to further enhance the performance of the CS-ResCNN method. Moreover, our cost-sensitive pattern can be applied and serve as an important reference for other imbalanced medical classification studies while smoothing the path for adopting artificial intelligence techniques in clinical applications.


Cohesin complexes have been extensively studied for their roles in sister chromatid cohesion during cell division, and in addition, regulate transcription through multiple mechanisms. Together with Nipped-B, a cohesin-loading factor that facilitates enhancer-promoter interactions, cohesins bind many activated enhancers but seem to preferentially associate with a subset of active genes linked to growth control and development . This isDrosophila homolog of TAR DNA-binding protein , which harbors nuclear localization and export signals , two RNA recognition motifs (RRMs), and a C terminus low complexity, prion-like domain [TBPH is the e domain .acrv1 gene by binding to TGTGTG sequences within its promoter. Deletion of RRM1 or disabling RNA binding compromise TDP-43’s repressor function, suggesting that an RNA intermediate may be involved in its role as a transcriptional repressor [Multiple lines of evidence led the authors to hypothesize that RNA-binding proteins may help define the repertoire of genes bound by cohesin and Nipped-B complexes. First, TBPH/TDP-43 has been shown to bind UG repeats within its RNA targets –8, whileepressor . Third, epressor . Lark waepressor .Drosophila cells and in wing epithelia, suggesting that these binding patterns are also present in vivo, in a developmental context. Next, the authors proceeded to decipher the mechanistic interactions between cohesion/Nipped-B and RNA-binding proteins using loss of function approaches. Depletion of TBPH by RNAi indicates that this RNA-binding protein facilitates the occupancy by cohesin and Nipped-B of most regulatory promoters, enhancers, and PREs with which they normally associate. In contrast, Lark appears to modify cohesin and Nipped-B binding sites differentially, depending on whether the sequences are contained within promoters, enhancers, or PREs. In the future, it will be interesting to determine what other molecular players may be mediating the complex effects of Lark on cohesin and Nipped-B and what the physiological consequences are of these seemingly differential interactions.To test this intriguing hypothesis, Swain et al.  used a CIn keeping with these complexities, RNAi depletion studies indicate that Nipped-B also facilitates the binding of TBPH and Lark to genes and their regulatory sequences. This underscores the interdependency between cohesin/Nipped-B on one hand and RNA-binding proteins on another, and highlights an intricate interplay between these DNA- and RNA-binding proteins that will be important to uncover in future studies. Co-immunoprecipitation experiments from nuclear extracts indicate that these proteins form a complex driven by protein–protein interactions, independent of the presence of DNA or RNA. Furthermore, transcription is not required to maintain the association of Nipped-B, TBPH, and Lark with chromosomes. Together with in vitro RNA–protein binding studies, these findings support a scenario where TBPH and Lark interact with nascent RNAs generated from cohesin-binding genes and help stabilize Nipped-B, which in turn loads cohesin onto chromosomes. Although more work is needed in the future to determine the precise order of assembly, perhaps using live imaging studies, the data presented by Swain et al.  providesDrosophila [What does the future hold for the interplay between cohesin/Nipped-B- and RNA-binding proteins in regulating gene expression? The report highlighted here  opens uposophila . It will


This study explored heterogeneous change patterns of South Korean older adults’ depressive symptoms by poverty status, focusing on health status and social engagement changes. We used data from four waves (2006–2012) of the Korean Longitudinal Study of Aging (KLoSA). Our sample contained 2461 poor and 1668 non-poor individuals. All were 65 years old or older at baseline. We used latent class growth analysis to identify trajectory groups’ depressive symptoms. Multinomial logistic regression was used to examine how a range of changes in health conditions and social engagement was associated with trajectories among poor and non-poor participants. Among the poor, five heterogeneous trajectories with clear patterns were identified: high-to-moderate, stable-high, slightly-increasing, steeply-increasing, and stable-low. Among non-poor, high-to-moderate, steeply-increasing, and stable-low groups were found. A decrease in health conditions was the most vulnerable subgroup’s (steeply-increasing) primary risk factor. Poor older adults who reduced participation in, or decreased contact with, social networks were likely to belong to the steeply-increasing group. Our study provides impetus for organizational and/or environmental support systems to facilitate social engagement among poor older adults. Future research should examine whether the significance of social engagement among poor elders applies in less-developed and developed countries. The influence of socio-economic disparity in later-year depressive symptoms has been well-established in both developing and industrialized countries . Guided In this new line of research, the inverse relationship between poverty and depressive symptoms is consistent: poor older adults show consistently higher levels of or increases in depressive symptoms. Existing evidence suggests older adults exhibit heterogeneous patterns of long-term change, partly because socioeconomic position (SEP) has a direct causal relationship with future status ,3 that vThe seemingly persistent effect of low income or poverty is an important concern, although not all poor older adults show consistent or increasing levels of depressive symptoms; some exhibit improving patterns or maintain low levels of depressive symptoms. To date, little is known about possible heterogeneous change patterns among poor older adults or how their change patterns may differ from non-poor peers. In this study, we examine change patterns of later-year depressive symptoms by poverty status.South Korea (hereafter Korea) provides a unique context in which to examine poverty-specific trajectories of depressive symptoms and their heterogeneity. Previous studies have found that while disparate patterns of depressive symptom trajectories exist in developed countries, such as the United States and France , elderlyThe positive relationship between low income and depressive symptoms has long been reported in cross-sectional  and longIn this study, we focused on two predictors of later-year depressive symptoms: health conditions and social engagement. A strong and consistent association between health conditions and later-year depressive symptoms is well-established. Predictors of higher symptom burdens include poor self-rated health ,8,15, chSocial engagement refers to the maintenance of social connections and productive participation in social activities . A largeThis study has two objectives. First, we attempt to identify distinctive trajectories of later-year depressive symptoms over a six-year observation period. We expect to find several subgroups with stable and unstable change patterns. Second, we examine the extent to which changes in health conditions and social engagement factors affect group trajectories of depressive symptoms. We expect deteriorating health conditions and reduced social engagement to be associated with subgroups demonstrating exacerbated depressive symptoms.To examine these questions, we explored separate models for poor and non-poor at baseline. Since no previous study has examined poverty-based group trajectories of depressive symptoms, we established no specific hypothesis. Based on socioeconomic inequality literature, however, we expect poverty to be associated with worse health conditions ,28, suchWe used data from four waves (2006–2012) of the Korean Longitudinal Study of Aging (KLoSA), a nationally-representative bi-annual study of older Korean adults. In the first wave, 10,254 (retention rate = 86.6%); community-dwelling respondents participated in the survey; 8688 in the second; 7920 (retention rate = 81.7%) in the third; and 7486 (retention rate = 80.1%) in the fourth wave. We selected the 4162 respondents aged 65 and older at baseline.To focus on low-income individuals, we calculated the income-to-needs ratio (INR), dividing disposable income by the poverty thresholds established by the Korea Ministry of Health and Welfare. The poverty thresholds are intended to represent the minimum cost of living and are estimated from data on household income and expenditure measures collected by the Korean National Bureau of Statistics (KOSTAT). The Korea Ministry of Health and Welfare provides detailed annual poverty thresholds varied by family size, age of each household member, and changes in the consumer price index.n = 33). Our final sample included 2461 individuals in the poor group and 1668 individuals in the non-poor group.We divided the sample into two groups: poor with INR of 1 or lower, and non-poor, excluding those who provided no information on depressive symptoms  . RespondHealth status: Health status was assessed for three categories: chronic physical disease, functional disability, and cognitive function. For all health conditions, we created dummy variables for change types. Chronic disease was measured by number of doctor diagnoses. Three dummy groups were created: constantly not having disease (0); constantly having disease (1); and status changed (2). Functional disability was assessed by the sum of difficulties with seven Activities of Daily Living (ADL) skills and 10 Instrumental Activities of Daily Living (IADL) skills. Dummy variables of functional disability change were: not having disability (0); constantly having disability (1); increased disability (2); and decreased disability (3). To measure cognitive function, we used the Korean version of the Mini-Mental State Examination (K-MMSE) 0–30). A score of less than 17 indicates suspected dementia; 18–23 low cognitive function; and 24 more normal cognitive function [. A scoreSocial engagement: Social engagement was measured with two indicators: social participation and contact with a close social network. Social participation was assessed by asking whether the respondent participated in seven activities: religious services, social clubs at a senior center, regular sports or cultural clubs, alumni events, village/clan events, volunteer activities, and political or civic organizations. Following previous studies , particiContact with social network was assessed as the frequency of contact on a scale of 0–4  with close friends, neighbors, or relatives. Dummy variables for contact frequency change were created: no change in contact frequency (0); increased contact frequency (1); and decreased contact frequency (3).Covariates: Demographic variables included gender ; education was a binary indicator (more than six years of education (0) or lower (1)); using INR, we categorized respondents into four groups based on the length of income status: constantly non-poor or not (0/1); constantly poor or not (0/1); exit from poverty or not (0/1) if poverty status changed from poor to non-poor over the course of the study (0/1); and entry into poverty (0/1) if poverty status changed from non-poor to poor; and marital status . Depressive symptoms and log-transformed income values at baseline were included to control for possible reverse causality.To identify heterogeneous subgroups of depressive symptom change, we used longitudinal person-centered analysis. The growth mixture model (GMM) enabled us to identify information about inter-individual differences in intra-individual change involving unobserved heterogeneity within a larger population . As a spData analyses followed three steps. First, descriptive statistical analyses were conducted. Second, we used LCGA to identify distinctive clusters of individual depression trajectories. Given well-established evidence of gender-patterned later-year depressive symptoms ,17, a con = 212, 8.61%), slightly-increasing , stable-low , stable-high , and steeply-increasing  , steeply-increasing , and stable-low .Based on the five criteria indices and better interpretation of trajectories, we selected the five-group model as the best : high-to, 3.98%) . High-top < 0.05). Regarding social factors, constant non-participation in, and decreased participation and contact with, social networks were associated with the steeply-increasing group. In sum, results from bi- and multivariate analyses indicate that, among the poor, the two high-risk subgroups were stable-high and steeply-increasing. Risk factors for stable-high included constant chronic conditions and low cognitive function; and for steeply increasing, constantly having health problems, deteriorating health conditions, constant low cognitive function, and reduced social contact and participation.We examined the extent to which change patterns in health status and social engagement were associated with depressive symptom trajectories . In bothp < 0.05). Moreover, when compared with the slightly-increasing group, constantly having functional disability  and increased disability  were significant predictors of membership in the steeply-increasing group. Constant non-participation in social activities  and reduced participation in social activities  were positively associated with being in the steeply-increasing group.We carefully examined the diverging trajectory patterns of the two risk groups, stable-high and steeply-increasing, conducting additional analyses using a different reference group (results not shown). When compared with the high-to-moderate group, those with constant chronic disease were more likely to belong in the stable-high group , increased functional limitations , and constantly low cognitive function/dementia risk , together with declines in cognitive function  were significant factors associated with the steeply-increasing group. Regarding social engagement, only decreased contact with social networks was found to be a risk factor for this subgroup.Among the non–poor , constanThis study explored heterogeneous change patterns of depressive symptoms among Korean older adults by poverty status, with a focus on changes in health status and social engagement. To the best of our knowledge, this is the first study to examine group trajectories of later-year depressive symptoms by poverty status. Our study contributes three primary findings to the literature on subgroup trajectories of later-year depressive symptoms: 1) depressive symptoms evolve differentially among poor and non-poor older adults; (2) decreasing health conditions are risk factors for both poor and non-poor elderly; and (3) social engagement is an important factor in the development of depressive symptoms trajectories among the poor and non-poor. Given the global public health challenge of later-year depression [ depressiOur first research question looked at whether different patterns of later-year depressive symptoms would emerge in poor and non-poor groups. Consistent with existing studies, we found stability and change that were divergent and poverty-based.Underlying these common patterns are important insights: poor older adults show more dynamic patterns of change. The stable-low group was more prevalent among the non-poor (83%) than the poor (37%). As expected, subgroups with persistently high depressive symptoms were identified only among the poor, confirming their relative vulnerability; however, the more dynamic trajectories observed in this group challenge accepted understandings about poverty’s effects on later-year depressive symptoms.These trends suggest that poor older adults are not necessarily on a uniformly low or worsening, or both, trajectory of depressive symptoms; rather, certain subgroups diverge over time. The experiences of the stable-high and slightly-increasing subgroups drive up the overall level of depressive symptoms. This important empirical knowledge can be used to identify the most vulnerable subgroups among the poor.Our second research question asked to what extent health conditions and social engagement factors were associated with depressive symptom trajectories. In this discussion, we focus on the two most vulnerable subgroups, stable-high and steeply-increasing. Among the poor, compared to the stable-low group those in the stable-high group were vulnerable to multiple health conditions with persistent patterns of low health . Regarding the steeply-increasing group, worsening health problems  were significant; among the non-poor, this association held. Taken together, these results corroborate our earlier findings: not only is the steeply-increasing group present in both poverty groups, but a decrease in health conditions over time is a primary risk factor regardless of poverty.The effects of social engagement factors on the poor were pronounced in the most vulnerable trajectory groups, particularly steeply-increasing. Poor older adults who either did not participate in social networks or whose participation had decreased over time were more likely to be in the steeply-increasing group; in the non-poor, similar patterns held, except that the effect of social engagement seemed to be narrower, with only decreased social network contact significant. This finding provides important insight into research and practice. For example, older adults with limited resources are not as prone to engage in meaningful social activities , perhaps because they have fewer opportunities . Social The current study provides several important implications for future research. The first implication affects the analytical approach employed to model group trajectories of depressive symptoms. In the current study, based on the strong existing evidence for gender-specific depression in old age, we modeled a conditional LCGA to identify a gender-contingent heterogeneous developmental pattern of depressive symptoms. In the current study, we aimed to explore the associations between health/social factors and trajectories of depressive symptoms, in the absence of established evidence related to the specific dynamic relations. This study’s findings provide initial evidence for associations between health/social engagement and whether trajectory groups of depressive symptoms vary by poverty status. Future research in this line should seek to determine whether health and social engagement were included as determining factors in different patterns of depressive symptom trajectories and how the dynamic varies by poverty status.Additionally, this study provides meaningful direction for future cross-national research. Among less-developed countries, the empirical evidence regarding poverty and later-year depression is informing efficient strategies to address later-year depressive symptoms, particularly since, in many, poverty eradication is a national goal . PreviouIn Korea, poor older adults have been identified as the most vulnerable subgroup, which our findings confirm dynamic patterns among health, social engagement, and depression are manifestations of this. Future empirical inquiries should explore whether the significance of social engagement we observed applies in less and more developed countries with different cultural and social-support contexts. For example, would a different society find different sub-populations among older adults vulnerable to depression ?Another promising venue for future research is a detailed examination of disparate types of social engagement. In this exploratory study, we used a single indicator; however, mounting evidence suggests that the direction and strength of social engagement effects on depressive symptoms vary by type of social activity . Future Second, data exclude older individuals residing in long-term care facilities, which introduces selection bias risk, given higher rates of depression among older individuals in hospitals or long-term care facilities . Third, Findings identifying differential patterns of later-year health and social risk and protective factors among poor and non-poor adults are an important contribution to geriatric literature, advancing knowledge regarding later-year depressive symptoms and laying the foundation for an efficient, targeted policy approach to ameliorating the persistent poverty-depression link.


Tomato yellow leaf curl virus (TYLCV) and its host Nicotiana benthamiana. For this purpose, we expressed tagged versions of each of TYLCV-encoded proteins  in planta in the presence of the virus. Using a quantitative scoring system, 728 high-confidence plant interactors were identified, and the interaction network of each viral protein was inferred; TYLCV-targeted proteins are more connected than average, and connect with other proteins through shorter paths, which would allow the virus to exert large effects with few interactions. Comparative analyses of divergence patterns between N. benthamiana and potato, a non-host Solanaceae, showed evolutionary constraints on TYLCV-targeted proteins. Our results provide a comprehensive overview of plant proteins targeted by TYLCV during the viral infection, which may contribute to uncovering the underlying molecular mechanisms of plant viral diseases and provide novel potential targets for anti-viral strategies and crop engineering. Interestingly, some of the TYLCV-interacting proteins appear to be convergently targeted by other pathogen effectors, which suggests a central role for these proteins in plant-pathogen interactions, and pinpoints them as potential targets to engineer broad-spectrum resistance to biotic stresses.Viruses reshape the intracellular environment of their hosts, largely through protein-protein interactions, to co-opt processes necessary for viral infection and interference with antiviral defences. Due to genome size constraints and the concomitant limited coding capacity of viruses, viral proteins are generally multifunctional and have evolved to target diverse host proteins. Inference of the virus-host interaction network can be instrumental for understanding how viruses manipulate the host machinery and how re-wiring of specific pathways can contribute to disease. Here, we use affinity purification and mass spectrometry analysis (AP-MS) to define the global landscape of interactions between the geminivirus  Tobacco etch virus [Viruses reshape the intracellular environment during infection, both to co-opt processes necessary for the development of the viral infection and to interfere with antiviral defenses. Studies of virus-host interactions have proven instrumental for understanding how viruses manipulate the host machinery, and have provided a wealth of insight into the biology of the host cell, including how re-wiring of specific pathways can contribute to disease. Due to genome size constraints and concomitant protein coding limitations, viral proteins have evolved to achieve multifunctionality, partly through targeting diverse host proteins. The number of interactions of a viral proteome is therefore anticipated to be substantial, as indicated by recent high-throughput proteomics analyses of virus-host protein-protein interactions in mammalian cells ,3,4,5,6 ch virus . Viruses and other pathogens have been shown to target proteins that play a central role in the modular organization of protein interaction networks ,11,12,13Geminiviruses, insect-transmitted plant viruses with circular single-stranded DNA genomes, cause some of the most economically important diseases in major vegetable and field crops worldwide, including cassava, cotton, maize, bean or tomato ,20. TherTomato yellow leaf curl virus (TYLCV) and the host Nicotiana benthamiana through in planta expression of tagged viral proteins, in the context of the viral infection, followed by AP-MS. The results obtained through this approach were used to infer a network of TYLCV-N. benthamiana interactions, comprising six viral proteins and 728 host proteins. Network-based analysis revealed that TYLCV targets highly connected host proteins, or hubs, which would potentially result in larger effects on multiple cellular functions. Remarkably, some TYLCV-interacting proteins appear to be convergently targeted by effectors from fungi and oomycetes, which suggests an essential role of these proteins in plant biotic interactions and identifies them as potential targets to engineer broad-spectrum resistance to pathogens.In this work, we investigate the global landscape of interactions between the geminivirus Open reading frames (ORFs) from TYLCV (GenBank accession number AJ489258) corresponding to Rep, C2, C3, C4, V2, and CP, were polymerase chain reaction (PCR)-amplified and cloned in pENTR-D/TOPO (Invitrogen) with or without a stop codon, and verified by Sanger sequencing. Primers used in this work are listed in N. benthamiana leaves by agroinfiltration. Samples were taken two days post-infiltration; accumulation of the GFP-fused proteins was confirmed by confocal microscopy. After grinding the plant tissue in liquid nitrogen, total proteins were extracted by adding lysis buffer  and the extracts were cleaned by filtration; extracts were incubated with GFP-Trap beads  for one hour, and beads were subsequently washed using washing buffer with detergent  three times and washing buffer without detergent twice . Immunoprecipitated proteins were run in an acrylamide gel and stained with Coomassie blue, and bands covering each lane were excised with a scalpel and subjected to mass spectrometry analysis.AP-MS was performed as described in . In brieN. benthamiana draft genome sequence v. 0.4.4 , which was obtained from the International Solanaceae Genomics Project (SOL) (https://solgenomics.net/). A decoy database was constructed with those protein sequences originated from randomized and reversed raw N. benthamiana protein sequences. The decoy hits were used to estimate the false-positive rate in the peptide-identification process. Because there was a large amount of potential protein-protein interactions (PPIs) identified from the AP-MS data, we used the MiST software (Mass spectrometry interaction STatistics) [Mass spectrometry analysis was performed at the Proteomics facility of the Shanghai Center for Plant Stress Biology. Matching raw MS data to peptide sequences was performed using Mascot software with the annotated proteins from the tistics) ,23, remoN. benthamiana PPI network was visualized using Cytoscape [Before using the MiST score pipeline, the DECOY proteins and proteins detected with more than two peptides in the control sample (expressing free GFP) were removed. The retained bait-prey pairs were assigned scores with the MiST package, ranked based on the MiST scores (removing the specificity component), and the pairs with top 10% highest MiST scores were regarded as high-confidence PPIs. The TYLCV-ytoscape . Venn diytoscape .N. benthamiana interactome data, we inferred the connectivity of TYLCV-targeted N. benthamiana proteins and the properties of the TYLCV-N. benthamiana PPI network using those of the best homologs in A. thaliana in the existing A. thaliana interactome, which was constructed by combining PPIs from the TAIR10 database (http://www.arabidopsis.org/), the predicted interactome from Matthew Geisler [http://interactome.dfci.harvard.edu/A_thaliana/). This interactome dataset covers 9300 proteins and contains about 83,000 PPIs. 621 A. thaliana proteins were identified as the best homologs of the 728 N. benthamiana proteins included in the TYLCV-N. benthamiana interaction network. 436 out of these 621 target proteins were covered in the Arabidopsis interactome. The topology properties of the PPI network were calculated using an in-house R script. 77 target proteins had more than 50 connections in the network and were thus regarded as hub proteins. Because there is no experimental  Geisler , and theN. benthamiana proteins that were targets of TYLCV had the potential to interact with other pathogen effectors [N. benthamiana proteins, Arabidopsis proteins, and other pathogens was constructed with Cytoscape [Based on the properties of their best homologs in Arabidopsis, seven ffectors . The intytoscape .N. benthamiana, gene ontology (GO) enrichment analysis was performed for the interactors of each TYLCV protein using topGO with the GO annotations included in the N. benthamiana draft genome v0.4.4 (https://solgenomics.net/). A GO term with a p-value < 0.05 was regarded as significantly enriched.To understand the functional roles of the TYLCV-targeted proteins in N. benthamiana and potato (Solanum tuberosum) proteins were established by reciprocal BLASTP searches, and only reciprocal best hits were retained. The non-synonymous substitution/synonymous substitution rate (dN/dS) between a pair of orthologues was estimated using the maximum likelihood-based program PAML [N. benthamiana proteins. Orthologous relationships between ram PAML . The subN. benthamiana protein-protein interactome, we started by cloning all six genes from TYLCV  fused to GFP at their N- or the C-terminus , leadinThe protein-protein interactions (PPIs) between the viral proteins and the co-immunoprecipiated host proteins were quantified using a modified version of a scoring system called MiST, previously applied to infer the HIV/human protein-protein interactome ,23. The N. benthamiana proteins were identified with high confidence as part of the TYLCV-N. benthamiana interactome. More specifically, 65 proteins were identified as associated with GFP-Rep, 224 with Rep-GFP, and five were common to both; 296 proteins were identified as associated with GFP-C2, 129 with C2-GFP, and 79 were common to both; 52 proteins were identified as associated with GFP-C3, 217 with C3-GFP, and five were common to both; 31 proteins were identified as associated with C4-GFP; 21 proteins were identified as associated with GFP-V2, 12 with V2-GFP, and eight were common to both; eight proteins were identified as associated with GFP-CP, 21 with CP-GFP, and four were common to both. The total number of interacting host proteins identified as high-confidence interactors for each viral protein is represented in Following this approach, a total of 728 N. benthamiana had been identified, we proceeded to (i) infer the PPI interaction network containing all viral proteins and all associated host proteins, representing direct or indirect interactions; and (ii) contextualize this network in the protein-protein interactome experimentally determined for Arabidopsis thaliana (hereafter referred to as Arabidopsis) [N. benthamiana PPI network inferred is shown in Once the subset of TYLCV-interacting proteins from idopsis) . The TYLN. benthamiana interactors in Arabidopsis, therefore constructing an orthologous network; 621 proteins from Arabidopsis could be identified as best homologues of the 728 originally identified in N. benthamiana. Of these 621 proteins, 436 were contained in the Arabidopsis interactome  and two effectors from the fungus Golovimonyces orontii (Gor), and two best homologues in N. benthamiana are targeted by three proteins from TYLCV, suggesting a crucial role in plant-pathogen interactions.Convergent targeting of Arabidopsis proteins has been shown for pathogens from three kingdoms of life  ,17. Inteproteins . Four oup-value < 0.05. Interestingly, some of the enriched GO terms are in agreement with known functions of the corresponding viral proteins, and/or consistent with their localization. For example, the subset of Rep interactors shows an over-representation of proteins under the “DNA topological change” term; the subset of V2 interactors shows an over-representation of proteins under the “protein folding” term; the subset of C2 interactors shows an over-representation of proteins under the “ubiquitin-dependent protein catabolic process”. Given that Rep is involved in viral DNA replication [In order to gain insight into the cellular processes affected by each of the viral proteins, we performed functional enrichment analyses of their identified interactors in the host, with the aim of identifying GO terms  over-represented in these subsets of proteins . All sublication , V2 localication , and C2 lication , interacN. benthamiana proteome in terms of their divergence pattern with respect to the orthologues from potato (Solanum tuberosum), another Solanaceae species that is not a host for TYLCV. Our results indicated that the TYLCV-targeted proteins identified in this work were more conserved than other proteins on average (In order to investigate the evolutionary forces shaping the plant/virus interface, we decided to analyse the degree of conservation of the viral targets. We reasoned that, if TYLCV targets proteins with a central role in the cell physiology, the identified host interacting proteins might be highly conserved in plants; a high degree of conservation of viral targets has been described in the HIV/human interaction . For thi average , indicat average .N. benthamiana, followed by AP-MS, to identify plant proteins interacting with each of the viral proteins in infected cells. This approach yielded a set of high-confidence interactors, which we used to infer a network of virus/host protein-protein interactions. The validity of our approach was indicated by several findings. First, some of the interacting partners identified had been previously found, as exemplified by alpha-importins, known interactors of geminivirus CP and C2 [Given that viruses are intracellular parasites that rely on the host cell machinery for their functions, the inference of the host-virus protein-protein interaction network is a cornerstone to gain a comprehensive understanding of how the infection takes place or, in other words, what the molecular and physiological changes required for a successful viral invasion are. In this work, we used transient expression of tagged TYLCV proteins in TYLCV-infected leaf patches in the host P and C2 ,36. SecoP and C2 ,29, suggInterestingly, analysis of the network properties showed that the proteins targeted by the virus were highly-connected proteins, or hubs A. PreferTYLCV-interacting host proteins can potentially play a role in the viral infection, and therefore may be good targets to explore in the quest for sources of resistance to the virus in crops. The fact that TYLCV targets hub and highly conserved proteins suggests that TYLCV is co-opting plant proteins with a central role in plant cell functions, most likely essential for survival of the host. In this scenario, knocking out the genes encoding TYLCV-targeted proteins may result in lethality, and analysis of their variability in different species or accessions may be a strategy with higher chances of success. Analysis of the conservation of these target proteins between TYLCV host and non-host plant species may point towards the most promising candidates, which would be worthy of investigation. N. benthamiana have been previously described as targets of effectors from pathogenic fungi and oomycetes (Strikingly, the Arabidopsis best hits of some of the TYLCV-targeted proteins in omycetes . The intOur work represents a first step towards a global view of the virus/host protein-protein interaction interface. We selected a method to detect interactions in planta, and in the context of the infection, in an attempt to maximize the biological relevance of the identified targets. However, our approach provided only a snapshot of the interaction landscape during the infection; in order to expand our current view of the molecular events underlying viral invasion and generate a comprehensive model of the viral/host protein-protein interactions, additional parameters, such as tissue specificity and temporal dynamics, must be incorporated to future studies. Technical improvements allowing for isolation of native, untagged versions of the viral proteins from infected cells coupled to careful temporal dissection of the infection will pave the way for this much needed deepening of our understanding of the viral infection of the cell.