The traditional design of effective vaccines for rapidly-evolving pathogens, such as influenza A virus, has failed to provide broad spectrum and long-lasting protection. With low cost whole genome sequencing technology and powerful computing capabilities, novel computational approaches have demonstrated the potential to facilitate the design of a universal influenza vaccine. However, few studies have integrated computational optimization in the design and discovery of new vaccines. Understanding the potential of computational vaccine design is necessary before these approaches can be implemented on a broad scale. This review summarizes some promising computational approaches under current development, including computationally optimized broadly reactive antigens with consensus sequences, phylogenetic model-based ancestral sequence reconstruction, and immunomics to compute conserved cross-reactive T-cell epitopes. Interactions between virus-host-environment determine the evolvability of the influenza population. We propose that with the development of novel technologies that allow the integration of data sources such as protein structural modeling, host antibody repertoire analysis and advanced phylodynamic modeling, computational approaches will be crucial for the development of a long-lasting universal influenza vaccine. Taken together, computational approaches are powerful and promising tools for the development of a universal influenza vaccine with durable and broad protection. In the history of fighting infectious diseases, vaccinations are amongst the most cost-effective approaches available to prevent infection. Traditional approaches to vaccine design have been successful against many pathogens. But vaccines that target rapidly-evolving and genetically-diverse disease agents have frequently failed to generate long lasting protection for human populations. This is particularly true for influenza viruses, a single-stranded, negative sense RNA virus. One of the important weapons being developed to effectively prevent influenza virus infection is a vaccine that can provide durable and broadly-reactive protection against multiple subtypes, including those that may cause potential pandemics, that is, a universal influenza vaccine . The NatRecent approaches to vaccine design have taken advantage of large-scale viral sequencing platforms, phylogenetic frameworks, protein structural modeling and systems biology to design novel broadly-reactive vaccine candidates, which have been used for influenza and other pathogens . These nIn this review, we aim to briefly summarize the currently applied approaches of seasonal and universal influenza vaccine design and their disadvantages (part 2), gather information on new or potential computational approaches and challenges (part 3), and to propose necessary resources and efforts needed for computational approaches of universal influenza vaccine candidates (part 4). We will explore the important role of computational vaccine design to improve the identification of pathogen antigens and key components for designing and evaluating a universal vaccine design. Furthermore, we will discuss the potential of incorporating interactions of virus-host-environment to develop models that allow for precise prediction for viral evolution and vaccine candidates. This review provides a framework to integrate computational advances that could help in restructuring the existing seasonal influenza vaccine design and contribute to the development of universal influenza vaccine.To prevent infections from circulating seasonal influenza viruses, the annually administered influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin) and two influenza B virus components (Victoria-like and Yamagata-like) . The vacInfluenza vaccines selected from natural influenza virus strains predominantly elicit specific antibodies against the globular head domain of the surface protein hemagglutinin (HA) for each subtype or lineage, which is only effective to protect against closely-matched antigenic variants . The HA,Seasonal vaccines offer a little or no protection to emerging zoonotic influenza viruses with pandemic potential, as many species, especially wild aquatic birds, are recognized as the natural reservoir of all subtypes of influenza A viruses and have the potential to occur spillover and infect humans directly . As withTraditional approaches have failed to produce stable and protective vaccines for hypervariable and rapidly-evolving viral pathogens, including influenza viruses ,31. The Fortunately, the growth of databases containing genome sequences sampled throughout global epidemics ,36,37, iComputational approaches to identify candidates for universal influenza vaccine design have been used with a variety of novel vaccine production strategies in development. These approaches mainly focus on the ‘unnatural immunity’  induced The field of immunoinformatics or computational immunology received major attention in 2000’s from the research and governmental funding agencies ,50. ImmuThe study of immunomes coined as a new discipline “immunomics”, where the ‘immunome’ is quoted as “the detailed map of immune reactions of a given host interacting with a foreign antigen” . Immunomhttp://www.allelefrequencies.net/).Immunomics can aid in identifying optimal B-cell and T-cell epitopes directly from the pathogen proteomes, while the literature suggested that T-cell predictions are more advanced and reliable than that of B-cell epitope predictions ,58. A woT-cell immunity plays a critical role in viral clearance thereby reduction in disease severity. Particularly, memory CD4+ T-cells can provide substantial protection against influenza infection through direct effector mechanisms as well as indirect regulatory and helper functions ,67,68. IA major challenge in the design of epitope-based vaccines is to focus immune response onto multiple well-conserved epitopes in order to elicit broad protective/neutralizing immune responses. Epitope grafting or scaffolding, has been proposed as a solution for epitope-based vaccine design. In this method, minimal epitopes that are highly conserved in pathogen are grafted onto an appropriate heterologous-protein scaffold. Approaches for scaffold selection and design include single algorithm-based tools like MAMMOTH or meta-servers like TM-align and consensus-based designs . Three mThere are currently three promising epitope-based universal influenza vaccines, FP-01.1, Flu-v and Multimeric-001 (M-001) are at different stages of clinical trials . Each vaFP-01.1 vaccine , comprises six different synthetic peptides (length: 35 amino acids) each conjugated to the fluorocarbon moiety C8F17(CH2)2-COOH. These epitopes were derived from the nucleoprotein (NP), matrix protein (M), and polymerase basic proteins (PB1 and PB2) and have high level conservancy across H1-H9 influenza A subtypes with wider population coverage. The phase I clinical trial  results Flu-v vaccine contains multiple highly conserved T cell epitopes derived from NP, matrix proteins (M1 and M2) from influenza A and NP from influenza B viruses and are conserved across most influenza viruses with high population coverage ,82. The Multimeric-001 (M-001), a universal influenza epitope-based vaccine, is currently at the pivotal phase III clinical trial to assess the safety and clinical efficacy as a standalone universal flu vaccine in participants with age of older than 50 for a two-year follow-up . M-001 cThe positive note on the epitope-based universal vaccine efficacy in eliciting the robust immune responses at clinical trials underpins the immunomics in advancing the current vaccine development approaches to prevent infections from remerging or emerging highly evolving influenza viruses.Antibody repertoire analysis can be used to directly incorporate host immune response for vaccine design. It combines sequence analysis with structural modeling and machine learning to predict and analyze all the antibody affinity and specificities that can be produced by an individual, which can be a valuable tool for quantitative evaluation of vaccine-induced immune responses . Though The main limitation with this approach is that linear sequence may not accurately predict the conformational variations when these antigens are put back in a complete protein context . When thConsensus sequences are usually generated by aligning and comparing multiple sequences and selecting the most common residue at each position A. These The critical step in designing COBRAs is to use multiple rounds of consensus generation. Within each phylogenetic subclade of the influenza virus subtype, the primary consensus with the most common amino acid at each position is generated for each individual outbreak group that is defined based on geographic location and collection time. The secondary consensus is generated from the primary consensus to represent the subclade. The third or fourth consensus is generated based on previous round of consensus, until the final consensus is generated and termed COBRAs . in-vitro assays and animal models. This preclinical evidence has showed broad HA inhibition antibody titers that were cross-reactive with different strains within the same subtype . . 103]. AIn detail, Ducatez and colleagues  developeThe functional and structural domains of pathogen protein can be under disparate immunologic pressures and thus have impacts on the evolutionary phylogeny  and the The integration of protein functions and structures into evolutionary models has two main challenges: (1) published viral protein structural and functional information may not be available or sufficiently resolved based on current studies; and (2) The assumption of nucleotide site independence in the model cannot capture the biological reality that some sites are linked due to shared function . Some moHigh-throughput experiments quantify the effects of all single substitutions on gene function so that evolutionary model can adequately capture the heterogeneity of selection at different sites, which may improve phylogenetic inference and ancestral sequence reconstruction ,114. TheUncovering the important ecological, immunological and environmental determinants on viral evolution is very important to make predictions of the viral emergence, fitness, transmissions and circulating potential after new substitution is introduced . EvolvabFurthermore, the complements between phylodynamic modeling and experimental testing can be integrated together to improve prediction on influenza virus evolvability. For example, experimental studies designed to assess viral evolvability ,127 demoComputational models with incorporating host-pathogen-environment can efficiently facilitate the understanding of viral evolution and the selection on critical information for vaccine design. With the challenges summarized above, extra resources and efforts are needed for developing computational vaccine design. Computational vaccine design relies on the input data quality . To be sThe unequal sampling of geographical regions is caused by global and local resource allocation . PolicieThe affiliated sequence meta-data records have been improved with samples from recent years; however, the epidemiological information, viral phenotypic characteristics and host characteristics are not sufficiently recorded. With no accurate information on geographical region, host species and migratory pathways and viral characteristics, we do lose lots of power in our model inference , not to As shown in More complicated and realistic models previously limited by computing capability can be developed with the advances of computing power . For exaMachine learning is a subset of artificial intelligence in the field of computer science, which usually uses statistical techniques and mathematical models to make computers “learn” with data without being explicitly programmed, that is, performance on a specific task progressively improves . MachineFor decades, we have been using the traditional approaches to design and develop influenza vaccines. The rapid genetic changes and antigenic drift of influenza virus populations result in short-term protection necessitating continual vaccine updates with novel viral components based on analysis of globally circulating variants. Furthermore, these vaccines do not offer any immunological protection against potential pandemic zoonotic strains, one of the lessons learned through the unprecedent appearance of swine-origin 2009 pandemic H1N1 virus. Recent decades have witnessed the technological advancements in the viral genetic sequencing and computational modeling in tracing the complexities involved in the interactions of host- pathogen-environment that produced important insights into influenza disease dynamics across biological scales. Integrating these computational and technological pipelines into the vaccine design protocols can facilitate the development of a broadly cross-reactive, evolutionarily-resistant universal influenza vaccine.


We used dissimilarities between sampling sites to quantify TBD, FBD and PBD, which were response variables in multiple-linear-regression and redundancy analysis models. We show that patterns of snake community composition based on TBD, FBD and PBD are associated with environmental heterogeneity in the Amazon. Despite positive correlations between all β-diversity measures, TBD responded to different environmental gradients compared to FBD and PBD. Our findings suggest that multi-dimensional approaches are more informative for ecological studies and conservation actions compared to a single diversity measure.Mechanisms generating and maintaining biodiversity at regional scales may be evaluated by quantifying β-diversity along environmental gradients. Differences in assemblages result in biotic complementarities and redundancies among sites, which may be quantified through multi-dimensional approaches incorporating taxonomic β-diversity (TBD), functional β-diversity (FBD) and phylogenetic β-diversity (PBD). Here we test the hypothesis that snake TBD, FBD and PBD are influenced by environmental gradients, independently of geographic distance. The gradients tested are expected to affect snake assemblages indirectly, such as clay content in the soil determining primary production and height above the nearest drainage determining prey availability, or directly, such as percentage of tree cover determining availability of resting and nesting sites, and climate (temperature and precipitation) causing physiological filtering. We sampled snakes in 21 sampling plots, each covering five km Investigating how environmental gradients influence community structure is crucial to understanding mechanisms and processes affecting biodiversity at different scales . QuantifNumerous methods have been developed to quantify β-diversity, and each method potentially gives different insights into the mechanisms driving biodiversity . Values Estimates of functional β-diversity (FBD) may change across heterogeneous landscapes because function is mediated by phenotypes potentially affecting the species performance and fitness, such as morphological, biochemical, behavioral and phenological traits . FunctioEstimates of phylogenetic β-diversity (PBD) usually incorporate information on the evolutionary history that is shared among species throughout assemblages . High vaMeasures of β-diversity based on between-site differences in TBD, FBD and PBD are more effective at identifying factors shaping community structure than measures of α-diversity, such as number of species or functional groups at particular sites . This isAlthough much of the Amazon basin appears relatively homogeneous in satellite images, ecological studies at mesoscales have shown that subtle changes along environmental gradients influence patterns of co-occurrence of frogs , understIn this study we examine the influence of environmental gradients on TBD, FBD and PBD estimates for snake assemblages in the Amazon rainforests. Investigating multiple assemblage dimensions in the same study system potentially allows accessing broad pictures of factors causing and maintaining biodiversity. Snakes are useful organisms to test for multidimensional changes in assemblages over landscapes in the Amazon because of the exceptionally-high species diversity , great sWe evaluate the effects of environmental gradients on snake assemblages and investigate spatial structuring causing levels of TBD, FBD and PBD among sites. We test the general hypothesis that patterns of community composition in taxonomic, functional and phylogenetic spaces result from current environmental heterogeneity. Additionally, we hypothesize that different β-diversity measures should respond to different environmental gradients, because they carry complementary information on snake assemblages.2 (five km separated by one km). RAPELD is a Brazilian acronym to accommodate two study scales, rapid assessments and long-term ecological research . RAPELD sampling sites will be mentioned throughout the text as modules.We sampled snakes in 21 RAPELD sampling sites , 2013, e2 fragment of non-flooded primary rainforest, located on the northern outskirts of Manaus. Eleven modules were installed along the federal highway BR-319 that connects Manaus to western Brazil. The highway was largely abandoned in the 1980s, and the modules were installed mainly to enable multi-taxa impact assessments of the effect of the road on biodiversity. Along this road, modules were placed in areas covered by primary and old-secondary rainforest, with patches of flooded forest and campinarana (forest on white sand). The southern Madeira River region contains seven modules. The Madeira River was recently dammed by two large hydroelectric power plants in the Porto Velho region, and the modules were installed along the banks of the river for monitoring the effects of flooding on biodiversity. The data used in this study were collected prior to flooding (see details below). The region is covered by primary and secondary rainforest under increasing threat of human occupation.In each module, we sampled 10 250 m long by 10 m wide plots with center lines following the altitudinal contours. The plots were distributed along two parallel five km long trails (five plots per trail) with standardized distance of one km between neighboring plots. The modules were distributed almost linearly over 880 km  from cenWe sampled snakes by nocturnal active visual search, with two observers per plot, and standard searching time of 1 h per plot. In the Amazon, nocturnal search simultaneously allows finding foraging nocturnal species, and resting diurnal species (field observation). To increase the sampling effort and accuracy in species detection, we undertook four non-consecutive surveys of each plot between 2007 and 2014. We show below that temporal variation  in assemblage structure is unlikely, because species composition did not differ among the surveys. Snakes were collected under RAN-ICMBio/IBAMA  permanent license no 13777-2/2008 in the name of Albertina Pimentel Lima (coordinator of the sampling expeditions). The license includes ethical approval of all the procedures used.Detection probabilities of snakes are usually very low , and theWe represented TBD as the scores from a Principal Coordinates Analysis (PCoA) ordination of a matrix of Forbes’ similarity index  on speciWe constructed a trait matrix using 10 continuous and discrete traits, measured or observed for adult individuals only. These were maximum total length, tail length proportional to body length, diameter of the eye proportional to head length, maximum size of offspring, discrete habitat , period of activity , foraging mode , diet , defensive behavior  and reproductive mode . All the traits used have been described as ecologically relevant for snakes  R-package . This fuWe visually controlled the robustness of the FBD estimate by constructing a functional tree  based onWe estimated PBD based on a well-supported phylogenetic hypothesis proposed by We estimated PD using the phylosor function of the picante R-package , which eWe evaluated the influence of clay content in the soil on the diversity measures because this gradient affects primary production, which influences the overall trophic network . Clay cowww.dpi.inpe.br/Ambdata).The height above the nearest drainage (HAND) algorithm estimates the depth to the water table, which represents a gradient of relative water potential, in which higher values indicate large gravitational potential and lower values may reflect soil waterlogging in the absence of drainage. Here we tested the effects of HAND  on snake diversity because vertical and horizontal distances from drainage are correlated in a micro-watershed scale , and horWe used temperature of the coldest month and precipitation of the wettest month because climate is often considered as a primary factor determining distribution of biodiversity at numerous spatial and temporal scales. Climate may limit species distribution through physiological filtering, especially in ectothermic animals such as snakes . Climate2, and therefore we expect spatial autocorrelation in the environmental data. To reduce the effects of geographic distance on the environmental gradients we calculated Euclidean distances between centroid coordinates per module, and reduced dimensionalities using the first axis from a PCoA. The coordinates were used in linear regressions given by gradient = a + b(geographic distance). Residuals from these models were used as independent variables in inferential models to quantity proportions of snake diversity that are explained by environmental variation. We used this approach because multiple-linear-regressions using raw environmental data as independent variables returned slight spatial autocorrelation in the residuals for TBD , FBD  and PBD , which was negatively significant (P < 0.05) in 20–30% of 10 geographic distance classes  + b2diversity measure = a + b + 3 + b4 + b5b. The gradients measured are given in different units, so we scaled them using the scale function in R  on the gradients can be found in the To test for the effects of gradients on the diversity measures we used multiple-linear-regression models, that were built following the general formula ion in R . The resAlternatively, we used redundancy analysis (RDA) to test the effects of environmental gradients on raw distance matrices among sampling units, separately for each diversity measure. This approach was useful to verify the robustness of the results obtained by the multiple linear regressions, considering information loss by dimensionality compression through PCoA. We constructed the models using taxonomic, functional and phylogenetic distance matrices as dependent variables, and environmental gradients as independent variables, which is equivalent to multiple-multivariate linear regression models . We usedP = 0.03), and plots and surveys interacting with each other (P = 0.01), but not with surveys alone (P = 0.26). This finding demonstrates that the patterns of spatial assemblage structure shown in this study have no bias of assemblages changing over time.We also compared the subsets of species that were found per plot in each of the four surveys (2007–2014), to control any effect of temporal variation on the diversity measures. We calculated Forbes pairwise dissimilarities among each survey on each plot, summarized the resulting matrix using PCoA (axis 1), and tested differences among surveys using ANOVA. We set a two-factors model—plot and survey—to decouple assemblage compositions between space and time. We found that differences in assemblage composition are associated with plots . The models were set up with the pairwise distance matrices used to summarize TBD, FBD and PBD.P > 0.25 in all cases). Therefore, we used axis 2 to represent TBD. PCoA axis 1 captured 100% of the variance for FBD and 30% for PBD and were used as univariate versions of the diversity measures in the inferential models.The first axis of PCoA ordination captured 59% of the original variance in the raw data used to estimate TBD, and the second axis captured 39%. However, the variance captured by axis 1 was not related to the environmental variation quantified by the gradients tested (R2) of the variance in TBD . This finding was particularly associated to the effects of clay content (P = 0.002) and tree cover (P = 0.034) on β-diversity among the sampling modules. HAND, temperature and precipitation were not related to TBD . About 37% of the variance in FBD was explained by the multiple regression , which was mainly due to the effects of HAND (P = 0.023) on dissimilarities between modules. Clay content, tree cover, temperature and precipitation were not related to FBD . About 32% of the variance in PBD was explained by the gradients , but only HAND (P = 0.034) contributed to the model. The other gradients tested were not related to PBD . All the multiple regression models returned residuals that did not differ statistically from a normal distribution . A complete summary of the multiple-regression models can be found in The multiple linear regression explained 61 percent , 43% to FBD  and 36% to PBD . In general, the results were consistent with the multiple regressions (P = 0.24).The RDA models significantly captured 32% of the constrained variance to TBD  compare to the relationships between TBD and FBD , and between FBD and PBD .All diversity measures were positively related to each other . HoweverOur data indicate that environmental gradients affect snake co-occurrence in central-southern Amazonia, which results in levels of β-diversity identified by taxonomic, functional and phylogenetic dissimilarities among modules. These findings are consistent with temperate snake assemblages, which may be structured by phylogenetic species variability and trait variability . FurtherEnvironmental gradients affecting species composition have been found in many groups of organisms in the Amazon, such as frogs , understWe found that clay content in the soil predicted snake community assembly based on taxonomic dissimilarities among modules. Associations between soil texture and patterns of vertebrate community structure are often assumed as indirectly causal, because soil texture affects many factors driving regional species occurrence, such as vegetation density  and distImantodes lentiferus) and aquatic species . Lower values of HAND are often associated with seasonal surface waterlogging  composition among plots were proportional to changes in the snake taxonomic identities (TBD). Spatial congruence between estimates of phylogenetic and TBD has been suggested as often higher compared to α-diversity measures , especiaFrom the point of view of conservation, the positive relationships between diversity measures directs the focus of biodiversity monitoring programs and reserve planning to cover higher levels of phylogenetic diversity, because this measure reflects the maintenance of ecosystem processes operating over long timescales . FD is eOur findings are unlikely to be biased by the spatial distribution of sampling units see , becauseWe used an unprecedented standardized sampling effort to show that environmental heterogeneity is associated with β-diversity in Amazonian-forest snakes. Positive correlations between β-diversity measures estimated show that PBD alone may be sufficient to investigate spatial structure in Amazonian snake assemblages under taxonomic, functional and phylogenetic perspectives. However, TBD response to different environmental gradients suggests that testing the effects of a set of environmental gradients on at least two β-diversity measures can generate deeper understanding of factors causing spatial community assembly. This finding highlights the efficiency of using multi-dimensional approaches to quantify biodiversity in community-level conservation status assessments and decision-making on natural resources management.10.7717/peerj.5628/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5628/supp-2Supplemental Information 2Click here for additional data file.


DNA damaging agents are widely used in oncology to treat both hematological and solid cancers. Some commonly used modalities include ionizing radiation, platinum drugs , cyclophosphamide, chlorambucil, and temozolomide. By modifying the chemical structure of nucleic acid, these agents induce apoptosis to subsequently eliminate cancer cells from the body. Unfortunately, the efficacy of these agents can be significantly reduced by various factors that drive drug resistance. For example, increases in drug efflux and/or increased drug metabolism can lower the intracellular concentration of an anti-cancer agent thereby reducing its ability to inflict enough DNA damage to induce apoptosis. Another mechanism involves deletions or mutations in proteins associated with several DNA repair pathways that respond to damaged DNA. For example, cancers such as Li-Fraumeni Syndrome and Lynch Syndrome ) possess mutations in p53, a key regulator in DNA damage response or DNA mismatch repair, respectively , 2. In tin vitro nucleotide incorporation assays demonstrated that several high- and low-fidelity human DNA polymerases efficiently utilize the triphosphate form of 5-NIdR when replicating an abasic site. Collectively, these biochemical and cell-based studies provide evidence for the ability of the artificial nucleoside to inhibit the replication of DNA lesions produced by temozolomide.In a series of recent publications, we evaluated whether inhibiting TLS activity could improve the efficacy of DNA damaging agents such as temozolomide , 7. The Equally important, our pre-clinical studies using a xenograft mouse model demonstrated that 5-NIdR can function as an adjunctive therapeutic agent with temozolomide to treat GBM. Results showed that tumor-bearing mice treated with temozolomide displayed a ∼4-fold delay in tumor growth compared to vehicle-treated animal. However, combining 5-NIdR with temozolomide caused complete tumor regression in >65% of mice while producing a delay in tumor growth in the remaining 35%. In addition, preliminary toxicology studies showed that repeat dosing with high doses of 5-NIdR did not produce adverse effects on hematological or major organ function. Current investigations focus on pre-clinical studies using intracranial brain cancer models to confirm that 5-NIdR effectively crosses the blood brain barrier and potentiates the efficacy of temozolomide. These efforts are especially important as approximately 4,000 children and 20,000 adults in the United States are diagnosed with a brain tumor each year. Developing compounds that increase the efficacy of temozolomide may improve treatment of this disease. Clearly this is important as brain cancers are the deadliest of all cancers, having 5-year survival rates of less than 5% and a median survival time of less than 16 months .Another important question is whether or not this approach can be extended to DNA damaging agents other than temozolomide to provide better treatments against other cancers? A relevant is ionizing radiation therapy which is used in ∼50% of all cancer patients. The primary advantage of ionizing radiation lies in the ability to attack tumors that are typically inaccessible to surgical removal. This is achieved by precisely focusing the ionizing beams at the tumor which also minimizes injury to surrounding healthy tissue. At the cellular level, ionizing radiation primarily generates double strand DNA breaks, a non-instructional DNA lesion that is similar to an abasic site. Double strand DNA breaks are processed by different repair pathways that require the activity of several DNA polymerases. However the polymerases involved in processing double strand DNA breaks are distinct from those involved in replicating abasic sites. Regardless, it will be of significant interest to evaluate if artificial nucleosides such as 5-NIdR can inhibit these polymerases to potentiate the cell-killing effects of ionizing radiation.


Drosophila olfactory circuit . Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the  Nervous systems are composed of intricately structured assemblies of neurons. Indeed, their proper function requires highly specified circuit organization, wherein neurons make precise connections with their synaptic partners. The study of neural circuit assembly has generated an ever-expanding catalog of wiring molecules, whose biological roles ensure the fidelity of neuronal connections and thus of information transmission . While sPlexins, a conserved family of single-pass transmembrane receptors, play varied roles in the development and homeostasis of diverse tissues in both vertebrates and invertebrates. Through the effort of many laboratories in the past two decades , geneticOver 600 million years old, Plexin-family receptors display high degrees of conservation across evolutionarily distant species for both extracellular and cytoplasmic domains . StructuDrosophila olfactory map, with level-independent functions in the axon fasciculation of olfactory receptor neurons (ORNs) and level-dependent tasks in ORN axon trajectory choice and subsequent glomerular targeting . These point mutations did not affect PlexB’s expression and cleavage in vivo (A bipartite GTPase-activating protein (GAP) domain resides in the cytoplasmic part of Plexins. Recent studies identified the Ras homolog Rap as its substrate for signal transduction . With ho in vivo .mut, which has two amino acid substitutions  in the second site . Indeed, the two arginine mutations abolished PlexB cleavage and increased the presence of full-length PlexB , ORN axon defasciculation was observed in almost every antennal lobe with differing severity , ORN axons arrive at the antennal lobe and fasciculate with neighboring axons, forming two discrete bundles. Over the next 6 hr, these two axon bundles circumnavigate the antennal lobe . In Plexseverity . To quanculation . Howeverculation , suggestAs shown in Notably, uncleavable PlexB appeared to exhibit better rescue than wild-type PlexB, with only one severe defasciculation case out of 47 examined . ConsideTaken together, our data suggest that PlexB-dependent axon fasciculation is mediated by full-length PlexB but not the cleaved fragments. Moreover, fasciculation appears to not require any individual cytoplasmic signaling motif but relies on the extracellular Sema domain. Thus, PlexB-dependent axon fasciculation is likely an intercellular adhesion process, in which full-length PlexB proteins bundle axons together through Sema domain-mediated molecular adhesion.plexB null background. However, plexB null mutants exhibit axon fasciculation defects that cannot be completely rescued even by wild-type PlexB, as described above  or ventromedial (VM) – and then circumnavigate the antennal lobe in the next 6 hours . Importaed above . The abuAs described previously , we quanmut) only partially weakened the phenotype . By 48hAPF, the antennal lobe has been divided into roughly 50 proto-glomeruli, where the axons and dendrites of matching ORNs and PNs interact . We prevIn line with our previous observations, PlexB overexpression caused mistargeting of VA2 ORN axons stereotypically to the DM5 glomerulus . Like thPlexB without its Sema domain failed to induce glomerular mistargeting , emphasiOverexpression of the cleaved products, either independently or together, was insufficient to drive mistargeting , suggestCollectively, PlexB-mediated synaptic partner selection engages both extracellular and cytoplasmic modules for signaling, resembling trajectory choice but not axon fasciculation. Moreover, extracellular cleavage of PlexB does not appear to be critical in synaptic partner selection.Our systematic in vivo analysis shows the divergent engagement of different PlexB structural motifs in distinct neurodevelopmental processes , arguingThe extracellular Sema domain is highly conserved in all Plexins and Semaphorins . StructuDrosophila PlexB retains some GAP activity in our triple arginine mutant through an alternative mechanism.On the other hand, the catalytic site of the RapGAP domain appears to be dispensable for all PlexB-mediated wiring processes examined. Although structural and in vitro studies have pinned down Rap as the substrate for Plexin’s GAP domains , in vivoIt has been shown that the integrity of GTPase-binding sites is crucial for PlexB-mediated axon guidance of embryonic motor neurons . Here, wPlexB locus would overcome this caveat, at the expense of losing cell type specificity in genetic manipulation, which is of more concern for a widely expressed protein like PlexB. Despite the technical limitations, the comparative analysis here allowed us to functionally characterize PlexB structural motifs individually in vivo and reveals how the task-specific, combinatorial engagement of structural motifs enables a single molecule like PlexB to accomplish multiple distinct developmental tasks in neural circuit assembly   (Or92a-rCD2 (VA2 ORNs) (UAS-mtdTomato (KG00878plexB (PlexB mutant)  , Pebbledpan-ORN) , Or92a-rA2 ORNs) , UAS-mtdtdTomato , plexBKG mutant) , as wellpUAST-attB vector , extracted by QIAprep spin miniprep kit , and verified by full-length sequencing . Constructs were then injected into vas-int.Dm;;ZH-attP-86Fb embryos , rabbit anti-DsRed , mouse anti-rat CD2 . Donkey secondary antibodies conjugated to Alexa Fluor 405/568/647  were used at 1:250.Images were acquired by a Zeiss LSM 780 laser-scanning confocal microscope , with a 40x/1.4 Plan-Apochromat oil objective . Confocal z-stacks were obtained by 1 μm intervals at the resolution of 512 × 512.For quantification of fasciculation defects, a single scorer binned antennal lobes into three categories – ‘no defasciculation’, ‘mild defasciculation’, and ‘severe defasciculation’ – while blinded to the genotypes. Antennal lobes with clear trajectories and lacking axon invasion into the lobe were binned as ‘no defasciculation’. Both ‘mild defasciculation’ and ‘severe defasciculation’ indicate axon invasion into the antennal lobe, while the ‘severe’ cases also showed the loss of trajectories.We quantified ORN axon trajectories at 24hAPF as previously described . BrieflyImages were exported as maximum projections by ZEN  in the format of TIFF. Preview  was used for image rotation and cropping. Illustrator  was used to make diagrams and assemble figures.Drosophila medium  and snap frozen in liquid nitrogen before stored at –80°C. Samples were lysed on ice in pre-cooled RIPA buffer  with protease inhibitors  and then rotated for 2 hours at 4°C. After centrifugation for 30 min at 16000 RCF  at 4°C, the supernatant was collected and kept on ice. Laemmli sample buffer  and 20 mM dithiothreitol  were added to the sample, followed by heating at 95°C for 10 min. Precision Plus Protein Kaleidoscope prestained protein standard  was used as the molecular weight marker. Electrophoresis with the NuPAGE Tris-acetate gel and PVDF membrane transfer  were performed according to the manufacturer’s protocols. We note that the PlexB protein level is extremely low in vivo, even in the context of overexpression  was used for statistical analysis and plotting. Significance among multiple groups was determined by one-way ANOVA with Tukey’s test for multiple comparisons. Significance of contingency tables was determined by Fisher's exact test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila olfactory circuit assembly" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Kristin Scott as the Reviewing Editor and K VijayRaghavan as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Takahiro Chihara (Reviewer #1); Bing Ye (Reviewer #2).Thank you for submitting your article "Functional divergence of Plexin B structural motifs in distinct steps of The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Drosophila olfactory receptor neurons, ORNs. The authors generated a series of transgenic lines expressing mutated Plexin B and performed genetic rescue experiments for axon bundle formation (fasciculation) and overexpression in ORNs for axon trajectory choice and synaptic partner selection. The authors found differential requirements of the Plexin B domains. For example, the Sema domain is required for all the steps but the GTPase-binding domains are involved in trajectory choice and synaptic partner choice but not in axon fasciculation. The study nicely builds on the authors' previous work to elucidate novel concepts. It also offers an in vivo test of ideas based on previous biochemical findings . The study is well designed and the results are convincing. The paper is clearly written and requires only minor text changes.Guajardo et al. provide in vivo evidence for the functional requirements of Plexin B functional motifs in Essential revisions:Drosophila Plexin B mutant GAPmut ? The authors should indicate the relevant references or experimental evidence or consider the possibility that activity is not abolished in the text.1) The authors find that the GAP domain is not involved in any steps of ORN assembly. Is there experimental evidence showing GAP activity is really abolished in the 2) The findings and discussion regarding the cleavage of Plexin B are intriguing. Based on the results, the uncleavable Plexin B is more effective  than wild-type Plexin B (except glomerular selection). The authors discussed the potential physiological significance of Plexin B cleavage, stating that "Plexin B cleavage is a way of tuning the Plexin B level (function)". Can authors provide any genetic evidence or references supporting this claim? More discussion here is warranted. Essential revisions:mut ? The authors should indicate the relevant references or experimental evidence or consider the possibility that activity is not abolished in the text.1) The authors find that the GAP domain is not involved in any steps of ORN assembly. Is there experimental evidence showing GAP activity is really abolished in the Drosophila Plexin B mutant GAPDrosophila Plexin B and its loss in this mutant. These three arginine residues are highly conserved across species and across Plexin sub-families . Previous studies  assayed Plexins from different species and sub-families and found that these residues, individually, are indispensable for the GAP catalytic activity. Thus, the combined triple mutant  is likely to disrupt the GAP activity. However, we cannot exclude the possibility that Drosophila Plexin B uses unconventional and currently unknown residues in catalysis. We have added discussion on this possibility in the revised manuscriptWe agree with the reviewers that we do not have direct evidence on the GAP catalytic activity of 2) The findings and discussion regarding the cleavage of Plexin B are intriguing. Based on the results, the uncleavable Plexin B is more effective  than wild-type Plexin B (except glomerular selection). The authors discussed the potential physiological significance of Plexin B cleavage, stating that "Plexin B cleavage is a way of tuning the Plexin B level (function)". Can authors provide any genetic evidence or references supporting this claim? More discussion here is warranted.The western blot in Figure 1—figure supplement 1 shows elevated level of full-length Plexin B proteins when the uncleavable Plexin B is expressed, indicating that cleavage provides a way to down-regulate full-length Plexin B. Phenotypically, disrupting cleavage intensifies trajectory shifting caused by elevating the Plexin B level (via overexpression). We have modified the text to make these conclusions clearer.


Early diagnosis of HIV infection among infants and children is critical as prompt initiation of antiretroviral therapy prevents morbidity and death. Yet despite advances in the accuracy and availability of infant HIV diagnostic testing, there are increasing challenges with making an early definitive diagnosis. These challenges relate primarily to advances in prevention of mother-to-child transmission (PMTCT) of HIV. Although PMTCT programs have proven to be highly effective in reducing infant HIV infection, infants who are HIV-infected may achieve virological suppression and loss of detectability of HIV nucleic acid prior to diagnosis because of antiretroviral drug exposure. Hence, false-negative and indeterminate HIV polymerase chain reaction (PCR) results can occur, especially among high-risk infants given multi-drug prophylactic regimens. However, the infant HIV diagnostic landscape is also complicated by the inevitable decline in the positive predictive value of early infant diagnosis (EID) assays. As PMTCT programs successfully reduce the mother-to-child transmission rate, the proportion of false-positive EID results will increase. Consequently, false-negative and false-positive HIV PCR results are increasingly likely despite highly accurate diagnostic assays. The problem is compounded by the seemingly intractable prevalence of maternal HIV within some settings, resulting in a considerable absolute burden of HIV-infected infants despite a low mother-to-child transmission rate. However, making an early definitive diagnosis of HIV is becoming increasingly difficult despite improvements in the accuracy and availability of infant diagnostic testing. Importantly, the challenges associated with early infant diagnosis (EID) relate directly to improvements in prevention of mother-to-child transmission (PMTCT) of HIV.Early diagnosis of HIV infection among infants and young children is critical as prompt initiation of antiretroviral therapy (ART) markedly reduces morbidity and mortalityAs a result of the passive transfer of maternal HIV antibodies to infants in the third trimester and the persistence of these antibodies during infancy and early childhood, HIV antibody tests used to diagnose HIV in older children and adults cannot be used for EID. Rather, detection of HIV antibodies in infants, as determined by enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests, indicates HIV exposure  but not necessarily infant infection. Hence, to diagnose HIV infection in infants, nucleic acid tests, such as HIV polymerase chain reaction (PCR) assays, are used. These tests directly detect HIV DNA, RNA, or total nucleic acid and may be qualitative or quantitative, the latter referred to as HIV viral load assays.in vitro diagnostic use are now available. These range from fully automated high-throughput real-time PCR closed systems, designed for centralized laboratories, to single-test point-of-care devices with vastly reduced analytical turnaround times. Yet despite these advances, there are increasing challenges with making an early definitive HIV diagnosis among infants and young children. In this review article, developments and successes in the field of PMTCT, including nucleic acid and antibody testing and their implication for pediatric HIV diagnosis, will be presented.Whereas the first nucleic acid tests for HIV diagnosis were beset with challenges, including suboptimal amplification of HIV subtype C and carry-over contamination, assay refinements and development of fully automated closed systems led to marked improvement in EID accuracy. An increasing number of commercial assays approved for3. This reduction in mother-to-child transmission was achieved by increasing access to maternal treatment and infant prophylaxis regimens as well as lowering the threshold for maternal ART initiation. PMTCT prophylaxis, originally recommended only around the time of childbirth, has been progressively expanded to safeguard the pregnancy and postpartum period. The World Health Organization (WHO) currently recommends lifelong ART for all HIV-infected pregnant women regardless of CD4 count or clinical stage; this is referred to as WHO PMTCT Option B+. Hence, there is a growing population of women living with HIV who are initiated on suppressive ART regimens for months, if not years, prior to delivery. This in turn has altered the epidemiology of early infant HIV infection.EID not only is essential for clinical decision making  but also provides an opportunity to measure the effectiveness of PMTCT programs by documenting transmission rates. For example, routine laboratory data from South Africa’s National Health Laboratory Service demonstrated the successful reduction of early infant infection from more than 20% in 2004 to less than 2% by 2015 among HIV-exposed infants4. Therefore, routine HIV PCR testing at 4 to 6 weeks of age has been the mainstay of EID testing as both intrauterine and intrapartum infections can be detected at a single time point which coincides with a routine immunization visit 11.Mother-to-child transmission of HIV can arise from one of three routes: transplacentally (intrauterine infection), exposure to blood/secretions at time of delivery (intrapartum infection), or via breastmilk . Importantly, the risk of infection from each transmission route is directly related to maternal viremia. Prior to the ART era, intrapartum infections were the predominant mode of transmission among formula-fed infants and accounted for up to 50% of all HIV infections among breastfed infantsn visit . However12. In order to detect intrapartum infections early, a second EID test was also included in the national infant testing guidelines. Importantly, HIV testing occurs within the context of PMTCT practices and considerable exposure to antiretroviral drugs. In addition to recommending antenatal maternal ART, which results in significant intrauterine antiretroviral exposure13, WHO PMTCT Option B+ recommends that all HIV-exposed infants receive daily antiretroviral prophylaxis for 4 to 6 weeks regardless of feeding method14. Studies suggest that infant prophylactic regimens can result in virological suppression and loss of detectability by PCR assays in HIV-infected infants18. For these reasons, guidelines from the US recommend that infants at high risk of HIV transmission undergo repeat nucleic acid testing 2 to 4 weeks after cessation of antiretroviral prophylaxis19. Out of similar concern, South Africa’s testing guidelines recommend routine HIV PCR testing at 10 weeks post-delivery, instead of 6 weeks, for detection of intrapartum infections.South Africa introduced routine birth testing in 2015 and rapidly achieved a testing coverage of 95% within the first year of implementationIn addition to impacting on the sensitivity of EID screening, antiretroviral drug exposure can complicate confirmatory testing. Guidelines recommend that infants who test HIV PCR-positive be recalled as a matter of urgency to both confirm their HIV infection status and initiate ART at the same visit. However, in some circumstances, confirmatory testing is performed after prolonged exposure to antiretroviral prophylaxis and sometimes even after ART initiation. Unsurprisingly, the confirmatory HIV test may yield an indeterminate or even a negative result even when the infant is infected. Recalling the infant for testing to resolve the discordant HIV test results is further complicated by additional reductions in virus. Hence, the longer the intervals between testing, the higher the risk of viral suppression and discordant HIV test results. Among infants with discordant results who have already initiated ART, treatment interruption represents a last resort for making a definitive diagnosis provided that close monitoring and repeat HIV testing can be ensured.20. Since 2010, the WHO has recommended ART interventions to prevent postnatal transmission of HIV and has subsequently updated guidelines to report that mothers living with HIV should breastfeed for at least 12 months and may continue breastfeeding for up to 24 months or longer whilst supported for ART adherence21. However, there are concerns about maternal ART adherence among women initiated on treatment during pregnancy, and frequent viremic episodes are reported during the postpartum period22. Hence, there is an increasing need for HIV testing services and postnatal surveillance within PMTCT programs, especially in light of the fact that postnatal transmission accounts for the majority of infant HIV infections within some high-burden settings23.Delayed HIV detection is also reported among infants exposed to postnatal antiretroviral prophylaxis during breastfeeding24. However, as weaning does not necessarily coincide with a planned clinic visit, testing coverage at this time point has been poor. To address the deficit in postnatal testing, the WHO originally recommended a screening antibody test at 9 months of age for all HIV-exposed infants. Infants testing positive required a positive HIV PCR test to confirm infection. The rationale for using antibody testing was to optimize access whilst limiting the high costs of nucleic acid tests. With improved access to and reduced costs of virological tests in recent times, PCR has replaced antibody testing at 9 months of age25.As HIV infection can arise at any time during breastfeeding and duration of breastfeeding is variable, a programmatic approach to prompt postnatal infant diagnosis is challenging. The WHO recommends repeat HIV testing for all infants 3 months (previously 6 weeks) after breastfeeding cessation - if the infant/child is less than 18 months of age at weaning then PCR testing is recommended, whereas if the child is more than 18 months of age then antibody testing is recommended26. Considering this and the delay caused by ART in detecting perinatal transmission and decreasing immunization coverage with age, South Africa has positioned a third HIV PCR test at the 6-month instead of the 9-month immunization visit27.A nationally representative sample of South African HIV-exposed infants demonstrated 4.3%  cumulative mother-to-child transmission at 18 months of age in 2012/13, and 81% of all transmissions occurred by 6 months of age29. Delayed seroreversion beyond 18 months of age using ELISA testing is also reported, and detection of maternal antibodies up until 24 months of age was associated with maternal ART use and was thought to result from the transfer from healthier mothers to their unborn infants of higher concentrations of HIV antibodies that take longer to decay30. Accordingly, a recommendation to confirm the HIV infection status of serologically positive HIV-exposed infants with a nucleic acid test between the ages of 18 and 24 months has been made to avoid a false-positive diagnosis with the consequence of lifelong ART27.In the pre-ART era, the majority of HIV-exposed uninfected infants had seroreverted  by 9 months of age, and some infants remained seropositive up until 18 months. Recent data from South Africa suggest that seroreversion rates at 9 months, using the same rapid test, decreased from 82 to 50% among HIV-exposed uninfected infants between 2005 and 201631. Increased risk of HIV acquisition among women throughout pregnancy and the postnatal period has also likely caused some HIV-exposed infants to be incorrectly classified as unexposed32. To ensure that no case of vertical transmission goes undetected, high-prevalence settings like South Africa are recommending that all children undergo HIV antibody testing at 18 months of age regardless of documented HIV exposure status27. However, universal 18-month HIV testing may pose a challenge among certain populations. For example, children testing positive at or after 18 months of age may be vaccine trial participants  in whom a positive antibody test does not necessarily indicate HIV infection. Conversely, HIV-infected infants initiated on ART who achieve prolonged virological suppression can test HIV antibody–negative because of a lack of antigenic stimulation. Hence, if caregivers fail to disclose the status of children already initiated on ART or if health-care workers are unaware that HIV-infected patients can serorevert, false-negative results may arise which could negatively impact treatment adherence.Although routine HIV serology testing at 18 months of age has been standard of care for many years, identification of HIV exposure at the 18-month immunization visit has been problematic, leading to poor testing coverage33.The reduced diagnostic sensitivity of HIV assays as a result of ART exposure is only part of the unfolding narrative of infant diagnosis. As PMTCT programs successfully reduce mother-to-child transmission, the positive predictive value of EID tests is expected to decline, resulting in an increasing proportion of false-positive results. For example, where the specificity of a test is 99.9% and the HIV prevalence in the tested population is 5%, the expected positive predictive value of the test is 98% . However, when the same test is used in a setting with a 1% prevalence, the positive predictive value decreases to 91% . To address this, the WHO has proposed using an indeterminate range, defined as viral copy equivalents too low to confer an accurate positive result, to improve the accuracy of all nucleic acid–based EID assays34. The cycle threshold refers to the number of thermal cycles required for the fluorescence signal to cross the assay’s diagnostic intensity threshold and therefore should be inversely proportional to the amount of target nucleic acid present in the specimen tested35. However, whereas during the pre-ART era HIV-infected infants usually had high-level viremia, making a diagnosis straightforward36, there has been a significant decrease in pre-treatment viral load associated with PMTCT practices37. Hence, HIV-infected infants increasingly present for testing with low-level viremia and may even be aviremic35. This in turn can result in a high number of HIV-infected infants who test indeterminate; South Africa reported around 3000 indeterminate results per annum between 2013 and 201538, and birth cohorts found that half of infants with an indeterminate result were HIV-infected39. As both HIV PCR and viral load tests are recommended for the diagnosis of HIV in infants11 and global demand for HIV viral load assays is far greater than for PCR assays, the temptation to simplify logistics and reduce costs of virological testing by using only viral load tests needs to be tempered with the likelihood of an increasing number of aviremic HIV-infected infants from exposure to antiretroviral drugs.Proposed indeterminate cutoff criteria are based on laboratory findings of poor positive predictive value and irreproducible positive results associated with higher cycle threshold values of real-time PCR assays40. As a means of reducing the indeterminacy rate without compromising accuracy, alternative verification methods have been proposed. Although data are limited, specimens that yield a reproducible HIV-detected result on repeat testing, even at high cycle threshold values, predict a positive HIV status. This has led to recommendations that all specimens that yield an EID non-negative result be repeat-tested on the same assay and that reproducible HIV-detected EID results be verified as positive whilst irreproducible HIV-detected results be verified as indeterminate41. Furthermore, infants who repeatedly test indeterminate should be managed as HIV-positive.Similar to the challenges of confirming HIV infection status among infants who screen positive, the management of infants who test indeterminate can be complicated by antiretroviral drug pressure. Repeat indeterminate and false-negative PCR results can arise, straining clinical and laboratory services and the trust of caregivers. Not surprisingly, indeterminate results are associated with a high loss to follow-up rate and delays in making a definitive diagnosis and ART initiation in those who are HIV-infected42, zero transmission is an unrealistic target for PMTCT programs. Rather, impact targets for EMTCT have been defined as a less than 5% transmission rate among breastfeeding populations (<2% among non-breastfeeding) and an HIV case rate of less than 50 infections per 100,000 total live births43. Yet although some priority countries achieve (or nearly achieve) a 5% mother-to-child transmission rate, countries with a high maternal HIV prevalence are unlikely to achieve this case rate target. South Africa, for example, has struggled to effectively address a high HIV incidence among young women, and the antenatal HIV prevalence has remained around 30% for over a decade45. Consequently, a 1% mother-to-child transmission rate in South Africa, which has about a million live births per annum, equates to a case rate five times above the elimination target. Hence, despite a low mother-to-child transmission rate, South Africa can expect a considerable absolute burden of HIV-infected infants for the near future, emphasizing the continued relevance of accurate and timely EID services.Prompted by the effectiveness of antiretroviral prophylaxis, the WHO has defined criteria for validating elimination of mother-to-child transmission (EMTCT). Although there is evidence that zero intrauterine and intrapartum infections can be achieved among women living with HIV who remain virologically suppressed throughout pregnancy and via breastmilk, further reductions in mother-to-child transmission are anticipated. This not only is expected to impact on the positive predictive value of EID tests but also may reduce the diagnostic sensitivity of nucleic acid–based assays. Surveillance during the first 24 months of life to monitor the effect of DTG on detection of HIV infection is essential to understand whether infant diagnostic guidelines require a total overhaul. For example, definitive diagnosis may not be possible until sometime after weaning. In adults, smaller HIV reservoirs are associated with a longer time to viral rebound46. This is likely to apply to infants too where the combination of maternal ART and early ART for prophylaxis and treatment is associated with rapid decline of HIV-infected cells to low or undetectable levels47. Hence, HIV-infected infants who achieve rapid and continuous viral suppression soon after birth could experience a prolonged period of time to viral rebound after weaning. Although data from the Children with HIV Early Antiretroviral Therapy (CHER) study suggest that almost all HIV-infected infants will experience virological rebound within 8 months of treatment cessation48, these findings predate WHO Option B+ PMTCT practices and may not apply to intrauterine infected infants exposed to suppressive ART soon after infection. Therefore, documenting the age at which the majority of transmissions occur, and the timing of viral rebound among infected infants suppressed due to maternal and prophylactic antiretroviral drug exposure, will be necessary for future rational guideline development.Future PMTCT developments are likely to further impact on the accuracy of EID assays. Dolutegravir (DTG), a potent antiretroviral capable of rapid viral suppression, is poised for widespread introduction into maternal ART programs. Because DTG is readily transferred to infants, both transplacentally40. Consequently, prognostic indicators for rapid disease progression in infants, other than intrauterine infection or high viral loads (which may be masked by ART), will be required. With increasing postnatal versus perinatal transmission, consideration should be given to the probability that an unknown proportion of these cases represent previously suppressed perinatal infections.The distinction between intrauterine, intrapartum, and postnatal transmission has always been fairly crude but the boundaries have been further blurred by ART exposure as evidenced by case descriptions of infants who test HIV PCR indeterminate at birth followed by negative results and subsequent viral rebound after weaning, suggesting either suppressed intrauterine infection or postnatal transmissionThe introduction of a single HIV PCR test for all HIV-exposed infants in low- and middle-income countries in the early 2000s represented significant progress for pediatric HIV outcomes. Today, EID algorithms require at least three HIV PCR tests for every exposed infant as well as testing at 18 months and after weaning. With high maternal HIV seroprevalence rates, the costs of the EID program are escalating, yielding fewer HIV-infected infants and more complex diagnostic dilemmas. In the quest for EMTCT, striving for virological suppression of all women living with HIV of childbearing potential will compound the complexities of an early definitive diagnosis of HIV. Consequently, false-negative and false-positive HIV PCR results are increasingly likely despite highly accurate diagnostic assays. The introduction of ART with even higher viral suppressive properties will require a complete rethink of efficient diagnostic algorithms, paired with more sensitive assays, for HIV diagnosis of infants and children in the future.


Cell-penetrating peptides (CPPs) are typically prone to endocytic uptake into human cells. However, they are often inefficient at escaping from endosomes, which limits their ability to deliver cargos into cells. This review highlights the efforts that our laboratory has devoted toward developing CPPs that can mediate the leakage of endosomal membranes, and consequently gain better access to the intracellular milieu. In particular, we have identified a CPP named dimeric fluorescent TAT (dfTAT) with high endosomolytic activity. We describe how we have used this reagent and its analogs to develop efficient cytosolic delivery protocols and learn about molecular and cellular parameters that control the cell permeation process. Specifically, we discuss how late endosomes represent exploitable gateways for intracellular entry. We also describe how certain features in CPPs, including guanidinium content, charge density, multimerization, chirality, and susceptibility to degradation modulate the activity that these peptidic agents take toward endosomal membranes and cytosolic egress. Cell-penetrating peptides (CPPs) are often used as delivery agents for cell-impermeable macromolecular cargos of biological or synthetic origin ,2. Many A focus of our laboratory has been to develop endosomolytic reagents and use them as models that can reveal how endosomal escape is mediated in living cells. Based on several reports in the literature, we first investigated the membrane-disrupting capabilities of virus-derived peptides . These pIn this review, we describe what we have learned about how dfTAT works and what it does on both a cellular and molecular level. Some lessons may be specific to our reagents, but others may be of general relevance. Therefore, we hope that some of these insights may be useful to researchers in the field who are interested in designing next-generation delivery agents. It should be noted that the mechanisms of cell penetration often raise ambiguities, which is a result of how complex membrane translocation processes are. We do not describe these ambiguities herein, as we focus instead on the results obtained in our hands. In this context, we apologize if the valuable work of others is not described. dfTAT is composed of two copies of the prototypical TAT peptide derived from the protein HIV-1 transactivator of transcription ,23,24,25Using the simple co-incubation method, dfTAT has been successful at delivering cargos that are diverse in size, chemical properties, and function. These cargos include transcription factors, antibodies, and metal–organic framework (MOF) nanoparticles, as shown in The polycationic nature of dfTAT is critical for its function . As descThe penetration of dfTAT, as observed by fluorescence microscopy, appears to be a binary event . In partThe process of dfTAT-mediated delivery necessitates a minimum incubation period. dfTAT must be first taken up into the endocytic pathway. The peptide must then traffic to the organelles that constitute the specific site of membrane leakage . Notably, due to the low toxicity of dfTAT, it is also possible to repeat several dfTAT deliveries on the same cell population within a short time frame . We haveA limitation of using the endosomal pathway as a route of cell entry is related to degradation. Endosomes contain various hydrolases, and both dfTAT and cargo can be subjected to degradation while transiting within the lumen of these organelles. The degradation of dfTAT by endosomal proteases can significantly impact the membrane-leakage activity of the peptide, thereby diminishing delivery efficiencies . DegradaMacropinocytosis is the dominant route by which dfTAT uptake occurs . InhibitdfTAT-mediated endosomal escape requires the late endosome-enriched lipid bis(monoacylglycero)phosphate (BMP) ,32,33. TThe uniqueness of how BMP interacts with dfTAT is further highlighted by a partitioning assay. When testing several different negatively charged phospholipid species, only BMP is capable of transferring dfTAT into an organic phase that mimics the hydrophobic environment of a lipid bilayer  27]. Th. Th27]. Although the dominant route by which dfTAT enters cells has been identified, the possibility that dfTAT enters through other pathways at less detectable levels cannot be ruled out. Since BMP is also present in lysosomes, it is possible that dfTAT leakage is occurring through this organelle as well ,33. Howe2). The peptides are dimerized via a disulfide bond and, as such, can be disassembled into the monomeric units upon exposure to a reducible environment . dfTAT efficiently penetrates the cytosolic space of cells in a dose-dependent manner (dfTAT is composed of two copies of the tetramethylrhodamine-labeled peptide fTAT (sequence: CK(ε-NH-TMR)RKKRRQRRRG-NHt manner . Howevern = 4, 5, 6, or 7 Arg residues) were synthesized with a universal N-terminal CK(TMR) scaffold. The charge density of the dfRn peptides remain relatively unchanged compared with dfTAT by truncating the length of the monomeric peptide (+3.7–4.4). To determine cytosolic penetration efficiency, the peptides were assayed at different concentrations [dfTAT has a total charge of +18 (stemming predominantly from 12 arginine residues and four lysine residues) and a charge density (charge/kDa) of +4.4. To address the effect that charge density has on cytosolic penetration, the peptides dfR8 and dfK8 were generated . Each brtrations . The ana3+ of each lysine of the scaffold. Each peptide only differed in the number of TAT copies, which led to differences in charge and guanidinium density. When used to treat cells, 1TAT was incapable of entering cells. In contrast, 2TAT penetrated the cytosolic space of cells, albeit with only a modest efficiency. Moreover, 3TAT exhibited efficient cytosolic penetration after treating cells with as little as 1 µM of peptide . In fact, a four to seven-fold decrease in cytosolic penetration and membrane lytic activity was observed for non-fluorescent variants of 2TAT and 3TAT. This suggests that the incorporation of fluorophore(s) enhances the cytosolic penetration activity of guanidinium-rich membrane lytic agents. Additionally, cell permeability is also affected by the how the guanidinium groups are displayed in a structure. For example, we have found that both R12 (a linear chain peptide of 12 arginine residues) and dfR6 are both cell-permeable. Yet, the linear representation (R12) leads to direct membrane translocation, whereas the dimerized dfR6 undergoes endocytic-mediated cellular internalization . In partl- to d-amino acid conversion of dfTAT [d-amino acid variant of dfTAT (d-dfTAT) is resistant to proteolytic degradation  remain active upon successful penetration into the cytosolic space. This poses an issue, as the membrane lytic CPP can potentially interact with anionic membranes from within the cytosolic space, yielding deleterious effects and even cytotoxic activity. In principle, such long-lasting CPPs may also interact with nucleic acids once inside the cells, contributing to defects in transcription and translation. Overall, this may therefore be a catch-22 of cell penetration: delivery reagents lose useful activity if they are not stable enough to resist the degradative environment of the lumen of endosomes but gain unwanted and undesirable activities if they persist in cells after delivery is achieved. Although the bottleneck of endosomal entrapment can be overcome, the cytosolic delivery of cargos mediated by dfTAT and dfTAT-like reagents has limitations and drawbacks. The electrostatic interactions between the polycationic CPPs and anionic BMP appear essential for endosomal escape. Yet, electrostatic interactions can take place with other anionic molecules, thereby contributing to the inhibition of cell penetration. For instance, dfTAT is ineffective at delivering polyanionic cargos, as delivery agent and cargo simply cluster and aggregate. Another consideration is peptide degradation throughout the endocytic pathway and within the cytosolic space of the cell. The degradation of peptides along the endocytic pathway leads to a decrease in endosomal escape activity, as degraded products lack the necessary arginine content to contribute toward endosomal escape. Conversely, peptides displaying protease resistance (e.g., Despite these limitations, arginine-rich peptides such as dfTAT provide immediate usefulness for many cell culture-based applications. Structure–activity relationship should continue revealing more insights on how dfTAT gains activity. Investigations into the mechanism of membrane disruption should also reveal the molecular underpinnings of membrane permeation. Together, this knowledge should facilitate the design of the next generation of CPPs. It may also provide insights on how to tune the activity of other delivery agents, including liposomes or polymeric nanoparticles, as these systems also often suffer from endosomal entrapment.


To report three cases of congenital corneal opacity where intraocular pressure (IOP) readings were high despite the use of multiple anti-glaucoma eye drops and normalized after corneal transplantation.Three Korean infants presented with bilateral dense stromal opacification which had been present since birth. IOPs measured by rebound tonometer were high despite administration of multiple anti-glaucoma medications. One eye of each patient underwent penetrating keratoplasty (PK) because corneal opacity impaired visual development. Immediately after PK, IOPs were normalized and maintained normal without medication, whereas they remained high in the contralateral unoperated eye. On histology, stromal fibrosis was observed in the removed corneal button, and molecular assays revealed increased levels of type 1 and 5 collagens.The IOP measurement using the conventional applanation-based tonometry can be inaccurate in congenital corneal opacity which is marked by corneal fibrosis. Therefore, IOP values should be interpreted with caution in these patients, and the possibility of false-positive diagnosis of glaucoma considered. Congenital corneal opacity and congenital glaucoma commonly coexist in pediatric patients with anterior segment dysgenesis. The diagnosis of glaucoma in these patients is difficult because corneal opacification obscures visualization of intraocular structures such as angle and optic disc. Moreover, the measurement of intraocular pressure (IOP) using conventional applanation-based tonometry is inaccurate in the abnormal cornea .Herein, we report three Korean pediatric patients with congenital corneal opacity presenting as dense stromal opacification. In the patients, IOP readings were high despite the use of multiple anti-glaucoma eye drops. Immediately after corneal transplantation, IOPs were normalized and maintained within a normal range in the absence of anti-glaucoma medication. Histologic and molecular analysis showed high levels of collagen deposition in the corneal stroma.The study was approved by the Institutional Review Board of Seoul National University Hospital.A 7-month-old girl was referred to our clinic for corneal opacity and glaucoma that had been present in both eyes since birth. She was born at full-term weighing 2.7 kg and received a brief intensive care unit care due to tachypnea. Otherwise, the infant was systemically healthy, and all serological test results for TORCH  embryopathy were negative. Slit lamp examination showed dense stromal opacity in the central cornea in both eyes Fig. a, b. TheA 15-day-old boy visited our clinic because of bilateral corneal opacity present at birth. He was born full-term at 3.24 kg. At initial examination, dense central corneal opacity was observed in both eyes . Results demonstrated that the mRNA levels of type I and V collagens were 19.7 and 7.5 times higher in the patient than in the control , the corneal size was normal (≤ 11 mm of diameter) and there were no symptoms of epiphora, photophobia and blepharospasm  in our patients. Moreover, administration of multiple anti-glaucoma medications was not effective in reducing IOPs. For these reasons, we suspected that the increased IOP measurements in our patients might not be due to congenital glaucoma but derived from thickened and stiffened corneas, and performed PK to clear the visual axis from dense opacity. Consequently, IOPs were immediately normalized by corneal transplantation and thereafter controlled without medication, whereas the IOPs in the unoperated eye remained high with using anti-glaucoma medication. The corneal opacity in our cases was clinically dense central stromal opacification in both eyes. Histologically, stromal fibrosis was observed (case 1), and molecular assay revealed increased levels of type 1 and 5 collagens which are the main extracellular matrix components of the corneal stroma (cases 2 and 3). Although we did not directly measure CH in these patients, it is likely that corneal fibrosis might increase corneal resistance to applanation, thereby leading to overestimation of IOPs by rebound tonometry.In summary, our cases demonstrate that the conventional IOP measurement method using transcorneal approach might be incorrect and inadequate in congenital corneal opacity where CH is altered due to corneal fibrosis. In these patients, IOP values should be interpreted with caution, and the possibility of false-positive diagnosis of glaucoma considered. Eventually, new technology for measuring IOP independently of corneal biomechanical properties is needed for accurate IOP measurement and glaucoma diagnosis.


Texture also improved baseline models . Our findings suggest that incorporating breast anatomy information could augment imaging markers of breast cancer risk with the potential to improve personalized breast cancer risk assessment.We retrospectively analyzed negative screening digital mammograms from 115 women who developed unilateral breast cancer at least one year later and 460 matched controls. Texture features were estimated in multiple breast regions defined by an anatomically-oriented polar grid, and were weighted by their position and underlying dense versus fatty tissue composition. Elastic net regression with cross-validation was performed and area under the curve (AUC) of the receiver operating characteristic (ROC) was used to evaluate ability to predict breast cancer. We also compared our anatomy-augmented features to current state-of-the-art in which parenchymal texture was assessed without considering breast anatomy and evaluated the added value of the extracted features to breast density, body-mass-index (BMI) and age as baseline predictors. Our anatomy-augmented texture features resulted in higher discriminatory capacity (AUC = 0.63 vs. AUC = 0.59) when breast anatomy was not considered ( Full-field digital mammography (FFDM), routinely used for breast cancer screening3, has demonstrated substantial potential in providing quantitative image-derivable measures which relate to breast cancer risk7. Mammographic density has been the most established such measure and has been shown to be a strong independent risk factor for breast cancer9.Breast cancer risk assessment has become increasingly important for forming tailored breast cancer screening and prevention strategies10. This research field, aiming to translate mammographic images to computational imaging phenotypes of breast cancer risk, is rapidly evolving and several computerized methodologies have been developed to quantify the properties of the breast parenchymal pattern using texture descriptors10.Going beyond breast density, recent studies also suggest that parenchymal texture, which reflects the heterogeneity of the breast parenchymal pattern, plays a complementary role in breast cancer risk assessment by capturing information that is not reflected by mammographic density alone or other established risk factorsi.e., the shape, size, and tissue structure of the specific breast analyzed. Breast anatomy may, however, be an important component in quantification of breast parenchymal patterns. For instance, texture characterization adapted to the largely variable breast morphology would allow establishing anatomical correspondences across mammograms and, therefore, generate more comparable textural measurements across subjects. In addition, and perhaps most importantly, parenchymal characteristics from different areas of the breast may contribute differently towards the risk for developing breast cancer. For example, a recent study11 showed significant associations between breast cancer and the relative spatial distribution of dense versus fatty regions within the breast, with fatty tissue in the lower quadrants and the absence of density in the retromammary space being protective against breast cancer. Moreover, the central breast area (CBA) and the upper-outer area (UOA) of the breast have been associated with a higher frequency by location of breast cancers12 and with early changes in texture due to breast cancer development13. These findings suggest that inherent breast tissue properties predisposing women to higher risk of breast cancer may not be uniformly expressed in the breast parenchyma and, therefore, allowing breast morphology and anatomy to drive texture measurements could augment their predictive capacity.Although useful in risk prediction, parenchymal texture measurements do not generally incorporate breast morphology and anatomy — The purpose of our study was to evaluate whether incorporating breast anatomy information can strengthen the associations of mammographic parenchymal texture phenotypes with breast cancer risk. Towards this end, we introduce a computational breast-anatomy-driven approach for extracting parenchymal texture features and evaluate their predictive value in a case-control study with full-field digital mammograms.i.e., ‘FOR PROCESSING’) bilateral digital mammograms. Briefly, cases (n = 116) included all women diagnosed with unilateral breast cancer between July 2012 and December 2014 (biopsy and state registry confirmed cancer diagnosis) who also had negative screening mammograms available at our institution at least one year prior to their diagnosis. One case was excluded due to the presence of bilateral breast implants. For all the remaining 115 cases, we retrieved the earliest negative screening study with raw mammograms available (average time from screening to diagnosis: 1.9 years ± 0.7) which was used for our analysis. Eligible controls were women who had a negative routine screening exam during the same period and confirmed negative one-year follow-up. Following case-control matching without replacement, controls were matched to cases on age at screening , ethnicity and screening exam date (within 1 year) at a 4:1 ratio (n = 460), yielding a total sample of 575 women. Briefly, cases and controls were randomly ordered. Then, for each case, eligible controls were identified as controls matching to cases on the specified matching criteria and four of them were randomly selected. Since we were matching without replacement, matched controls were not available to serve as matches for subsequent cases. This process was repeated until four matches were found for each cancer case.In this IRB-approved , HIPAA-compliant study under a waiver of consent, we retrospectively analyzed a case-control sample of raw (14 which provides area-based breast PD (APD) measurements, and the commercially available software package Quantra TM , which allows for APD and volumetric breast PD (VPD) assessment.For the purposes of our study, we used the mediolateral-oblique (MLO) view of the available digital mammograms as it visualizes the maximum amount of breast tissue and can potentially better define anatomically the breast quadrants. All images were acquired using Selenia Dimensions  units. Breast Imaging-Reporting and Data System (BI-RADS) density classification and body-mass index (BMI) were extracted from the archived clinical records and the radiologists’ screening reports for all women. Fully-automated quantitative measurements of breast percent density (PD) were also obtained for all images in this study, using the publicly available “Laboratory for Individualized Breast Radiodensity Assessment”  softwareTo incorporate breast anatomy in quantitative computational metrics, first several landmarks and sub-regions of the breast parenchyma were defined in each 2D FFDM image: (a) the air-breast boundary, (b) the pectoralis muscle, (c) regions of predominantly dense versus predominantly fatty tissue, (d) the nipple, and (e) the UOA and CBA Fig. .Figure 115. Specifically, LIBRA delineates the air-breast boundary using automated gray-level thresholding, and extracts the pectoralis muscle region by applying a straight-line Hough transform16. To identify dense versus fatty tissue areas, LIBRA uses adaptive fuzzy c-means clustering to partition the breast into density clusters (DCs) of similar gray-level intensity, which are then aggregated into the final dense tissue segmentation.The anatomical components (a)–(c) were identified using LIBRArP equal to the perpendicular distance from the pectoralis muscle to the nipple, the CBA was defined as that part of the breast at a radial distance less than f *rP from the nipple, and the UOA as that part of the breast lying above the nipple and further than f *rP from the nipple. The parameter f, was implemented to range within  so that CBA adequately covers the central part of the breast, defining the relative sizes of the CBA and the UOA.For landmarks (d)–(e), the breast image was first rotated, and then flipped for right breasts, so that the pectoralis muscle was vertically aligned on the left side of all images. Then, the nipple was approximated as the rightmost circular region of the breast bounded by a rectangle of maximum size 1.5 cm in both dimensions. Lastly, two breast sub-regions were identified: for D, centered on the nipple, was then overlaid on the mammographic image , including gray-level histogram, co-occurrence, run-length, and structural features, all of which have been previously used for mammographic pattern analysis and breast cancer risk assessment10. Briefly, gray-level histogram features are common first-order statistics which describe the distribution of gray-level intensity. The co-occurrence features also consider the spatial relationships of pixel intensities in specified directions and are based on the gray-level co-occurrence matrix (GLCM) which encodes the relative frequency of neighboring intensity values. Run-length features capture the coarseness of texture in specified directions by measuring strings of consecutive pixels which have the same gray-level intensity along specific linear orientations. Using the polar grid, the axes for calculating the co-occurrence and run-length features were aligned with the structure of the parenchymal tissue and ductal structures  was designed as a combination of weights due to the region’s anatomical position (S), and weights due to the underlying tissue composition (T) of each region As the final step, we generated a weight map which assigned a weight to each region; this weight map was, then, region-wise multiplied to the 34 original texture feature maps to generate a set of 34 weighted texture feature maps. Our design of the weight map was motivated by studies associating the CBA and UOA with potential specific roles in breast cancer developmentFor each weighted texture feature map we estimated the mean value of the corresponding weighted feature across all regions (labeled “TF#_mean”), and the standard deviation (labeled “TF#_std”). These constituted the breast’s texture signature: a 68-element feature vector corresponding to the average and the variation of each of the weighted texture feature maps.D, f, a, b, and c) and determining the most discriminatory subset of covariates out of the 68 available texture features. To this end, for different parameter combinations  of the receiver operating characteristic (ROC) (see Supplementary Note N3 for feature optimization details).The optimization of our anatomy-driven approach for parenchymal texture analysis and breast cancer prediction involved two inter-related tasks: automatically configuring the tunable texture analysis parameters 25.The model corresponding to maximum cross-validated AUC  was compared with a model built using the same statistical approach as described above but where textural features were generated using a current state-of-the-art algorithm based on a simpler lattice approach to sample the breast without incorporating breast anatomy23 were also evaluated in terms of their ability to augment established risk factors in breast cancer risk prediction.Finally, we assessed the potential of augmenting established risk factors by evaluating four baseline models including breast density  adjusted by BMI and age at screening, and tested the added value of incorporating our breast-anatomy-driven texture features. Baseline and augmented logistic regression models were fitted to obtain estimates of AUC, odds ratios (ORs), and statistical significance of predictor variables. For comparison, conventional lattice-based texture measurementsp = 0.05 level. The breast-anatomy-driven approach was developed in Matlab R2014b  and statistical analysis was performed in Stata 13 .All tests of statistical significance were at the standard There was a significant difference in Quantra VPD and a marginally significant difference in age between cases and controls in our sample Table .Table 2SD = 6.3 mm) and for a weight map in which dense tissue regions were assigned higher weights than fatty regions (b = 1 in weights T), and regions in the CBA were weighted more heavily than in the UOA (a = 0.8 in weights S). Each region’s location (weights S) was slightly less important than the region’s underlying tissue composition (weights T) in calculating the final weight (c = 0.4 in weights W). After excluding 17 features with low IQR, elastic net regression selected 30 of the 51 textural features for inclusion in this optimal model, including nine gray-level histogram, nine co-occurrence, eight run-length, and four structural features; among these, 14 represented mean values and 16 the variation  in texture feature maps (Supplementary Table The best model performance for the anatomy-driven features was AUC = 0.63 (95% CI [0.59 0.69]). This was achieved for a spatially dense polar grid (p = 0.021 by DeLong’s test)   (p = 0.p = 0.029 by DeLong’s test)  Table . The latt) Table .Table 3AOur findings suggest that incorporating breast anatomy information in mammographic phenotypes of parenchymal pattern could augment imaging markers of breast cancer risk with the potential to improve personalized breast cancer risk assessment. Interestingly, the configuration of the weight map in our breast-anatomy-driven approach as indicated by the optimization experiments also suggests that the textural properties of different regions in the breast may contribute differently towards breast cancer risk, with dense tissue regions and the central breast quadrant having potentially a more important role. Further, the heterogeneity in textural measurements within the breast may also be important, as more than half of the features selected as strongest covariates in our model represent the variation  of the corresponding texture feature distribution within the breast.without the incorporation of factors describing breast anatomy structure and variability23. We postulate that the improvement observed in this preliminary comparison is due to including factors that capture the wide variety of breast morphology and anatomy, found not only across the screening population but also within a single woman due to differences in positioning for FFDM. Incorporating such information, therefore, allows for also establishing anatomical correspondences across breasts of the same or different women, which in turn results in standardized imaging features and more comparable texture measurements. The observed improvement in discriminatory capacity may also be due to our polar grid, which allows for more granular texture measurements to be obtained in the retroareolar breast area where some of the most complex parenchymal tissue patterns usually appear17, and the ability to consider different contributions of the different sub-regions within the breast in the overall parenchymal texture signature. With this improvement, our anatomy-driven approach achieved a promising performance in this challenging task of breast cancer risk prediction with prior screening mammograms.In addition, our anatomy-driven approach to breast parenchymal texture analysis outperformed parenchymal texture features assessed 26, where case-control classification models considering parenchymal textural features in addition to established risk factors and breast density achieved AUC values of 0.62 up to 0.7810. Together, these findings consistently support independent associations of parenchymal texture with breast cancer and, therefore, create a strong argument for incorporating quantitative breast textural features in models estimating breast cancer risk. The improvement of breast cancer risk estimation models can have substantial clinical implications, as it would allow for more informed recommendations for supplementary breast cancer screening  and prevention tailored by individual risk profiling. Hence, the potential of parenchymal texture to leverage breast cancer risk assessment might ultimately affect the chance of early cancer detection or prevention in women categorized as being at low breast cancer risk based on conventional risk factorsOverall, adding the breast-anatomy-driven features to baseline models with established breast cancer risk factors led to a significant increase in discriminatory capacity, suggesting a promising role in augmenting breast cancer risk assessment models. Similar conclusions have been reported in related studies27, different FFDM representations, and vendors28, in our future studies we will plan to more thoroughly test the robustness of our method by incorporating multiple FFDM vendors from larger populations. In addition, larger studies will allow us to more rigorously evaluate the added discriminatory capacity of such imaging biomarkers when considering additional demographic and clinical risk factors , potentially also expanding our feature set into higher phenotypic representations, including deep learning. Finally, while 2D FFDM images were analyzed as a first step for the purposes of this proof-of-concept study, we ultimately envision extending our algorithm to volumetric texture analysis for digital breast tomosynthesis images , as tomosynthesis is increasingly being clinically implemented due to its reported improvements in sensitivity and specificity as compared to conventional 2D FFDM29. This new pseudo-3D imaging technology may, therefore, also result in superior imaging phenotypes of breast cancer risk.Important limitations of our study must also be noted. To avoid potential confounding effects of proprietary FFDM post-processing algorithms in textural measurements, our study focused only on “For Processing”  FFDM images from a single vendor. In addition, our analysis was confined to a fixed feature set and, although elastic-net regression was used to alleviate model over-fitting, our reported model performance may be over-estimated due to the relatively small sample size as a single-institution evaluation. Considering the reported substantial differences in textural measurements across image acquisition settingsIn conclusion, our study provides evidence that incorporating breast anatomy strengthens the associations of mammographic parenchymal phenotypes with breast cancer risk and suggests that anatomy-driven measurements of parenchymal texture could complement current established risk factors and quantitative breast density measures. This additional information reflecting breast anatomy structure has the potential to further refine individualized risk assessment and, therefore, advance tailored screening and prevention strategies for breast cancer.Supplementary Information