Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus affecting approximately 90% of the world population. HCMV causes disease in immunologically naive and immunosuppressed patients. The prevention, diagnosis and therapy of HCMV infection are thus crucial to public health. The availability of effective prophylactic and therapeutic treatments remain a significant challenge and no vaccine is currently available. Here, we sought to define an epitope-based vaccine against HCMV, eliciting B and T cell responses, from experimentally defined HCMV-specific epitopes.We selected 398 and 790 experimentally validated HCMV-specific B and T cell epitopes, respectively, from available epitope resources and apply a knowledge-based approach in combination with immunoinformatic predictions to ensemble a universal vaccine against HCMV. The T cell component consists of 6 CD8 and 6 CD4 T cell epitopes that are conserved among HCMV strains. All CD8 T cell epitopes were reported to induce cytotoxic activity, are derived from early expressed genes and are predicted to provide population protection coverage over 97%. The CD4 T cell epitopes are derived from HCMV structural proteins and provide a population protection coverage over 92%. The B cell component consists of just 3 B cell epitopes from the ectodomain of glycoproteins L and H that are highly flexible and exposed to the solvent.We have defined a multiantigenic epitope vaccine ensemble against the HCMV that should elicit T and B cell responses in the entire population. Importantly, although we arrived to this epitope ensemble with the help of computational predictions, the actual epitopes are not predicted but are known to be immunogenic. Human Cytomegalovirus (HCMV) seroprevalence is 50–90% in the adult population. HCMV can be transmitted via saliva, sexual contact, placental transfer, breastfeeding, blood transfusion, solid-organ transplantation or hematopoietic stem cell transplantation. The main risk factors for HCMV infection, reactivation and disease are: immune-naive state, immunosuppressive regimens, organ transplants and co-infection . The preHCMV, or human herpesvirus 5, is a beta herpesvirus consisting of a 235 Kpb double-stranded linear DNA core. HCMV genome is among the longest and most complex genomes of all human viruses, due to the diversity of wild-type strains in intrahost and interhost HCMV populations. The HCMV genome is translated in 3 overlapping phases  giving rise to RNAs and proteins with a structural and/or a functional role in different stages of the viral cycle [Although HCMV can reside in both, myeloid and lymphoid lineages, monocytes are its primary target. HCMV reactivation and dissemination may occur after infected monocytes migrate into tissues and differentiate into macrophages since, unlike monocytes, they are permissive for viral gene expression . InitialHCMV-specific adaptive immunity is required for long-lasting protective immunological memory, which prevents from reinfection, reactivation, uncontrolled replication and serious disease. Protection against HCMV is correlated with high frequencies of CD8 cytotoxic T lymphocytes (CTLs) specific for immediate-early 1 protein (IE-1) and 65 KDa phosphoprotein (pp65) as well as type 1 CD4 T helper (Th1) cells specific for glycoprotein B (gB), TLR14 and UL16, which also exhibit cytotoxic activity . Unlike Despite eliciting strong immune responses, HCMV has a large evasion armoury that is responsible for the resilience of the virus and its prevalence in the population. HCMV interferes with cytokine pathways, NK cell activation and antigen processing and presentation . In addiDespite much effort, an effective treatment for HCMV disease remains a significant challenge. The most effective approach to prevent infection, transmission or reactivation in immune-naive or immunosuppressed individuals will be a multifunctional HCMV vaccine . CurrentHere, we designed multi-functional epitope-based vaccine for HCMV through an approach that combines legacy experimentation with immunoinformatic predictions –31. The Compared to other organisms, viruses have a high replication rate, displaying great sequence variability. This feature facilitates immune evasion and can hinder the development of vaccines providing protection to all strains. Such immune evasion can be better countered back with vaccines consisting of non-variable epitopes . We analhttps://www.iedb.org/) 20 experimentally verified HCMV-specific CD8 T cell epitopes from 499 available epitopes after the following search criteria: A) recognition by human subjects exposed to the virus and B) induction of epitope specific CD8 T cells with killing activity over cells infected with HCMV. This type of selection guaranties that CD8 T cell epitopes are appropriately processed and presented by both, dendritic cells priming epitope-specific CD8 T cells and infected target cells. Of those, we discarded any peptide with variable residues and size out of the 9–11 residue-range as they are unlikely to bind class I human leukocytes antigen (HLA I) molecules. Thus, we retained 9 conserved CD8 T cell epitopes with a size between 9 and 11 residues that were subjected to HLA I binding predictions and population protection coverage (PPC), analyses (details in Methods). We found that just a single epitope (QYDPVAALF) could reach a PPC that is at the least of 66.71% (Table We retrieved from IEDB (https://www.iedb.org/) 291 experimentally validated HCMV-specific CD4 T cell epitopes recognized by humans exposed to the HCMV. Of those, we selected 91 epitopes belonging to structural proteins for size and conservation analysis. Thus, we identified 77 conserved epitopes with a size between 9 and 21 amino acids, the usual length of peptides restricted by class II HLA (HLA II) molecules. These 77 epitopes belonged to pp65 (UL83) and gB (UL55). No conserved epitopes were identified in other structural proteins. Although these 77 epitope peptides were unique, some were largely overlapping. Therefore, we applied a clustering-based procedure (details in Methods) to identify shared epitopes defined by overlapping peptides. Thus, we proceeded with 37 CD4 T cell epitopes, 15 derived upon clusters, for HLA II binding and PPC analyses. In Table We obtained from IEDB , glycoprotein L (UL115), glycoprotein B (UL55), glycoprotein M (UL100), glycoprotein UL4 (UL4), glycoprotein UL1 (UL1), TLR10 (IRL10) and TRL12 (IRL12). We clustered these epitopes to identify common overlapping epitopes, finding only two epitopes from 2 sets of 4 and 7 overlapping epitopes (see Methods). All remaining 90 epitopes were fragmented into 9mers overlapping 8 amino acids, sought for conservation and clustered to identify the longest conserved fragment. Thus, we identified 15 conserved epitopes for which we computed their flexibility and accessibility (Table Since only one epitope (AFHLLLNTYGR) had a flexibility ≥1.0 and an accessibility ≥48%, determining their location in highly flexible and solvent-exposed regions , we sougThere have been considerable efforts to develop a vaccine against HCMV, ranging from using attenuated viruses to various viral subunits . HoweverThe main advantage of the epitope-based formulations is their exquisite selectivity as well as the possibility of inducing immune responses to subdominant epitopes and to various antigens at the same time. Moreover, they have been proposed to be safer than traditional vaccines , 35. DevThe epitopes obtained from the initial selection steps were subjected to different analysis for vaccine inclusion. The final epitope ensemble vaccine that we propose consists of 6 CD8 T cell epitopes, 6 CD4 T cell epitopes and 3 B cell epitopes  only 40% of the selected T cell epitopes and 15% of the selected B cell epitopes are conserved. These results indicate that sequence variability enables HCMV to escape the immune response, particularly the Ab response. They also highlight the crucial role of T cell responses in the control of HCMV in infected individuals.Our epitope ensemble vaccine is multiantigenic, targeting 4 different HCMV proteins: pp65 (UL83), 150KDa phosphoprotein , envelope gL (UL115) and envelope gH (UL75). There are 2 antigens represented in the CD8 T cell epitope component (pp65 and pp150) and 2 antigens in B cell epitope component (gL and gH). However, CD4 T cell component only contains epitopes from the pp65. Arguably, it would have been better to include epitopes from some other antigens in the CD4 T cell component. However, the selected CD4 T cell epitopes do provide the maximum PPC and ought to offer effective help to both CD8 T cells and B cells.Three of the targeted antigens  have been included in other vaccines currently undergoing clinical trials, highlighting the importance of these antigens as components of a HCMV-specific vaccine. The viral protein pp65 (UL83) is delivered to infected cells as a virion component and rapidly moves to the nucleus where it antagonizes the cellular antiviral response through the NF-κB pathway . The virA potential adverse effect of vaccines is that of inducing immune responses cross-reactive with self-antigens. Thereby, we verified that none of the included epitopes matched exactly human proteins or human microbiome proteins. The sequence similarity of all epitopes with human proteins is less than 80%; only two epitopes have a similarity over 80% with microbiome proteins. Since immune recognition is exquisitely specific, it can be disrupted by single amino acid mutation , and it We have assembled a HCMV vaccine consisting of 6 CD8 T, 6 CD4 T and 3 B cell epitopes from 4 different HCMV antigens. The epitopes do not match self proteins, are conserved and all but 2 B cell epitopes are experimentally verified and reported to be recognized by humans exposed to HCMV. This epitope ensemble was built using a knowledge-based, computer assisted approach aimed at identifying epitopes that are likely to induce protective adaptive immune responses. Thus, the T cell epitopes are predicted to provide a PPC over 90% and include CD8 T cell epitopes mediating cytoxicity against HCMV infected cells. The B cell epitopes are all in highly flexible and accessible regions of the ectodomain of gH and gL proteins which makes them suitable for inducing Abs cross-reactive with the relevant native antigens. Moreover, they are proximally located to regions involved in the assembly of key complexes for viral entry. Thus, Abs induced by these epitopes could be neutralizing and block infection.We have sought to identify optimal epitope components for making a protective HCMV vaccine, but there remains a long road ahead prior to deploying a preventive vaccine. Epitope peptides are known to be poorly immunogenic and the epitope ensemble will have to be contained within a formulation capable of inducing potent innate and adaptive immune responses. An attractive formulation will be to encapsulate the T cell epitopes along with appropriated adjuvant on liposome-based nanoparticles, displaying the B cell epitopes on the outer surface .51Cr cytotoxic assays with cells infected with HCMV (relation between epitope and antigen is source organism).Experimentally confirmed HCMV-specific epitopes were obtained from IEDB . We onlyWe used CD-HIT  to clustH) [Pi is the fraction of residues of amino acid type i and M is the number of amino acid types. H ranges from 0 (only one amino acid type is present at that position) to 4.322 . Following these calculations, we masked in the reference HCMV proteome (NC_006273) any site with H ≥ 0.5, thus generating consensus sequences. HCMV epitopes that matched entirely with the consensus HCMV sequences were retained for subsequent analysis.Sequence variability of the MSA was analysed per site/position using the Shannon Entropy (H) , as the We used CD-HIT  to identhttp://imed.med.ucm.es/Tools/episopt.html) [http://tools.iedb.org/mhcii/). The reference set includes HLA II molecules belonging to HLA-DP, HLA-DQ and HLA-DR genes and a 5% percentile rank was used to assess binding. As the prediction method, we selected “IEDB recommended”. This method provides a consensus prediction which combines matrix and neural network-based models, when the relevant predictors are available, otherwise returning predictions provided by NetMHCIIpan [http://tools.iedb.org/tools/population/iedb_input) [We predicted binding of CD8 T cell epitopes to 55 HLA I molecules using EPISOPT (pt.html) . EPISOPTpt.html) , 52 and pt.html) . For lonpt.html)  with IEDMHCIIpan  for CD8 b_input) . EPISOPTb_input) , and ideb_input) .α B-factors, ZBi (Eq. The flexibility and accessibility of B cell epitopes was predicted using the relevant Protein Data Bank (PDB) files, when available, as described elsewhere . Briefly ZBi Eq. , after ti is the B factor of the Cα from residue i, obtained from relevant PDB, μB is the mean of Cα B factors, and ∂B is the corresponding standard deviation. Likewise, we used NACCESS [In Eq.  NACCESS  to compuFb) and accessibility (Ab), respectively, for each B cell epitope.Subsequently, we used Eq. where n is the total number of residues encompassed by the B cell epitope.Fb and Ab values with predicted B and RSA values of the relevant residues . We visualized 3D-structures and produced molecular renderings using the PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.We used BLAST searches  against


The cohorts consisted of 333 Czech, and 235 Norwegian OMT–exposed newborns, and 106 and 294 newborns in the comparison groups, respectively. In both countries, the neonatal growth parameters were similar in the OMT and the comparison groups. In Norway, OMT exposure prolonged gestational age  while the odds of preterm birth and Apgar score at 5 minutes were lower than in the comparison group . Newborns of women in OMT had similar growth parameters as newborns of women with opioid use disorders who were not in OMT during pregnancy. Overall, our findings do not suggest that OMT results in worse neonatal outcomes.There is lack of knowledge about the safety of treatment with methadone and buprenorphine as part of opioid maintenance treatment (OMT) during pregnancy. The purpose of this study was to examine neonatal outcomes concerning the use of OMT during pregnancy. We used nationwide registry linkages from the Czech Republic (2000‐2014) and Norway (2004‐2013). We compared prenatally OMT–exposed newborns with (a) newborns of women hospitalized with opioid use disorder during pregnancy in the Czech sample and (b) newborns with neonatal abstinence syndrome (NAS) in Norway. We performed multivariate linear and binary logistic regression exploring the associations between OMT and neonatal outcomes . Regression coefficients ( ATCAnatomical Therapeutic ChemicalNASneonatal abstinence syndromeOMTOpioid maintenance treatmentORsodds ratiosSGAsmall for gestational age1Opioid use disorder is characterized by the persistent use of opioids despite the adverse consequences of its use. Opioid maintenance treatment (OMT) can stabilize women and reduce the risk of relapse to illicit opioid use.Previous research has emphasized benefits with OMT during pregnancy. If left untreated, opioid use disorders during pregnancy are associated with a range of obstetric and neonatal complications, such as spontaneous abortions, intrauterine growth retardation, preterm birth, and low Apgar score.OMT is, however, also associated with disadvantages. Neonates exposed to opioid agonist therapy have higher rates of adverse neonatal outcomes compared to neonates in the general population. There is evidence of high rates of neonatal abstinence syndrome (NAS), lower birth weight, length, and head circumference as well as increased rates of perinatal mortality.With an increasing focus on preventing harm to the unborn child, the low evidence behind OMT has resulted in critical questions from pregnant woman, clinicians, researchers,Previous studies have several weaknesses, including one major concern of unmeasured confounding. Most studies only compare newborns of women in OMT during pregnancy with newborns of women from the general population. Women in these two groups have very different confounder distributions, and comparison between them is therefore not suitable. Using appropriate comparison groups is one way to come closer to an unbiased association. Many of the studies were also conducted several years ago, and often in selected populations. Many were performed in countries where the quality of health care differs for different patient populations, making a comparison between groups difficult.Moreover, OMT has changed substantially since methadone was first introduced and since the early studies were conducted: more pharmacological options have been introduced, and inclusion and exclusion criteria for OMT have changed. In light of these changes, exploring these questions in new settings and using new approaches may contribute to shed more light on the problem. Use of data from nationwide health registries is a new approach that makes it possible to address several of the limitations from earlier studies.To increase the knowledge about the safety of OMT treatment during pregnancy, we utilize nationwide registry data from two different European countries to study associations between OMT exposure and adverse neonatal outcomes in newborns. To reduce the problem of unmeasured confounding, we compared newborns born to women who were in OMT during pregnancy to newborns whose mothers had indications of opioid use disorders but who were not in OMT during pregnancy.22.1The study is an observational cohort study with two national cohorts based on linkages of nationwide registries.2.2OMT became available for pregnant women in 1997 in the Czech Republic and in 1998 in Norway.2.3Both the Czech Republic and the Norway have nationwide registries with personal identification numbers. This enables linkages of data between different registries on an individual level, and on family levels such as between mother and child. A more detailed description of the data sources is provided elsewhere.2.4In the Czech Republic, physicians are obliged by law to report data to the national health registries.2.4.1The NRRH includes information about all women and their children during pregnancy, delivery, and puerperium. For instance, maternal variables such as demographic and socioeconomic data, alcohol, tobacco, and illegal drug use during pregnancy and information about the delivery are included in the registry. Information about the newborn, such as birth parameters, congenital malformations, and death, is also included.2.4.2The NRAT contains data on all patients starting and terminating different addiction treatments. It includes, for instance, information about patients who receive OMT, such as date of treatment initiation and termination and type of OMT drug.2.4.3The NRIT includes information on every episode of all types of hospitalizations, including information on dates of admission and discharge from the hospital. Diagnoses on the discharge summary are coded according to the International Statistical Classification of Diseases, 10th Revision (ICD‐10).2.52.5.1The MBRN is based on a compulsory notification of every birth or late abortion from physicians or midwives attending the birth. The MBRN includes information concerning all births and late abortions from the 12th gestational week and onwards. The registry includes information concerning pregnancy, delivery, and the newborn. Maternal data are also collected, such as demographic and socioeconomic backgrounds of the pregnant women and also tobacco smoking during pregnancy. Information about neonatal outcomes, such as gestational age, birth parameters, NAS, and congenital malformations, are also included.2.5.2Pharmacies are obliged by law to forward prescription data to the NorPD. The NorPD includes information about all prescription drugs, including OMT drugs, dispensed at pharmacies to patients in ambulatory care. For each prescription, the dispensing date and detailed information on the drug is registered. The drugs are classified according to the Anatomical Therapeutic Chemical (ATC) classification system.2.5.3From Statistics Norway we included information about maternal education. Educational institutions are obliged to report completed education on an individual level to SSB.2.6The study populations were pregnant women and their children born during the study period: 2000‐2014 in the Czech Republic (N = 1 547 273) and 2004‐2013 in Norway (N = 554 310). From this population, we identified women who had indications of opioid use disorders during pregnancy, who were either in OMT during pregnancy or not in OMT.2.7Exposure was defined as the use of the OMT drugs methadone or buprenorphine during pregnancy. In the Czech Republic, data on initiation and termination of drug treatment from the NRRH were used to identify if women were receiving OMT drugs during pregnancy. Of the 333 women who were in OMT treatment during pregnancy, 192 (58%) were in OMT throughout the entire pregnancy, while 141 (42%) were in OMT for only parts of pregnancy .In Norway, NorPD was used to identify women using OMT drugs. Those who were dispensed OMT drugs at least once during pregnancy were defined as exposed to OMT during pregnancy. More than 95% of all OMT women receive more than one prescription of an OMT drug during pregnancy. Pregnant women in OMT receive, on average, a total amount of buprenorphine corresponding to approximately 10 mg/day in pregnancy, while women who use methadone on average receive a total amount of about 65 mg/day. Approximately 80% of the OMT drugs are dispensed at pharmacies both early and late in the pregnancy. This suggests that they used these drugs throughout their pregnancy.2.8The outcomes studied were neonatal outcomes identified in the NRRH or MBRN, and included: gestational age, preterm birth (<37 weeks of gestation), growth parameters , small for gestational age (SGA),2.9We obtained information on sociodemographic variables and lifestyle from the NRRH in the Czech Republic and from the MBRN and SSB in Norway. This information included age and marital status . Information about education was provided in the following categories: primary, secondary, university, or unknown. Information about occupation was only available in the Czech Republic, and included the categories unemployed, employed and unknown. Information on recreational drug use during pregnancy, that is, use of alcohol and illicit drugs was only available in the Czech Republic. Information about tobacco smoking was available in both countries and categorized as yes, no, or unknown.2.10To be able to study the safety of the OMT in pregnancy we created relevant but different comparison groups in the two countries by identifying pregnant women with indications of opioid use disorders who were not in OMT during pregnancy.For the Czech comparison group, we selected pregnant women who, according to the NRIT, had been hospitalized during pregnancy with a diagnosis at discharge indicating that she had an opioid use disorder . Furthermore, the women could not have received OMT during pregnancy, according to the NRAT.The Norwegian control group comprised of women who, according to data in the MBRN, gave birth to a child with NAS. Newborns with NAS were recognized if they have received an ICD‐10 diagnosis of “neonatal withdrawal symptoms from maternal use of drugs of addiction” (P96.1 according to ICD‐10). When reporting to the registry, the midwife or physician could also tick a box for abstinence if neonatal irritability and neurological symptoms had been observed and there was documentation of maternal abuse of prescription drugs, alcohol or illicit drugs during pregnancy, or the woman herself gave such information. Furthermore, the women in the comparison group could not have received OMT during pregnancy, according to the NorPD.2.11In a previous study using the same nationwide registry data as in this study,In supplementary tables, we show maternal socioeconomic characteristics, as well as neonatal outcomes in the OMT–exposed newborns compared to newborns of women in OMT before and after (but not during) pregnancy and compared to all the children in the general population.2.12The study was approved by the Institutional Review Board of the General University Hospital in Prague (IRB00002705), the Regional Committees for Medical and Health Research Ethics , and the Norwegian Data Inspectorate.33.1In the Czech Republic, 333 women were in OMT during pregnancy, while 106 had opioid use disorder without being in OMT during pregnancy. In Norway, we identified 235 women who were in OMT during pregnancy, while 294 had indications of opioid use disorder but were not in OMT during pregnancy. Tables 3.2Table 3.3b = 0.93 weeks), preterm birth (unadjusted OR = 0.44), and low Apgar score at 5 minutes (unadjusted OR = 0.26). After adjustment for confounding factors the risk estimates of OMT exposure compared to no exposure were still significant; gestational age was nearly one week longer in OMT‐exposed . OMT exposure also reduced the risk of preterm birth preterm birth  and the risk of low Apgar score at 5 minutes . OMT was not significantly associated with a difference in any of the neonatal outcomes in the Czech cohort.Table 3.4Tables 4In this study, we aimed to increase knowledge about the safety of OMT during pregnancy by examining neonatal outcomes concerning use of OMT during pregnancy using relevant comparison groups in two nationwide cohorts of pregnant women. We found no significant differences in neonatal growth parameters  between OMT–exposed newborns and newborns of drug‐using pregnant women not in OMT during pregnancy. These findings were consistent in both countries. In the Norwegian sample, gestational age was nearly one week longer, and the odds of preterm birth and low Apgar score were lower in the OMT–exposed group. In the Czech Republic, these differences were not observed.Our results on growth parameters are in contrast to previous research,Performing studies on safety of prenatal OMT exposure in another setting than in the United States, where the majority of early research has been conducted, might reduce the role of unmeasured confounding. In Norway and the Czech Republic, socioeconomic differences in the population are smaller than in the United States, and health care services are freely available for everyone. Thus, while opioid–dependent women in these countries have low socioeconomic status, they do not live in pronounced poverty.In this study, we tried to identify comparison groups with quite similar risk factor profiles as women in OMT. The comparison group in the Czech Republic did not differ from the OMT group concerning background characteristics. In Norway, the women in OMT smoked more and had lower education. If background characteristics play an important role in affecting neonatal outcomes, the similar characteristics of the OMT and the comparison groups in our study suggest that the selected comparison groups were suitable for studying adverse outcomes of OMT during pregnancy. However, it should be noted that the comparison groups were identified by different criteria. In the Czech Republic, women with at least one diagnosis indicating drug dependence during pregnancy constituted the comparison group, while women who gave birth to a newborn with NAS comprised the comparison group in Norway. This difference combined with different treatment settings There were differences in gestational age, preterm birth, and Apgar score between the OMT–exposed and the nonexposed group in Norway. Recent reviews report that gestational age should be considered as a continuum concerning the risk and severity of adverse outcomes.4.1Using information from the nationwide registries reduces the risk of selection and recall bias. By using registry data, all pregnant women are identified and followed‐up unless they move out of the country. This reduces the problem of selection both due to inclusion and to loss to follow‐up that might be especially important for pregnant women with opioid use disorders who more often have a transient lifestyle.The Norwegian Prescription Database only includes information on prescription drugs dispensed to outpatients. If a pregnant woman received all her OMT drugs during pregnancy at hospital or institution, this would not have been registered in the prescription database, and we would not have identified the woman as in OMT. We assume that this is the situation for a limited number of women in OMT.In Norway, we used women who gave birth to children with NAS as the comparison group. Although it is not possible to attribute the cause of NAS to opioid exposure alone, opioids are the most common cause of NAS.Neonatal growth parameters in the OMT–exposed newborns were similar both to the outcomes in newborns of drug–dependent women not in OMT during pregnancy and to the groups of women using OMT outside of pregnancy, but not during pregnancy. However, when compared to the general population, all the newborns of women who had any indications of drug abuse before, during or after pregnancy, seem to have worse neonatal outcomes – regardless of whether the woman received OMT during pregnancy or not. Taken together, this might suggest that it is not the OMT drugs themselves that are associated with worse neonatal outcomes, but other factors related to opioid use, such as comorbidity, socioeconomic, and lifestyle factors.Some critics have questioned the rationale for public health care services offering opioid maintenance treatment (OMT) during pregnancy. Our findings did not suggest that OMT results in worse outcomes for the newborns compared to no treatment. Moreover, we observed some important positive neonatal outcomes from OMT versus no OMT in the Norwegian sample. Seen in conjunction with the beneficial effects for the pregnant woman such as improved prenatal care adherence and obstetrical outcomes,None of the authors has anything to disclose. Click here for additional data file. Click here for additional data file.


GRMZM2G419655 and GRMZM2G511067, which could be used as functional markers in screening starch granule size for different commercial uses.Starch is an important nutrient component of maize kernels, and starch granule size largely determines kernel waxiness, viscosity, and other physiochemical and processing properties. To explore the genetic basis of maize starch granule size, 266 tropical, subtropical, and temperate inbred lines were subjected to genome-wide association analyses with an array of 56,110 random single nucleotide polymorphisms (SNPs). In the present panel, the kernel starch granule size ranged from 7–15.8 µm long and 6.8–14.3 µm wide. Fourteen significant SNPs were identified as being associated with the length of starch granules and 9 with their width. One linkage disequilibrium block flanking both sides of a significant SNP was defined as a quantitative trait locus (QTL) interval, and seven QTLs were mapped for both granule length and width. A total of 79 and 88 candidate genes associated with starch length and width, respectively, were identified as being distributed on QTL genomic regions. Among these candidate genes, six with high scores were predicted to be associated with maize starch granule size. A candidate gene association analysis identified significant SNPs within genes  Maize starch is not only an important food source but is also one of the most industrially used resources3. Its uses include the preparation of soups, sauces, baked goods, dairy, confectionery, snacks, pasta, coatings, and meat-containing products5, as well as adhesives, paper, and textiles6.Starch is a widely and naturally occurring biopolymer. It is composed of D-glucose units that form two types of polymers: amylose and amylopectin. Many cereals and tuber crops produce different types of starch. For instance, maize is an important crop worldwide7. Developing seeds synthesise storage compounds from imported sucrose during their maturation phase8. The assimilation of sucrose, which is imported through the phloem, by the endosperm may involve sucrose synthase to form ADP-Glc or UDP-Glc and fructose or, alternatively, involve invertase to form free hexose. Many studies have focused on the physicochemical properties of maize starch and their influencing factors12. Starch type13 and amylose content14 have important effects on starch properties. Other influencing factors include granular structure , molecular structure , and the presence of non-starch materials15.Starch formation in cereal grains involves the synthesis of ADP-glucose (Glc) by ADP-Glc pyrophosphorylase and the incorporation of ADP-Glc into starch by ADP-Glc starch synthase16 and ultimately determines the industrial application17. Small starch granules can be used to replace fat in food applications because of their fat mimetic properties18. In food production, granule size affects the pasting properties of starch, with smaller granules showing lower peaks, troughs, and final viscosities than larger granules19. The starch granule size may also influence gelatinisation temperature20, viscosity19, and enzymatic susceptibility23. Additionally, it determines the grain milling yield in hard wheat24. In maize, the size of the starch granules varies according to chemical composition26.The size of the starch granules, which depends on the plant species, is an important factors affecting starch characteristicsTriticeae crops have been identified, including a major QTL related to the A:B ratio of wheat starch granules on chromosome 4S27 and a QTL on barley chromosome 228. Recently, genome-wide association studies (GWASs) have been proven to be useful tools for the identification of candidate loci associated with traits in animal and plant species29. For example, an analysis of maize oil biosynthesis identified 74 loci significantly associated with kernel oil concentration and fatty acid composition in a GWAS using 1 million single nucleotide polymorphisms (SNPs) characterised in 368 inbred maize lines30. Furthermore, a GWAS and QTL mapping were found to be complementary, overcoming each other’s limitations, in Arabidopsis31.Quantitative trait loci (QTLs) of starch granule sizes in 34, few studies have investigated the unimodal starches, particularly that of maize35. Although the sizes of maize starch granules are highly linked to the end-use quality of the products, many studies on maize starch have focused on its processing and nutritional properties35, with little attention paid to the study of granule size34. Here, we used a set of associated populations to identify significant SNP markers for starch granule size with the aim of predicting associated candidate genes.Compared with starches having a bimodal size distributionA total of 266 maize lines were used for association mapping. Although the starch granules of these inbred maize lines varied largely in size, more than 75% of the granules were 10–13.5 µm long and 9.7–11.8 µm wide Table . The inbThe rapid visco analyser  profile revealed the paste viscosity characteristics of maize starch Table . Seven p−4; Fig. The average data from different replicates of each inbred line were used for association analysis Figs  and 4. FPZE-103182712) on chromosome 3, one (PZE-106103012) on chromosome 6, and five  on chromosome 7.Nine significant SNPs were identified to be associated with starch granule width, as well as seven QTLs and 88 candidate genes Fig. , Table 2GRMZM2G180104 was located between 75,849,776–75,850,502 bp on chromosome 7 and 3,503 bp upstream of significant SNP30343, while GRMZM2G419655 and GRMZM2G419660, also on chromosome 7, were identified as being associated with starch granule size.The QTLs analysis led to the indentification of 108 candidated genes that were either associated with granele length or width. Among these, six genes with higher scores were located close to associated SNPs Table . GRMZM2GGRMZM2G167673, was predicted to be involved in gibberellin synthesis and electron transport as a p450 cytochrome. GRMZM2G419655 and GRMZM2G419660 on chromosome 7 were predicted to encode phytosulfokine receptor precursors. GRMZM2G511067 and GRMZM6G663759 on chromosome 3 were predicted to encode a zinc finger CCCH domain-containing protein that binds metal ions and may repress the inhibitor of the phytosulfokine receptor protein kinase.The Blast2Go program was used to predict the functions of these candidate genes Tables , S4. TheThe results of the GWAS analysis revealed that maize kernel starch granule size is a typical quantitative trait determined by multiple genes.GRMZM2G419655 genomic sequence and the SNP at 58 bp of the GRMZM2G511067 genomic sequence were significantly associated with maize starch granule length and width 37. LDS for particle size analysis is simple to perform; however, the starch granule’s oblate shape can cause the laser to diffract from the flat surface or narrow edge, or at obtuse angles to these surfaces, leading to system errors. Comparing LDS with IA, Wilson et al.38 reported that LDS underestimated A-type granule’ diameters by ~40% and B-type granule’ diameters by ~50% in wheat. Edwards et al.24 revealed that LDS measurements underestimated C-, B-, and A-type granules by maximum averages of 0.83, 3, and 23 mm, respectively. Additionally, LDS requires a prior starch extraction, which may cause artefacts to develop during extraction or precipitation39. Therefore, LDS is more suitable for the analysis of totally spherical granules. Thus, LDS has often been used in the study of wheat starch but rarely in the study of maize starch.Starch is the major storage carbohydrate in cereal seeds, and the size of the starch granules is strongly associated with its end use. However, it is difficult to accurately determine the size of starch granules. To date, two techniques have been developed to analyse granule sizes: laser light scattering (LDS)et al.40 analysed the size of starch granules in Brachypodium distachyon by SEM, while Zhang et al.26 used a light microscope with the Zeiss software AxioVision to observe the starch granule size in potato. Compared with LDS, IA coupled with light microscopy or SEM is more direct and more readily distinguishes among individual granules, agglomerated granules, and non-starch particles. It can also simultaneously record the surface features of individual particles. Considering the shape diversity of maize starch granules, IA with SEM was chosen as a more accurate method of obtaining direct data in this study.Starch granule size has been measured by other direct methods, such as light microscopy and scanning electron microscopy (SEM). Chen 17. Variations in starch granule size mainly exist among inbred lines of maize, which allows for the selection of different commercial hybrids having the required granule size, resulting in improved industrial use.In cereal, the size of the starch granules is an important property affecting the appropriate industrial use42. In the current study, 14 and 9 SNPs were significantly associated with maize starch granule length and width, respectively. Among them, seven significant SNPs and five QTLs were shared between starch granule length and width  714D (CYP714D). In plants, CYP is involved in several cellular processes. A homologous gene in rice encodes the CYP protein, which regulates the embryo to endosperm ratio and increases the proportion of kernel endosperm43. More recently, the insertion of a 247-bp transposable element into the 3′-untranslated region of ZmGIANT EMBRYO 2  was associated with an increased embryo to endosperm ratio44. In the present study, the gene ontology analysis revealed that GRMZM2G167673 has monooxygenase activity. Subfamily members of CYP, including CYP78A in maize also have monooxygenase activities, with CYP78A5, CYP78A7, and CYP78A9 regulating organ size by generating mobile growth signals that stimulate cell proliferation46. Thus, GRMZM2G167673 may regulate endosperm growth, determining the size of the starch granules.In a candidate gene analysis for starch granule size, GRMZM2G419660, encodes a protein with serine/threonine kinase activity. Serine/threonine kinases are a subfamily of calcium-dependent protein kinases (CDPKs) in plants47; moreover, overexpressing and silencing the CDPK gene OsCPK31 indicated that it regulates grain filling and early maturation in the Taipei 309 rice cultivar. A SEM examination showed that the starch granules increased in size when OsCPK31 was overexpressed compared with in non-transformed controls48. Thus, GRMZM2G419660 in maize may also be an essential factor for the phosphorylation of sucrose synthase, which is a major enzyme involved in the starch biosynthetic pathway, similar to protein kinases in rice.Another candidate gene associated with starch granule size, The size of the starch granules is an important factor influencing the industrial applications of starch. In the present study, inbred maize lines with different starch granules sizes were evaluated. Maize lines with different sized starch granules can be used in different industries. Moreover, SNPs or candidate genes identified in this study could be used as molecular markers to accelerate the breeding and production of plants with starch granules appropriate for different commercial purposes.50. Because some tropical germplasm cannot mature in temperate zones, affecting the starch content and granule size, all of the inbred lines were cultivated in Sanya  during the winters of 2012 and 2013. The field experiment followed a randomised complete block design with two replications. Plots were 4 m × 0.67 m and comprised 16 plants at a density of 65,250 plants per hectare. During the growing seasons, plants were irrigated and underwent common field management practices to avoid any stress.The investigation was based on a set of 266 inbred maize lines, containing a wide range of temperate, subtropical, and tropical germplasmAll of the inbred lines were self-pollinated by hand in the field, harvested when physiologically mature, and dried under natural conditions; those ears that showed abnormal development were subsequently discarded. Kernels in the middle part of each ear were then hand-threshed for starch granule size evaluation. Ten representative matured and dried kernels were selected (five kernels from each ear) and affixed to aluminium specimen stubs using double-sided adhesive tape. The samples were then sprayed with gold powder and screened using SEM  at the Centre of Biotechnology, Henan Agricultural University, Zhengzhou, China. The sizes of 20 randomly selected maize starch granules were evaluated for length and width. Data were analysed using the analysis of variance method with SPSS software . A frequency map was constructed by Origin 8.0 software . Sample means were used as phenotypic data for an association mapping analysis.et al.51.Four maize lines were selected to extract starch, and the pasting properties of the starch were measured using a rapid visco analyser according to Hao www.r-project.org). Frequency plots were also constructed by the plot function in R. Averaged data for each inbred line were used in the association analysis.All statistical analyses were performed using the R statistical environment  between SNPs on each chromosome was estimated with r2 using TASSEL 5.053. A mixed linear model with the obtained SNPs, principal components, kinships, and the mean starch granule sizes was used for the GWAS. The relative distribution of −log10p-values was observed for each SNP association and compared individually with the expected distribution using a quantile–quantile plot. The adjusted p-value threshold of significance in each trait was corrected. SNP loci in significant LD regions were identified by revealing significant contributions to the phenotypic variations of the agronomic traits with the highest r2 values (magnitude of marker–trait association) and lowest adjusted p-values (threshold p < 1 × 10−4).Selected inbred lines were genotyped using two genotyping platforms (RNA-sequencing and SNP array) containing 56,110 SNPs according to the method described by Yang 54, thus a 100-kb region flanking the left and right sides of a SNP was defined as a QTL. If several SNPs were located closely within one LD block, the middle coordinate was chosen.The overall LD decay across the genome of this panel was 100 kbp < 1 × 10−4). The Blast2Go program was used to predict the functions of corresponding genes (http://www.geneontology.org/).The available maize genome sequence (B73) was used as the reference genome for candidate gene identification. SNP probe sequences of ~120 bp  were used as queries in a BLAST algorithm-based search against the reference genome sequence in MaizeGDB (http://www.maizegdb.org/gbrowse). Based on the LD decay, a 200-kb window for the significant SNPs (100-kb upstream and downstream of the lead SNP) was selected to identify candidate genes. Genes within the region were identified according to the position of the closest flanking significant SNP  contained 1 µl of NEB  Taq DNA Polymerase, 4 µl of 5× NEB PCR Buffer, 0.5 µl of dNTP mixture, 0.5 µl each of the two primers, and 1 µl of template DNA. The PCR reaction was carried out in a Bio-Rad Thermal cycler with an initial denaturation at 94 °C for 3 min followed by 34 cycles of denaturation for 10 s at 94 °C, annealing for 1 min at 64 °C and extension for 1 min at 68 °C, with a final extension for 10 min at 68 °C. SNPs within the three candidate gene sequences were selected for the association analysis. Primers for amplification of the three genes are listed in Table Three candidate genes, In total, 30 maize lines with different starch granule sizes  were selected from the association panel to study the expression levels of candidate genes. Maize kernels (20 d after pollination) were used for RNA extraction according to the manufacturer’s user manual . The primers for the selected 10 candidate genes are shown in Table Supplementary Information


Feather holes are small (0.5–1 mm in diameter) deformities that appear on the vanes of flight feathers. Such deformities were found in many bird species, including galliforms and passerines. Holey flight feathers may be more permeable to air, which could have a negative effect on their ability to generate aerodynamic forces. However, to date the effects of feather holes on flight performance in birds remained unclear. In this study we investigated the relationship between the number of feather holes occurring in the wing or tail feathers and short term flight performance traits – aerial manoeuvrability, maximum velocity and maximum acceleration – in barns swallows, which are long distance migrating aerial foragers. We measured short-term flight performance of barn swallows in a standardized manner in flight tunnels. We found that acceleration and velocity were significantly negatively associated with the number of holes in the wing flight feathers, but not with those in the tail feathers. In the case of acceleration the negative relationship was sex specific – while acceleration significantly decreased with the number of feather holes in females, there was no such significant association in males. Manoeuvrability was not significantly associated with the number of feather holes. These results are consistent with the hypothesis that feather holes are costly in terms of impaired flight. We discuss alternative scenarios that could explain the observed relationships. We also suggest directions for future studies that could investigate the exact mechanism behind the negative association between the number of feather holes and flight characteristics. Lagopus muta  in the barn swallow. Barn swallows are small (ca. 20 g), aerially insectivorous long-distance migratory birds that spend winter in the southern hemisphere Møller . We measThe study was carried out in two barn swallow colonies (ca. 20–30 breeding pairs each) located in two nearby horse stables in the Łomianki commune near Warsaw , central Poland, during the breeding seasons 2010 and 2014. Further details on the study area and the study population are given in Matyjasiak  and MatyBirds were captured by mist-netting conducted from early spring as they returned from Africa, sexed according to Svensson , ringed 2/wing area. To measure wing area – an index of the wing size – wing drawings were made by making a tracing of the outlines of fully extended wings. Wing drawings were subsequently analysed in MultiScan ver. 14.02 . Wing area was estimated as the area of both wings including the area of that part of the body that was included between the wings  and outermost tail feathers was measured to the nearest 1 mm with a ruler. Keel and tarsus lengths were measured to the nearest 0.1 mm using a pair of callipers. Body mass was measured to the nearest 0.5 g with a Pesola spring balance. Body mass measurement was repeated on the day of flight tests. Paired measurements were averaged to calculate mean wing, tail and tarsus length. Aspect ratio – an index of the overall shape of the wing – was calculated as (wing-span)Aerial manoeuvrability was tested by releasing birds through a flight maze measuring 18 m × 4 m × 1.6 m (length × width × height). The maze consisted of a metal frame covered in a double layer of fine-mesh garden sunshade netting. The west end of the maze was closed and contained the release box (where the bird was kept before being released in the maze) while the east end was open. The first 9 m section of the maze (on the release box side) was free of obstacles and acted as an acceleration zone. The remaining 9 m section towards the exit acted as a test zone. It contained 16 successive panels of vertical white cotton strings suspended from the roof of the maze and weighed. Both the distance between the strings within a panel and the distances between consecutive panels decreased towards the exit. Further details of the arrangement of the fight maze's test zone used in this study are given in Matyjasiak .−1) obtained with the use of angled mirrors positioned in line with the first and last panels of strings. A bird's image was reflected in the first mirror as it entered the test section and the second image was reflected in the other mirror when it left the maze. The flight time was determined by counting the number of frames between the two images and converting the result into seconds (accuracy of 0.04 s). Videos were analysed by viewing them frame-by-frame in Edius Pro 3 .Birds were released (after 2 min of acclimation) from the release box at the closed end of the flight maze and flew through the maze to escape from the open end. The front side of the box was opened remotely with a string. We measured the time taken for a bird to negotiate the test zone of the maze, which was used as measures of the bird's ability to cope with the obstacle course. A faster flight time indicate greater manoeuvrability  . Birds were captured at dawn, between 0430 and 0530 h. Before flight tests the feather hole count was performed and body weight was measured. We selected for flight tests birds that did not suffer from flight feather breakage. Flight tests were conducted the same morning at 0700–1000 h. First, birds were tested for aerial manoeuvrability in the flight maze and recaptured in a mist-net positioned at the maze exit (the distance between the last panel of strings and the mist-net was ca. 50 cm). Immediately after the manoeuvrability test birds were released through the second tunnel for acceleration and velocity performance, after which they instantly regained freedom. These measurements of flight performance are significantly repeatable, and hence they are sufficiently precise to allow use in statistical analyses  or they were indistinguishable from background noise on radar during the initial (about 0.5 s) phase of flight (2 cases).ps > 0.05), including the tail length, which is an indicator of quality of male barn swallows . Furthermore, these variables were not associated with body condition index of individual barn swallows . The sexes did not differ with respect to the numbers of wing or tail feather holes . Morphological, flight performance, and feather hole count variables were log transformed before analysis.The feather hole counts were not significantly correlated with morphological predictor variables  as a function of the number of holes in wing feathers and the number of holes in tail feathers as predictor variables. The initial model contained also covariables – age, sex, year, colony, wing area, aspect ratio, tail length, keel length and body weight on the day of the flight tests. Insignificant variables were removed from the models, which were then rerun. However, those variables that significantly interacted with other variables in the model were not removed regardless of their significance. We checked the residuals for normality and homoscedasticity at each step of model reduction. Statistical analyses were made in STATISTICA ver. 12 .r2 = 0.22, d.f. = 1, 40, F = 11.07, p = 0.002, slope = −0.35, 95% CI −0.62 to −0.07) and was significantly positively associated with keel length . The model accounted for 28% of the variation in acceleration performance of females , and the number of feather holes explained 18.7% of the variation. In contrast, male acceleration performance only was significantly negatively related to aspect ratio .In our final general linear model, time to copy the flight maze was not significantly related to the number of feather holes in the wing flight feathers or in the tail feathers . This suMaximum velocity was significantly negatively associated with the number of holes in wing feathers, and this relationship was independent of sex . MaximumIn the present study of barn swallows we found that two short-term flight performance traits – maximum acceleration and maximum velocity – were significantly negatively associated with the number of holes present in the wing flight feathers. The number of holes present in the tail feathers was not significantly associated with any of these flight variables. In the case of acceleration, the negative relationship between the number of feather holes and flight performance was sex specific. While acceleration significantly decreased with increasing wing feather hole numbers in females, there was no such significant association in males. To our knowledge, this is the first study to show the existence of a relationship between flight performance and the number of feather holes. Furthermore, we found that aerial manoeuvrability was not significantly associated with the number of holes, whether in the wing flight feathers or in the tail feathers.Flight performance trials used in our study assumed that birds were escaping from flight tunnels. In the case of flight maze, the crowded stringed section forced the birds to perform increasingly tight turns around obstacles. Flight performance displayed by the birds in such conditions may be close to the maximum of manoeuvrability and acceleration Lind . Short-tOur study is non-experimental, however, and alternative explanations of the results cannot be excluded. According to an alternative scenario, the correlation between the number of feather holes in wing feathers and acceleration and velocity may result from the fact that poor quality individuals with low quality plumage are worse fliers and, due to compromised plumage, are more susceptible to structural damage of flight feathers. Consequently, there may be no causal relationship between the number of feather holes and flight performance. This scenario does not seem to be supported by the results obtained in this study. First, we found no significant relationship between the number of feather holes and condition indices of male and female or tail length of male barn swallows. Second, we found no significant relationship between the number of feather holes and wing size or aspect ratio. The latter result may suggest that there is no relationship between the quality of flight feathers and the number of holes in these feathers. We would like to suggest, however, that future studies should take into account measures of feather microstructure (e.g. Pap et al. Tachycineta bicolor, reported no effect of feather holes on migration performance or annual survival (Lombardo et al. Results obtained in this study are consistent with the results of other studies on barn swallows, which showed that feather holes had a significant effect on migration efficiency and fitness, as males and females with more feather holes delayed arrival from spring migration and survival rate of females was significantly reduced (Møller et al. The mechanism behind the negative association between the feather hole intensity and flight performance is unclear. One possible explanation is that holes may worsen air tightness of flight feathers, which in turn may affect the aerodynamic performance of these feathers Videler . A futurIn conclusion, two short-term flight performance traits in barn swallows – maximum acceleration and maximum velocity – were significantly negatively associated with the number of feather holes occurring in wing flight feathers. We found stronger negative effect of feather holes of acceleration in females than in males. These relationships are consistent with the hypothesis that feather holes are costly in terms of impaired flight. A future study on this issue could take into account the independent effect of feather microstructure on flight performance. Also, the exact mechanism behind the negative association between the number of feather holes and flight traits remains to be investigated.


International Journal of Telerehabilitation (IJT) is a biannual journal dedicated to advancing telerehabilitation by disseminating information about current research and practices. The journal is PubMed indexed, and was recently accepted into Scopus, with indexing to follow.The International Journal of Telerehabilitation (IJT) explores five diverse topics that fall under the unifying umbrella of telerehabilitation: telehealth privacy and security; the usability of an immersive augmented reality based system with haptics for synchronous remote musculoskeletal examination; parent perspectives of occupational therapy delivered through telehealth; the use of telehealth in early intervention in Colorado; and the use of telerehabilitation as an effective platform to help manage Low-Back Pain (LBP). We thank our authors and are honored to publish their work.The current issue of the multi-disciplinary We are grateful for the generous efforts of the IJT reviewers, many of whom have reviewed prospective IJT articles for over a decade. Sections Editor, William E. Janes, OTD, MSCI, OTR/L has been a very impactful reviewer and support to authors.Thanks also to the publisher of IJT, the University Library System, University of Pittsburgh. IJT is currently sponsored by the Rehabilitation Engineering Research Center on Information and Communication Technology Access at the University of Pittsburgh. The RERC is funded by the National Institute on Disability, Independent Living, and Rehabilitation Research (NIDILRR). Volumes 1–7 were sponsored by the Rehabilitation Engineering Research Center (RERC) on Telerehabilitation at the University of Pittsburgh.We cordially invite submissions to the Fall 2019 issue by mid-September 2019. IJT accepts original research, case studies, viewpoints, technology reviews, book reviews, and country reports that detail the status of telerehabilitation.Sincerely,Ellen R. Cohn, PhD, CCC-SLP, ASHA-FIJT EditorJana Cason, DHSc, OTR/L, FAOTASenior Associate Editor


Tcea3. Myogenin binds to the Tcea3 promoter and is required to recruit RNA polymerase II. TCEA3 can bind to both myogenin and MyoD and is co-recruited with the MRFs to promoters dependent on the MRFs. Depletion of myogenin inhibits the recruitment of TCEA3, suggesting that the interaction of TCEA3 with the MRFs serves to aid in recruitment to target promoters. Like TFIIS, we show that TCEA3 interacts with RNA polymerase II. TCEA3 travels with the elongating RNA polymerase II in the coding region of genes and depletions of TCEA3 inhibit the recruitment of RNA polymerase II to promoters. In proliferating cells, TCEA3 expressed at low levels and is present in both the nucleus and cytoplasm. However, upon differentiation, TCEA3 is upregulated and transported exclusively to the nucleus. Thus, our data show that TCEA3 is a required co-factor for MRF driven gene expression during myogenesis.The transcription elongation factor TFIIS is encoded by a three member gene family in vertebrates. Here we show that one member of this family, TCEA3, is upregulated during skeletal muscle differentiation and acts to promote gene activation by the myogenic regulatory family of transcription factors, which includes MyoD and myogenin. We show that myogenin is a direct regulator of  TCEA1, TCEA2 and TCEA3 . ThWe also see that TCEA3 can modestly promote the activation of MYOD1, which activates MYOG. Thus, TCEA3 reinforces its own expression through this feed forward loop. Our results also suggest that maintaining productive elongation by RNAPII is a critical function in myogenesis. While genome wide gene expression was not assayed, every MYOG dependent gene examined in this study was severely down regulated in the absence of TCEA3, suggesting that the ability to overcome transcriptional arrest sites is required for many muscle specific gene coding units. Skeletal muscle cells also express the ubiquitous TCEA1, but clearly TCEA3 seems to have unique function as a MYOG dependent co-factor for the MRFs in promoting skeletal muscle differentiation that cannot be compensated for by additional transcription elongation factors such as TCEA1. The translocation of TCEA3 upon differentiation is intriguing and suggests that this an additional mechanism to prevent precocious activation of the MRFs and assure precise control of myogenic differentiation.S1 Table(DOCX)Click here for additional data file.


One of the approaches by which the scientific community is seeking to cure HIV is the use of therapeutic vaccination. Previous studies have highlighted the importance of the virus-specific CD8+ T cell cytotoxic responses for the immune control of HIV and have oriented research on vaccine constructs based on CTL epitopes from circulating HIV-1 strains. The clinical trials with therapeutic vaccines to date have had limited success likely due to (i) a discrepancy between archived CTL epitopes in the viral reservoir and those in circulating viruses before antiretroviral therapy (ART) initiation and (ii) the lack of strong affinity between the selected CTL epitopes and the HLA grooves for presentation to CD8+ cells. To overcome these limitations, we launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles of aviremic patients, most of whom are under ART. The near full-length genomes or Gag, Pol and Nef regions of proviral DNA were sequenced by Sanger and/or Next Generation Sequencing (NGS). The HLA-A and B alleles were defined by NGS or molecular analysis. The TuTuGenetics software, which moves a sliding window of 8 to 10 amino acids through the amino acid alignment, was combined with the Immune Epitope Data Base (IEDB) to automatically calculate the theoretical binding affinity of identified epitopes to the HLA alleles for each individual. We identified 15 conserved epitopes in Pol (11), Gag (3), and Nef (1) according to their potential presentation by the dominant HLA-A and B alleles and now propose to use the corresponding conserved peptides in a multi-epitopic vaccine . HIV-1 infection is a chronic infection that induces a steady decrease in CD4+ T lymphocytes and leads to acquired immunodeficiency syndrome (AIDS). The availability of combination antiretroviral therapies (cART) has made it possible to control viral replication in treated patients, resulting in at least partial reconstitution of their immune defenses and allowing them to lead a largely normal life. However, these treatments are costly and have side effects. Trials in which treatment was discontinued were followed by a rebound of viral replication  and hideThe scientific and medical communities are now seeking to cure HIV. Several pathways have been or are being explored ,4, one oSome comments can be made at this point: (i) most of the vaccine constructs, whether they are based on recombinant viruses, plasmid DNA or lipopeptides, are designed on generic HIV-1 viruses and circulating strains may exhibit variations (depending on subtypes or not) especially regarding the CTL epitopes (ii) the virus resuming after discontinuation of cART originates from the archived proviral DNA and our team ,16 plus We previously raised the hypothesis of a vaccine strategy where the CTL epitopes of the archived HIV DNA plus the HLA alleles of every patient would be identified, followed by theoretical matching of the epitopes for their affinity to the HLA grooves before the synthesis of “patient-specific lipopeptides”. However, this strategy may pose significant hurdles from an industrial point of view as personalized vaccines would require individual immunogen sequences to be synthetized.Gag, Pol as well as Nef where some potentially beneficial epitopes have been described . A summary of the patients’ main characteristics together with the technical strategy used in the study is presented at the end of “Materials and Methods”.With the exception of eight elite controllers in Baltimore, all patients  have been on successful cART for at least 6 months. The patients in Montreal were close to primary infection and their biological follow up has been previously presented : the DNAThe most frequent HLA A and B alleles in the population studied are noted in The HIV-1 B subtype was globally predominant but with some local differences: while B was the only subtype noted in Baltimore, Lima and Montreal, other viruses were observed in Rio de Janeiro  and in Bordeaux, where the pattern was more complex . In Bordeaux, CRF02_AG accounted for more than 10% of the samples tested.50 values for the affinity between epitopes and HLA alleles were above the threshold of 50 nm for MHC IC50. The 15 epitopes have a length ranging from 8 to 10 residues, most of them (9 out of 15) being composed of 9 residues. Pol02 (FLWMGYEL) is not described in the Los Alamos database; Pol03 (YELHPDKWTV), Pol06 (TVQPIVLPEK) and Pol07 (DVGDAYFSV) are largely overlapping with described epitopes with a shift of one or two residues compared to epitopes noted in the HIV database; Pol07 (DVGDAYFSV) has a theoretical affinity for A*68:02 (MHC IC50: 20.2 nM) while it is listed in the database as having affinity for A*02:01; Pol10 (SMTKILEPF) was found to have an affinity for the HLA allele B*15:01 (MHC IC50: 24.8 nM) in our in silico study, while no HLA allele is mentioned in the database. The other epitopes are already referred to in the database along with a specific HLA class I restriction and published in vitro functional immunological data.From all the data obtained, we designated 15 conserved epitopes; 11 in RT, three in Gag and one in Nef according to their potential presentation by the A*02:01 (5 epitopes), A*24:02 (one epitope), A*03:01 (2 epitopes), A*11:01 (2 epitopes), A*68:02 (one epitope) and HLA-B*07:02 (2 epitopes), B*08:01 (one epitope), B*35:01 (2 epitopes), B*15:01 alleles (one epitope). The dati.e. coding for conserved CTL epitopes according to the HLA groove) was not frequently associated per patient with variants exhibiting an MHC IC50 > 50 nm, we analyzed these areas directly using the NGS Illumina sequences from Rio de Janeiro, while, in Bordeaux, we analysed first the Sanger data followed by further investigation using NGS (PGM) 50 < 50 nm and some variants close to this threshold (e.g. Gag 01). In some rare cases, there were some variant epitopes (e.g. Gag 02) with a predicted IC50 > 500 nm but with a low percentage (around 1%) in the subspecies per individual. Globally, the results seemed encouraging so we validated the above mentioned list to synthesize the corresponding peptides, which will be used in the following part of the research program. Since there is an inversed relationship between similarity of HIV to its host and its immunogenicity [To be sure that the variability at sequence areas of interest . The epitope KLVDFRELNK at RT 73–82 (Pol05) can host DRM 74 V/I to abacavir and tenofovir (in association with other DRMs), but the switch of residue 74 L to V or I has no theoretical consequence on the affinity to HLA-A alleles A*03:01 and A*11:01. The epitopes RT 107–115 (Pol11) and 110–118 (Pol07) may host the mutation 115F to abacavir but without any negative consequence on presentation by alleles B*35:01 and A*68:02, respectively.Five epitopes identified here overlapped positions that have been related to drug resistance mutations (DRMs), here exclusively to NRTI and NNRTI since they are located in RT: Pol01, Pol02, Pol05, Pol07 and Pol11. There were few DRMs in these areas of the investigated proviral DNA and, when observed, they had no consequence on the theoretical affinity of the epitope for the HLA groove. We then simulated some DRMs in selected epitopes. For example, epitope RT 181–189 (Pol01) may face some DRMs to 3TC/FTC and to NNRTIs at residues 181 (Y to C/I/V), 184 (M to V/I) or 188 (Y to C/H/L). The 184V DRM alone does not impair the affinity of the epitope for the HLA-A*02:01. Cumulated DRMs 181C, 184V plus 188C theoretically cancel this affinity  for HLA-A*02:01. As a consequence, our conservative approach may have missed some potential epitope candidates which may limit the breath of responses upon vaccination. It would be certainly interesting to explore the immunogenicity of epitopes with higher binding affinity, including those up to 500nm threshold which has been suggested to provide a relevant cut-off for effective CTL epitopes [Another issue is the conservation of epitopes. The fact that most of the conserved CTL epitopes in RT are located in the thumb or palm of the enzyme implies some potential functional constraints that limit their variability. The range of variation of the epitopes in Gag, Pol and Nef might also be limited by other unknown constraints, as it has been pointed out for Nef . Furtheron phase . In factepitopes .Furthermore, it remains to be shown that T cell responses to the conserved epitopes identified here have antiviral activity in vivo. As mentioned above, at least some HIV-infected CD4+ cells in cART-treated patients are able to translate Gag, Pol and Nef proteins although they do not contain a fully replication competent provirus . This woAnother issue is the efficacy of CD8+ T-cell stimulation in cART-treated patients. It is now accepted that “T-cell exhaustion” occurs in chronic untreated HIV infections, which impairs the response of CD8+ T-cells to antigens and maybe those cited above ,26. Duriversus the GALT is unclear. Some published data seem to indicate at least that HIV-1 does not evolve in this compartment under efficient cART [An additional factor that may hamper the in vivo efficacy of response to the above defined epitopes is the compartmentalization of host immune responses to HIV. For example, the GALT is an important anatomic reservoir of the virus and it has been demonstrated that resident CD8+ T-cells (Trm) are expanded in the tissues and do not circulate, so they cannot be investigated in the blood for their functional properties . As yet ent cART . At presIn conclusion, we have screened more than 200 patients’HIV proviral sequences for conserved CTL epitopes presented by the most common HLA class I alleles of the studied population. Some of these epitopes have been described in the listing of optimal CTL epitopes of the Los Alamos Immunology database; others are newly described epitopes resulting from our in-silico analysis. The reasons for the conservation of these epitopes may be due to functional constraints. Alternatively, however, they may be conserved since they are not targeted or “underutilized” by the immune system during the natural infection  and are The study was authorized by the “Comité de protection des personnes du Sud-Ouest et Outremer” (DC 2012/48). Patients provided informed consent for participation and the written form of consent; samples were anonymized.Samples from 202 patients were obtained from sites in Europe, North America and South America. Their characteristics are detailed in Extraction of total DNA and quantitation of proviral DNA were carried out as in our previous studies ,16. CD4+The method used for the molecular characterization of HLA alleles A and B in the Bordeaux and Montreal cohorts has been described previously ,16. For 1s protocol. Libraries were quantified by fluorescence using the Qubit dsDNA HS Assay Kit  and diluted to 4 nM according to the mean size and quantification of each library. Products were sequenced in an Illumina MiSeq platform using 1% denatured PhiX DNA as a sequencing control. The HLA alleles were typed, commonly at the six-digit resolution (some with eight-digit), using the commercial software HLA-Twin .In Brazil, HLA class I typing was carried out by Illumina NGS sequencing. Briefly, separate amplifications of the HLA-A, B and C loci comprising the promoter region and exons 1–7 were performed; the fragments obtained have 5.4, 4.6 and 4.8 kB, respectively. They were subsequently quantified and pooled per sample at a final concentration of 4 nM for the construction of libraries using the Nextera XT DNA Sample Preparation kit  according to the manufacturerNef 1–5 (5’-3’: GCCACAGCCATAGCAGTAGCTGAGGGG) and Nef 1–3 (5’-3’: CCAGTACAGGCAAAAAGCAGCTGCTTATA) for outer PCR and Nef 2–5 (5’-3’: CCTAGAAGAATAAGACAGGGCTTGGAAAG) and Nef 2–3 (5’-3’: CGCCTCCCTGGAAAGTCCCCAGCGG) for inner PCR.Proviral DNA Gag and Pol sequencing was carried out followed by Sanger sequencing, as described in previous Provir studies ,16. NearThe sequencing of samples from Bordeaux and Montreal using 454 Roche technology has already been described ,16. For 50 value according to each HLA allele of each patient to identify theoretically new HLA-epitope pairs. In order to identify potential epitopes with high binding affinity and conserved sequence across the available DNA sequence dataset, a conservative cut-off was applied so that only those epitopes with a theoretical MHC IC50 < 50nm binding affinity and observed in >80% of all sequences analyzed were included.Using our TutuGenetics software , we were


New international enterprises that are referred to as Born Globals have become the subject of research due to the success of their global operations, despite their early internationalization and limited resources. Given the importance of analyzing the characteristics that contribute to the success of Born Globals, our study examines the influence of international orientation on export performance. Additionally, we consider internal and external drivers for early and accelerated internationalization such as the Born Global's innovative capacity, the dynamism of the market and the favorability of the environment. By estimating a structural equation model, the results show that international orientation is a strong driver of the export performance of Born Globals. However, this relationship is moderated by innovative capacity and the dynamism and favorability of the environment, thus achieving a greater effect on export performance when international orientation is complemented by these variables. Business; Market dynamism; Export performance; Innovative capacity; International orientation One of The internal capabilities and competences are the most important aspects for small firms to succeed in international markets . This isConsidering both the internal push and the external pull effects of internationalization , we are Altogether, in this study we are interested in exploring how BGs achieve superior international performance from a capability perspective. We thereby focus on international orientation and its relationship to export performance considering the impact of internal and external drivers on this relationship. Doing so, we answer calls for more research that relates to: 1) Providing explanations of how BGs are able to achieve precocious internationalization and superior international performance ; 2) AnalIn the following section we derive the conceptual model based on the review of literature. This is followed by a description of the methodology in section 2Exporting remains an important strategic option for firms to internationalize and is the most frequently used entry method due to its cost-effectiveness and high flexibility as compared to other foreign market entry modes such as foreign direct investments . ExportiStudies on the export performance in BGs focus on capabilities for entering and operating in international markets that include entrepreneurial skills to exploit resources and capacities, acquiring new technologies and entering new markets, and adapting such resources and capabilities to take advantage of market opportunities .Taking into consideration that we are interested in understanding how BGs achieve superior international performance, we are adopting a capability perspective. Earlier research shows that internal capabilities are able to offset the resource-disadvantage of smaller firms for achieving early and rapid internationalization particularly regarding tangible resources . This peAccording to the resource-based view of the firm , in ordeAmong the strategies that are adopted by BGs in order to achieve early and rapid internationalization, an international orientation is a critical factor as it represents the mentality and the attitude of the entrepreneur and decision-maker for international expansion . An inteInternational orientation refers to entrepreneurs' attitudes and the assignment of resources to international activities . InternaIn our study, we adopted the definition by Innovative capacity is considered to be a strategic element in firms as it allows the development of practices that are able to promote innovative activities among employees . InnovatRelated to the external drivers for firm performance, According to 2.1Previous research proposes a positive relationship between international orientation and export performance , suggestStudies find that an international orientation provides the ability of a firm to enter international markets . SorenseHypothesis 1An international orientation has a positive effect on the export performance of BGs.The international orientation of the founders, managers and employees of BGs allows them to acquire the abilities that are necessary to maintain their competitive advantage. This makes them more willing to assume risk when they expand into new markets and improves their export performance . Given t2.22.2.1The resource-based view proposes that the importance of internal resources is a key factor for achieving a competitive advantage. Hypothesis 2Innovative capacity positively moderates the relationship between an international orientation and export performance in BGs.2.2.2Besides the resource-based view that focuses on the internal factors that drive export performance, our study also considers institutional theory. We postulate that the influence of environmental factors on the BG's behavior is based on the influence of market dynamics and the favorability of the environment in the relationship between international orientation and export performance. The social, economic and political factors that frame society determine the survival of companies in the market and their decision-making processes . Thus, mHypothesis 3Market dynamism positively moderates the relationship between an international orientation and export performances in BGs.BGs can be confronted with greater problems in dynamic markets because such firms may not have yet developed response mechanisms to changes due to their recent creation and early internationalization. These problems may also stem from the fact that they are still at the stage of learning about the market . Given t2.2.3As noted previously, markets are subject to constant change and firms must evaluate their capacity to adapt to these changes. In international business environments, changes in global markets tend to be more dynamic because firms must adapt their processes to the regulatory framework of each country and the varying preferences of consumers . In thisHypothesis 4The favorability of the business environment positively moderates the relationship between an international orientation and export performance in BGs.Previous research identified that favorable environments mean better access for firms to international markets. Firms often promote strategies that focus on obtaining better results because managers’ positive perceptions about their business environment can help them to acquire new commitments and to create better conditions within which to improve management practices in international activities . Thus, i33.1Our study focuses on 112 exporters in major cities in Colombia (South America). The firms were classified based on three requirements for BGs defined by 3.1.1To collect the data, a questionnaire was developed that consisted of a set of general information questions on the firms, followed by the measurement scales that were taken from the literature. To measure international focus, we considered the scale that was developed by Independent of the described variables, the other specific characteristics of a firm can also assert an impact on export performance. Therefore, we used firm size and international experience of the firm as control variables. We assumed that both variables positively affect export performance. Firm size was measured using the number of employees, and international experience was determined using the number of years that a firm has operated in international markets. For the regression analysis, we used a logarithmic scale to transform these variables and to correct asymmetries .3.22(485) = 799.82; GFI = 0.88; RMSEA = 0.041; SRMR = 0.047; CFI = 0.95; TLI (NNFI) = 0.95. To analyze the psychometric properties of the scales that were used in this study, we conducted a correlation analysis and an exploratory factor analysis for each scale to identify its fit and unidimensionality. We then conducted a confirmatory factor analysis to assess the convergent and discriminate validity of the constructs. The factor analysis indicates that the data have a reasonable fit, and all of the measures show adequate reliability as measured by compound reliability indices (SCR) that exceed 0.6  and aver4To rule out the presence of multicollinearity in the indicators that make up each construct value's factor inflation variance (VIF), condition indices that are well below the threshold of 10 and not as close to 0 are used (greater than 0.1); they indicated no multicollinearity problems . AdditioA Harman factor was used through the potential barrier of common method variance, which is a frequent problem when all of the variables are measured with the same instrument (questionnaire), which can potentially lead to the variance being attributed to the method and not the constructs . TherefoMoreover, it is important to note that the survey respondents expressed interest in the study and its subsequent results, and therefore their responses may prevent response bias. Additionally, a high response rate was achieved due to the development of surveys and forecasts some respondents found to be personally unacceptable or they were unable to answer or they simply were not available to respond. In this sense, a subset of the companies that met the conditions of being born global was selected.However, although we made a great effort to avoid problems of bias, it is clear that these challenges are inherent in studies of this nature, but they do not invalidate the results.2 = 0,28; R2 adjusted = 0,28). When we included the moderating effects, the R2 value increased by 8,9% (p < 0.001). The results of the final model that includes the main effects and the moderating effects are presented in The model estimation was performed using a hierarchical linear regression see . Before To interpret the results of the main effects and the moderating effects, we followed the procedures that were proposed by Figs. The results show that IO is more effective on EP when it is accompanied by high levels of IC. Thus, the variables seem to complement each other, as postulated in Moreover, for high values of IO (+1.4) and a low MD (-0.862), the effect of IO on EP is -2.924 with a standard error of 1.23 and a t-value of -2.38 (P < 0.05). For high values of MD (+0.862), the effect of IO on EP is 0.22 with a standard error of 0.944 and a t-value of -0.23 (P > 0.1). In all of the IO levels are considered, we obtain a difference in the effect of IF on EP between the low levels and high levels of MD. This difference is 3.14, and t = 3.309 (P < 0.01). This result allows us to confirm Finally, according to Moreover, for high values of IO (+1.4) and low values of FE (-0.957), the effect of IO on EP is -3.045 with a standard error of 1.46 and a t-value of -2.08 (P < 0.05). For high values of FS (+0.957), the effect of IO on EP is 0.55 with a standard error of 0.996 and a t-value of -0.55 (P > 0.1). At all of the IO levels considered, we obtain a difference in the effect of IO on EP between low levels and high levels of FS. This difference is 4.15, t = 3.819 (P < 0.01). This result allows us to confirm In summary, this study contributes to the literature because it enriches our knowledge of BGs and export performance and their determining factors. Additionally, the study allows us to shed light on other research that assumes that an international orientation is a strategy . It furt5The purpose of our study consisted in exploring empirically how BGs achieve superior international performance from a capability perspective. Particularly we were interested in the relationship between the BGs international orientation and its export performance mediated, on the one hand, by its innovation capacity , and, on the other hand, by the market dynamism and the favorability of the business environment .Through the conceptual model we have chosen and the empirical context of our research, we make the following contributions to research on the phenomenon of BGs. Firstly, our research is the first to use the construct of international orientation in the context of BGs. We therefore provide important explanations of how BGs are able to achieve superior export performance through particular capability antecedents . SecondlIn the following we present a summary of the results, followed by a discussion of the theoretical contributions, and conclude with the managerial implications of our findings.5.1We predicted a positive relationship between the BGs international orientation and its export performance and the result supports this prediction. The results further show that international orientation has a greater effect on export performance through the internal and external mediator variables introduced into the model. Related to the innovative capacity of the BG as an internal firm capability, our results indicate that international orientation is more effective on export performance when it is accompanied by high levels of innovative capacity. Related to the market dynamism as an external market factor, our results indicate that the positive relationship of the international orientation on export performance are subject to the high market dynamism where BGs operate in. Related to the favorability of the business environment as an external factor, our results show that the more favorable the business environment in which BGs operate, the greater the positive influence of an international orientation on export performance. Hence, the results confirm the moderating effect of all internal and external variables on the relationship between international orientation and export performance.5.2Our results confirm the findings of literature that establish a strong and positive relationship between the firm's capabilities and its export performance . To the The international orientation of the BG therefore constitutes an important intangible resource that contributes to the sustained competitiveness of the firm . FurtherBesides, establishing the international orientation as an important internal capability able to contribute to superior export performance of the BG, we provide insights into its moderating effects. Moderating effects are important as both internal and external drivers are found to impact the early and accelerated internationalization of new ventures .In line with previous export literature  and BGs The effect of a diminishing return is most evident for high levels of international orientation at high levels of market dynamism see . This meThe differences in these diminishing effects is best explained by the nature of markets where BGs are operating in. Market growth is generally considered to have a positive impact on the firm's international performance . A favouOur current understanding on BGs focuses largely on the description of their formation and to a lesser extent on the determinants of their performance . Our resThe country context of our BGs also contributes to a better understanding of this type of firm from a developing country perspective (i.e. Colombia). As noted by many , we lack5.3Developing a global vision, where the firm sees the world, not just Colombia, as its market, is most probably the most important determinant for export success of Colombian BGs. Founders and managers of these firms are advised to develop a global outlook for their operations and to promote vigorously the exploration of new business opportunities on export markets. Policy makers can contribute to the development of an international orientation by promoting exports and facilitate international trade fair participation and export missions to potential firms. The development of an international orientation as a firm capability should most probably be one of the most important development objectives for export promotion agencies in Colombia as it helps to offset the resource scarcity of these small and medium-sized firms in order to make these firms more competitive in export markets.However, the sole commitment towards export markets is not sufficient. The firm also needs to develop an innovative capacity and target appropiate markets. Compared to the external factors of the environment, the development of a capability for innovation is more challenging for the BG. It is similar to the international orientation, an intangible resource that needs to be developed over time and requires constant attention in order to be maintained as a capability. The environmental drivers are easier to control. Nevertheless, spotting attractive international markets requires significant and constant attention on behalf of the managers of BGs. Policy makers can assist firms by providing necessary market intelligence in order to survey international markets for their trends and growth potential.Due to the diminishing effects on export performance we observed in our results, founders and managers of Colombian BGs should be aware that the development of an international orientation reaches a saturation point. Beyond that point, the contribution of additional international orientation does not contribute significantly to more export performance. Hence, the development of an international orientation reaches a tipping point and no further investments for strengthening the international orientation are necessary.Our study, like all empirical quantitative research, is subject to limitations. We are recognizing that our quantitative method have two main limitations. First, hierarquical model is a transverse method and does not include a dynamic behavior for our variables and this information might be associated a punctual economic effect. Other hand, this method has a high tendency to increase our results, especially R2 adjust, due to it has more variables.Future research should consider the following aspects building on the findings of our study. First, we researched capabilities and external drivers applying a static perspective. The survey instrument we used allowed us to construct a one-shot image of the current relationship between international orientation ad export performance. However, we were not able to provide insights into the dynamics of capabilities. Capabilities are subject to change and development . Future Second, the focus of our research has been on a particular capability – the international orientation of the BG. Other capabilities such as a learning orientation, market orientation, international entrepreneurial orientation, and international growth orientation have shown to be positively related to the international performance of BGs . These cThird, our sample consists of BGs from Colombia. In Latin America, Colombian and Chilean entrepreneurs particularly show a high international orientation . TherefoFinally, the institutional environment is important to keep into consideration for the study of BGs . The impD. Escandon-Barbosa, J. Rialp-Criado, S. Fuerst, G. Castro-Aristizabal: Conceived and designed the analysis; Analyzed and interpreted the data; Wrote the paper.A. Rodriguez-Orejuela: Contributed analysis tools or data.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.