Pitp, Pi33, Pi54, Pib, Pi20, Pi38, Pita2, Pi1, Piz, Pi9, Pizt, and Pi40) with linked as well as gene-specific markers, identified 84 resistant landraces possessing different gene(s) either in singly or in combinations and also identified seven resistant landraces which do not have the tested genes, indicating the valuable genetic resources for blast resistance. To understand the molecular diversity existing in the population, distance and model based analysis were performed using 120 SSR markers. Results of both analyses are highly correlated by forming two distinct subgroups and the existence of high diversity  was observed. To practically utilize the diversity in the breeding program, a robust core set having an efficiency index of 0.82 which consists of 33 landraces were identified through data of molecular, blast phenotyping, and important agro-morphological traits. The association of eight novel SSR markers for important agronomic traits which includes leaf and neck blast resistance was determined using genome-wide association analysis. The current study focuses on identifying novel resources having field resistance to blast as well as markers which can be explored in rice improvement programs. It also entails the development of a core set which can aid in representing the entire diversity for efficiently harnessing its properties to broaden the gene pool of rice.North-East (NE) India, the probable origin of rice has diverse genetic resources. Many rice landraces of NE India were not yet characterized for blast resistance. A set of 232 landraces of NE India, were screened for field resistance at two different hotspots of rice blast, viz., IIRR-UBN, Hyderabad and ICAR-NEH, Manipur in two consecutive seasons. The phenotypic evaluation as well as gene profiling for 12 major blast resistance genes ( Magnaporthe oryzae. It infects rice leaves, nodes, color, and panicles at different stages of crop growth thus, decreasing the overall yield. In India, neck blast is becoming severe in many agro-ecological zones and causing more threats to the rice production (Pb1) has been reported for neck blast  controlling key traits and the genotype–phenotype associations in germplasm. For the successful application of association mapping, understanding of population structure is essential to avoid type I and type II errors between molecular markers and traits of interest . DetermiIn the current study, we have systematically performed phenotyping for blast resistance in a span of two years. Phenotyping along with gene profiling for major blast genes identified novel genetic resources which might contain the novel resistance genes. For clear understanding, the genetic diversity of NE landraces were analyzed with distance as well as model based diversity approaches. To apply the existing diversity in breeding programs, a core set was derived. Using 232 landraces, the marker–trait association was identified for blast resistance as well as yield-related traits. The information generated in the present study has a lot of value in the current rice improvement programs.Figure 1) for the current study. Along with the landraces, three varieties of Basmati, five varieties of Indica, seven varieties of Temperate Japonica, and five varieties of Tropical Japonica were also selected for the comparison of landrace alleles. The line harboring particular blast resistance gene was considered as a positive control, and the susceptible cultivars namely BPT, Swarna, and CO39 which do not have any blast resistant gene were considered as negative controls for gene profiling.A total of 232 landraces  for phenotyping of leaf blast disease at ICAR-IIRR, Hyderabad, India. The entire nursery block, as well as each entry, was surrounded by HR12 (susceptible cultivar) on four sides for spreading the disease evenly. A mixture of elangana  were useelangana . Scores  syringe . Disease syringe . The higThe landraces were grown in a block prepared by uniform distribution of soil enriched with nutrients required for the plant growth. A spacing of 20 cm between each row and 15 cm between plants was adopted at the time of planting. Twenty plants of each landrace were sown in rows and the data was recorded for five selected plants. Data was taken on various parameters like plant height, presence and absence of awns, seed color, tiller number, and spikelet number. At the time of harvesting, biomass and yield per plant were also recorded.DNA was isolated from the leaf tissue by following Murray’s  protocolPitp, Pi33, Pi54, Pib, Pi20, Pi38, Pita2, Pi1, Piz, Pi9, Pizt, and Pi40 using molecular markers as described in Pi54, Pi40, Pita2, Pi9, Piz, and Pizt and the remaining genes were profiled with tightly linked SSR markers. The markers used for the gene profiling and positive controls for respective genes are listed in Supplementary Table PCR analysis was done for landraces together with the positive controls and susceptible checks to identify the presence of 12 major known blast resistance genes namely For diversity analysis, all the alleles derived from each SSR marker were scored as “1” for the presence of amplicons and “0” for its absence in the respective allele position. The size of the amplicons was also determined based on their migration relative to standard molecular weight marker (50 bp DNA ladder from Fermentas). Allele number, allele frequency, heterozygosity, and polymorphic information content (PIC) values for all the markers were calculated using Power marker V3.25 software .For clustering of genotypes, a pairwise distance matrix was computed using DARwin V6 software. An unweighted neighbor-joining tree was constructed using dissimilarity index method. The genetic distance was calculated by Jaccard’s coefficient with bootstrap analysis of 1000 iterations using the SSR marker data .K value. For accurate determination of subpopulations, admixture model was followed. In this model, the burnin period length was set to as high as 150,000 with Markov Chain Monte Carlo (MCMC) 150,000 replications. Each K value was run for 10 times with K value varying from 1 to 25. To obtain clear peak at the ΔK (ad hoc quantity) to K value, the output generated from structure software was loaded in structure harvester (http://taylor0.biology.ucla.edu/structureHarvester/).The structure of the population was determined by a Bayesian-based approach using Structure V2.3.4 software . The numFST and the principal component analysis (PCoA) to characterize the subgroups of the germplasm set was computed using GenAlEx 6.5 software . The estimation of AMOVA based on Nei’s distance matrix  along wisoftware .A mini-core collection was developed using Powercore V1.0 software . For botK value in order to minimize the false positive associations  and mixed linear model (MLM) using TASSEL V3.0 . GLM wasciations . The sigThe stringent blast screening of 232 landraces at two locations in the year 2012 revealed 166 landraces showing resistance to leaf blast and 148 to neck blast. Whereas in 2013, 172 landraces were resistant to leaf blast and 154 to neck blast. On average, 153 landraces showed resistance to leaf blast and 135 landraces resistance to neck blast and interestingly, 91 showed resistance to both leaf and neck blast. The mean scores of resistant landraces ranged between 0 and 3 while the susceptible check HR12 gave the score of 9.Pi9 was found in the maximum number of landraces, i.e., 102 (43.9%), while the Pi20 gene is present in the least, i.e., 11 landraces (4.74%). One unique landrace Apaghi june found to be a rich source for blast genes as it has seven genes, viz., Pi9, Piz, Pi33, Pib, Pi38, Pi40, and Pi54. Another landrace, i.e., Chalhtssia has six genes, viz., Pi1, Pi9, Pi54, Pib, Pitp, and Piz. Seven landraces have the combinations of five genes whereas 14 landraces have four gene combinations. Similarly, the landraces containing three gene combinations, two gene combinations and single genes were also identified; these details are given in Supplementary Table Figure 2). Among the 91 blast resistant landraces, seven landraces, viz., Meghalaya lakang, Chingphourel, Manuikhamei, Kemenya kepeyu, Wainem, Thekrulha, and Koyajang do not have any of the tested major blast resistant genes .The gene profiling of landraces revealed that Diversity analysis with the allelic data generated from 120 SSR markers detected a total of 360 alleles, as the alleles per loci ranged from 2 to 8 with an average of 3 alleles per locus. Among the total alleles, 15% rare alleles  were found. The SSR marker RM22837 (Chr-8) recorded the highest number of alleles (8), which indicates its high polymorphic nature, while many markers showed two alleles. The major allele frequency ranged from 0.31 to 0.98 with an average of 0.59 per locus, and the average observed heterozygosity was 0.09. Where as expected heterozygosity ranged from 0.05 to 0.80 with an average of 0.51 per locus. The relative informativeness of each marker has been identified by PIC value, highest PIC value (0.77) was found for RM22837 while least (0.05) for RM21052 with an average PIC value of 0.44 for markers .The neighbor-joining tree showed the presence of two distinct major clusters, of which 59 landraces grouped in cluster-1 and 173 landraces in cluster-2. The cluster-1 is again divided into four subclusters—1A, 1B, 1C, and 1D, whereas cluster-2 divided into five subclusters, i.e., 2A, 2B, 2C, 2D, and 2E. The landraces present in the subcluster 1B shares alleles with Japonica and Tropical Japonica varieties while landraces present in 1C shares with Japonica and Basmati accessions. The landraces present in the subcluster 2D shares with Indica, Japonica, and Tropical Japonica varieties while landraces of 2E shares with Japonica and Basmati accessions. The sub-clusters 1A, 1D, 2A, 2B, and 2C formed exclusively with landraces (K) maximized to the highest value of 2260.4 at K = 2 , showing a clear peak indicating the classification of entire population into two distinct subgroups SG1 (subgroup1) and SG2 (subgroup2) . Among the 232 landraces, 33(14.2%) landraces were categorized under SG1 whereas the majority of the landraces that is 158 (68.1%) were grouped in SG2, and the remaining 41(17.7%) landraces were grouped as admixture. Subgroup2 which has a high number of landraces (158) was further subjected to structure analysis. This analysis revealed the classification into four sub-subgroups (SSG) .The distribution of molecular variance exposed significant genetic differentiation between and within populations among the landraces. Molecular variation of 75.1% was observed among the individuals within the subgroup (population) and 24.9% variation was observed among the subgroups. The pairwise The PCoA was performed for characterizing the subgroups of landraces. The first two-dimensional scatter plots described the PCoA axes accounting for 12.48 and 4.04% of the genetic variation among subpopulations formed from 252 accessions  that represent the core set  present on chromosome-5 showed association with leaf blast with a significant r2 value of 0.21 and at this location, blast gene Pi26 was reported. Similarly, RM5401 located on chromosome 5 having an r2 value of 0.21 is associated with the neck blast resistance where no single QTL for blast resistance was reported. Two markers (RM21936 and RM3787) located on chromosome 7 and chromosome 9, respectively were associated with both leaf and neck blast resistance. About 15 markers were associated with yield-related traits namely number of tillers, a number of spikelets, plant height, yield per plant, and color. The marker–trait associations and their significance were listed in Table 2. The relative kinship estimates based on the 120 SSR data resulted in 83.3% of the pairwise kinship estimates ranging from 0.25 to 1.0 and the remaining estimates ranged from 1 to 2, with a low number of pairs filling in the higher estimate categories .To identify the marker associated with the blast resistance as well as with other traits, we used 232 landraces for association study. Association analysis of landraces based on GLM and MLM using Tassel software revealed 20 marker–trait associations with a significant Successful rice breeding programs require the presence of genetic diversity in the existing germplasm. The current study focuses on ascertaining this phenomenon in NE Indian landraces. We used a completely unique and unexplored germplasm for identifying the best donors and markers associated with blast resistance. We demarcate the diversity into a manageable core set.Pb1) was identified (Systematic screening for blast disease at two different locations for two consecutive seasons led to the identification of 91 landraces resistant to both leaf and neck blast. Blast screening at different locations increases the consistency of phenotyping as different virulent blast isolates have diverse pathogenic behavior . Previouentified  and manyPi9 was major gene followed by Piz and Pizt existing in landraces. The Pi9 is predicted to be persistent in landraces from a long time as it was originated from the wild species (Oryza minuta) of rice. Piz among 32 NE Indian landraces by gene profiling with STS and SNP markers was reported  were also explored by Figures 2, 3). Identification of new genes having durability and its transfer into a cultivable variety may increase its resistance toward the disease since most of the identified genes are losing their effectiveness to blast disease , which is comparable to most of the diversity panel consisting of global accessions (gene diversity of 0.5–0.7) , 2016. GFigure 4). indica and japonica types based on the distance based clustering . Further population structure analysis in SG2 classified the landraces into four subgroups indicating that independent structure analysis defines the precise genetic structure of the accessions. The results of the unweighted neighbor-joining tree constructed on the basis of UPGMA and PCoA harmonized with the structure analysis of the landraces. The PCoA cited the resistant and susceptible genotypes into four different quadrants at different positions.The distance based clustering analysis grouped the landraces into two major clusters  than the variation present between the populations (24.9%). The Figure 6). The core set developed indicates the homogeneous distribution ranges of the phenotypic traits as MD% is less than 20% and CR% is larger than 80%  with leaf and neck blast disease (Table 2). These markers may be helpful for mapping genes for neck blast with durable resistance which does not exist in the currently available germplasm. Exploring novel gene pool for neck blast resistance and identification of novel genes which can offer resistance to leaf and neck blast resistance has become quite essential. P < 0.01) in a set of 276 indica landraces from China and few from different parts of the world were reported  having resistance to leaf and neck blast can be explored for the discovery of novel genes for broadening the gene pool to combat the pathogen competition. Distance and model based diversity analysis are highly correlated and genetic architecture of diverse material was effectively determined. The rich diversity existing in the landraces were divided into manageable subgroups and identified the molecular variance present among the populations and individuals which can be harnessed in the selection of distant individuals. To utilize the diverse landrace germplasm, an efficient core set which represents the entire diversity was made. Further, association analysis using a core set identified novel eight markers associated with the blast and other agronomically important traits, these markers are the candidates for the validation in the larger populations. The information emanating this work will help in conservation and utilization of valuable rice germplasm of NE India.MSM designed all experiments, developed the structure and arguments for the paper, made critical revisions, and approved the final version. BU and BV carried out the major work and prepared manuscript and contributed to the refinement of the manuscript. PSK helped in analyzing the data and SRD helped in data collection of agro-morphological traits. PS reviewed and approved the final manuscript. VPB, SK, SKS, and PKS helped in the collection and phenotypic screening of rice landraces for neck blast resistance at the North-East region. MSP helped in the screening of leaf blast resistance at IIRR. All authors have read and approved the final manuscript.The authors declare that the research was conducted by the support of DBT, Government of India.


The prefrontal cortex is the superlative structure of brain that needs the longest developmental and maturational duration that highlights the region of attention deficit hyperactivity disorder (ADHD) in neuroimaging studies. Prefrontal cortex functions generate enormously complex and its abundant feedback neurocircuitries with subcortical structures such as striatum and thalamus established through dual neural fibers. These microneurocircuitries are called corticostriatothalamocortical (CSTC) circuits. The CSTC circuits paly an essential role in flexible behaviors. The impaired circuits increase the risk of behavioral and psychological symptoms. ADHD is an especial developmental stage of paediatric disease. It has been reported that the CSTC circuits dysfunctions in ADHD are related to homologous symptoms. This study aimed to review the symptoms of ADHD and discuss the recent advances on the effects of the disease as well as the new progress of treatments with each circuit. Currently, it has been able to establish the corresponding relationship between brain areas and mental symptoms or functional abnormalities and to locate the symptoms in the dimension of the symptoms by neuroimaging techniques. As the most advanced part of brain development, the prefrontal lobe has attracted a large number of related studies including the executive function located in the dorsolateral prefrontal cortex (DLPFC) ; emotionThe prefrontal lobes are not functional separately. They combine with the striatum, thalamus, and cortex structure through the contact fibers to establish the loop structure, playing the overall function. Neurons in the cerebral cortex are connected with many other neurons to form the cortical neural circuits which play the priming effect of brain function. Particularly, the prefrontal cortex has an important impact on mental behavior. This neural network can transform a simple signal into a complex signal, eventually regulating the function and behavior of the brain. The psychiatrist can use a drug or treatment that regulates the function of a neurotransmitter on a particular neural circuit to affect the patient's clinical symptoms, therefore having a better understanding of the pathophysiology of the disease. Meanwhile, accumulating evidence has shown the abnormalities of the corticostriatothalamocortical (CSTC) circuits in ADHD patients –8, whichThe CSTC circuit mediates the transmission of information to “downstream” and leaves the cortex; meanwhile the cortex gets a feedback and determines how to process the information. Neural information is projected from the prefrontal cortex to the striatum and then from the thalamus to the striatum. The thalamus produces local interactions only with specific regions of the cortex. The neural circuits that pass through the striatum can be synapse-linked to the part of the striatum that leaves the striatum to the thalamus, returning to the initial region of prefrontal cortex finally; sometimes, it can return to the original pyramidal cells . The neuWith analysis of the representative nerve circuit in CSTC, we discover that they all initiate and end in cortical pyramidal cells. Since the pyramidal cell is involved in the neural circuitry of the cortical circuit, the neurotransmitters in these pyramidal cells will be affected when receiving some medicines or physical therapies and directly affect the function of these neurons, subsequently providing significant diagnostic and therapeutic effects . TherefoRecently, a total of five circuits in CSTC with vital research value have been summarized . In thisThe DLPFCSTC circuit is involved in regulating sustained attention and problem-solving. It is also known as sustained attention or executive function circuit. Neural impulses in DLPFCSTC circuit originate in the DLPFC and project into the superior caudate nucleus in the striatum, then spread to the thalamus, and finally return to the DLPFC shown in 1H-magnetic resonance spectroscopy (1H-MRS) demonstrates that N-acetylaspartate/creatine (NAA/Cr) value in right DLPFC is positively correlated with learning disabilities of ADHD, while NAA/Cr value in left DLPFC is negatively correlated with the morning behavior. It is indicated that the DLPFC neurometabolities between cerebral hemispheres of ADHD are correlated with different ADHD symptoms and each hemisphere controls its special executive functions [In the study of the circuit, unctions . ADHD paunctions .The VLPFCSTC circuit, also known as emotional circuit, participates in the emotional processing , 19. The1H-MRS confirms the severity of ADHD symptom is negatively correlated with myo-inositol/creatine (ML/Cr) in the right VMPFC and positively correlated with choline/creatine (Cho/Cr) value in the left subcortical striatothalamic area and is negatively correlated with glutamate-glutamine-GABA/creatine (Glx/Cr) value in the left putamen. These results indicate that there is widespread abnormality on the circuit; the significantly decreasing metabolic rate of nerve cells leads to a tendency of serious symptoms [Individuals with congenital malformation in the VLPFC perform the specific ADHD-like symptoms: egocentricity, lack of empathy, lack of respect for authority, impaired moral judgment, poor frustration tolerance, and many apathy symptoms . In the symptoms , 24. Thesymptoms .The ACCSTC circuit, also known as selective attention circuit, is responsible for the emotional regulation and selective attention. The signals of the circuit generate from the ACC and project onto the inferior striatum, then reach the thalamus, and finally return to the ACC shown in Stroop test normally activates the ACC but cannot activate the region in ADHD patients accordingly, and the right ACC thickness is negatively related to the diversity of symptoms . This ciThe MC plays a critical role in regulating motor activity. The MC is classified into primary motor cortex (M1) and secondary motor areas including the premotor cortex (PMC) and supplementary motor area (SMA) . The MCSCommon symptoms of activity in ADHD include fidgeting, leaving one's seat, running/climbing everywhere, and constantly playing without purpose and troublemaking. fMRI has shown that the extent of neural activation in ADHD decreases in the left M1, bilateral PMC, and SMA . FurtherThe OFCSTC circuit, which is called impulsive/compulsive-related circuit, controls impulsive behavior . The nerEither impulsive behavior or impulsive choice is related to the transfer function of dopamine and adrenaline neurotransmitter in the OFC regions which are the therapeutic target of stimulant. Impulsive symptoms of ADHD including hyperlogia, interrupting without thinking, blurting out, and unwilling to wait in sequence are associated with the circuit. Structural covariance network (SCN) reveals that the gray matter volume significantly loses in the right lateral OFC of ADHD . Further1H-MRS treatment for stimulant treated in the DLPFCSTC circuit. In contrast, in treatment-naïve ADHD patients, the lower corpus striatum Glx was significantly associated with more severe symptoms of inattention, and the differences in Glx levels were not due to the use of stimulant medication [Some drugs (methylphenidate and atomoxetine), which aim to increase the dopamine (DA) and noradrenaline (NE) receptor activation levels, have been widely used for ADHD. In the study of PT on the DLPFCSTC and VLPFCSTC circuits, compared with treatment-naïve ADHD, no differences were shown in dication . NIRS indication . Methylpdication , even stdication .1H-MRS showed that the ACC glutamate-glutamine-GABA/myo-inositol (Glx/ML) in ADHD patients who were treated with methylphenidate was significantly lower than that of patients without PT. The central stimulants make ADHD patients able to activate the ACC and thereby interfere with the emotional regulation and selective attention [In the study of medicines for the ACCSTC circuit, ttention .Only few reports have shown that methylphenidate and atomoxetine can activate the SMA , 47, sugStimulant may provoke abuse that impacts on the OFCSTC circuit. Impulsive control is associated with stimulant abuse, thus contributing to ADHD treatment. Atomoxetine directly inhibits NE concentration in the OFCSTC circuit, therefore reducing dopamine function in the same area, and nucleus accumbens has too little NE neuron to increase NE and DA in that region; that is the main reason why atomoxetine differs from methylphenidate for susceptibility of stimulant abuse .It has been reported that NPT can also improve and normalize cortical and subcortical function of ADHD . TranscrIt has not been reported that tDCS is as a therapy in ADHD, but the effect of tDCS has been reported on the other participants. ATDCS in VMPFC and pre-SMA in MC improve participants' inhibitory control and accelerated the stopping efficiency and stopping speed . Also hiTDCS treatment for subcortical structures is limited. Deep brain stimulation (DBS) has been applied for replacing the stereoscopic neurosurgery for the treatment of neural damage. DBS creates high-frequency electrical simulation that is similar to the damage effect of reversible functional nerve block. Clinical effects are obtained by activating nerve axon fibers networks across CSTC circuits; targets could be reachable to the striatum and thalamus . ADHD, O


P < 0.05). Overall, Jungle Fowl were the most tolerant to a rye-based diet, and both the modern and 1995 broilers were significantly affected by the high rye-based diet. However, the 1995 broilers consuming the rye-based diet appeared to experience more permanent effects when compared with the modern broiler. The results of this study suggest that chickens have a great potential as a nutritional rehabilitation model in human trials. The 1995 broilers line was an intermediate genetic line between the fast growing modern line and the non-selected Jungle Fowl line, suggesting that it would be the most appropriate model to study for future studies.This article is the first in a series of manuscripts to evaluate nutritional rehabilitation in chickens as a model to study interventions in children malnutrition . Inclusion of rye in poultry diets induces a nutritional deficit that leads to increased bacterial translocation, intestinal viscosity, and decreased bone mineralization. However, it is unclear the effect of diet on developmental stage or genetic strain. Therefore, the objective was to determine the effects of a rye diet during either the early or late phase of development on performance, bone mineralization, and intestinal morphology across three diverse genetic backgrounds. Modern 2015 (Cobb 500) broiler chicken, 1995 Cobb broiler chicken, and the Giant Jungle Fowl were randomly allocated into four different dietary treatments. Dietary treatments were (1) a control corn-based diet throughout the trial (corn–corn); (2) an early phase malnutrition diet where chicks received a rye-based diet for 10 days, and then switched to the control diet (rye–corn); (3) a malnutrition rye-diet that was fed throughout the trial (rye–rye); and (4) a late phase malnutrition diet where chicks received the control diet for 10 days, and then switched to the rye diet for the last phase (corn–rye). At 10 days of age, chicks were weighed and diets were switched in groups 2 and 4. At day 20 of age, all chickens were weighed and euthanized to collect bone and intestinal samples. Body weight, weight gain, and bone mineralization were different across diet, genetic line, age and all two- and three-way interactions ( Malnutrition is a growing concern as the global population continues to increase due to the increased global demand for food and the long-term effects of malnutrition. Malnutrition is due to the lack of, inadequate nutrition, or inadequate absorption of nutrients. It has been identified as the underlying cause of death in one-third of children under 5 years of age. Of these deaths, 83% are attributed to mild-to-moderate malnutrition compared with severe malnutrition , suggestThe most common manifestation of chronic malnutrition is stunted growth. It is estimated to affect 165 million children under the age of 5 years in low- and middle-income countries  and can Clostridium perfringens and Clostridium difficile proliferation when compared with corn-based diets , which impairs nutrient digestion and absorption due to little or no intrinsic enzymes capable of hydrolysis of NSP in the small intestine. The increased NSP provide more nutrients in the ceca and large intestine which serve as a nutrient source for bacteria. The altered nutrient source can lead to dysbiosis within the gut. In poultry, rye-based diets increased both viscosity and ed diets . Additioed diets , 10–17. Undernutrition of children has profound effects on health and development; nevertheless, the issue is not simply caused by a lack of food, but results from complex interactions of intra- and intergenerational factors , 18. Resad libitum feed intake . For the 1995 broiler chickens, 112 1-day-old mixed broiler chicks, from the random bred line initiated from 1995 Cobb broiler chicken line (n = 28 chickens/group). And for the Jungle Fowl chickens, 160 1-day-old mixed Giant Jungle Fowl (n = 40 chickens/group). On the day of hatch, chickens were neck-tagged, weighed, and randomly allocated to one of four dietary treatment groups in floor pens containing new pine shavings in an environmentally controlled room. All diets were antibiotic-free and formulated to meet or exceed the current broiler nutritional requirements according to the National Research Council  where tP < 0.0001) regardless of dietary treatment and genetic line. There was a significant difference in BW between all treatment groups, where the corn–corn group (P < 0.0001) had the highest BW, and the rye–rye group (P < 0.0001) had the lowest BW. Modern broilers were the heaviest, followed by the 1995 broilers and then the Jungle Fowl (P < 0.0001).The results of the evaluation of a nutritional rehabilitation model on BW in three genetic chicken lines fed rye or corn at varying time points are summarized in Table P < 0.0001), where modern broilers in the corn–corn group were heaviest (P < 0.0001) and Jungle Fowl in the rye–rye group were the lightest (P < 0.0001). Across genetic line at both day 10 and day 20, the modern broiler were heavier than the 1995 broiler or the Jungle Fowl for all treatment groups (P < 0.0001). Also, 1995 boilers weighed more than the Jungle Fowl for all treatments groups (P < 0.0001). Genetic line had a significant effect on BW at 9 days of age where regardless of diet, modern broilers weighed more than the 1995 broilers and Jungle Fowl (P < 0.0001) and 1995 broilers weighed more than Jungle Fowl (P < 0.0001).Regardless of dietary treatment, there was a significant increase in BW from day 1 to day 10 and day 10 to day 20 in the Jungle Fowl, 1995 broilers, and the modern broiler. Overall, there was a significant dietary treatment by genetic lines interaction (P = 0.0016) and corn–rye (P = 0.0106). There was no difference in BW between treatment groups consuming the same diet . Surprisingly, there was also no difference in BW between rye–corn treatment group and the corn–corn (P = 0.2104) and the corn–rye (P = 0.2979) treatment groups. However, during the first phase of the experiment, chicks in the corn–corn and corn–rye were both consuming a corn-based diet, just as chicks on the rye–rye and rye–corn groups were both consuming a rye-based diet. Therefore, when BW of chicks consuming the same diets were combined (corn–corn with corn–rye and the rye–rye with rye–corn), corn-fed chicks weighed significantly more than rye-fed chicks  and corn–rye (P = 0.0008). There was no difference in BW between the corn–corn and corn–rye treatment groups (P = 1.000) nor was there a difference in BW between the rye–rye and rye–corn treatment groups (P = 0.8282). Similar to what was observed in the modern broilers, there was also no difference in BW between rye–rye treatment group and the corn–corn (P = 0.412) and the corn–rye (P = 0.6993) treatment groups. However, when BW of chicks consuming the same diets were combined (corn–corn with corn–rye and the rye–rye with rye–corn), corn-fed chicks weighed significantly more than rye-fed chicks  but weighed significantly more than those fed rye in the second phase of the experiments (P < 0.0001). Also, modern broilers in the corn–rye group weigh significantly more than the rye–rye fed chicks (P < 0.0001). BW of 1995 broilers at 20 days of age was significantly different between all treatment groups, where the corn–corn group weighed significantly more than those fed rye at any phase of the experiment (P < 0.0001). The rye–corn group weight significantly more than the rye–corn and the rye–rye group (P < 0.0001). 1995 Broiler in the rye–rye group had significantly lower BW than the corn–rye group (P = 0.0007). At day 20, Jungle Fowl in the corn–corn group weigh significantly more than the rye–rye group (P = 0.0002); however, there were no significant difference in BW between the other treatments. Within each dietary treatment, there was a significant difference in BW, where modern broilers weighed more than both 1995 broilers and Jungle Fowl (P < 0.0001), and 1995 broiler weighed more than Jungle Fowl (P < 0.0001).At day 20, modern broiler in the corn–corn and rye–corn groups did not differ in BW (P < 0.0001). Dietary treatment had a significant effect on BWG (P < 0.0001), regardless of day or genetic line, where chicks in the corn–corn group had significantly higher BWG than those fed rye at any phase of the experiment (P < 0.0001). The rye–corn group had the second highest BWG which was significantly higher than those chicks fed rye in the second phase of the experiment (P < 0.0001). Also, the corn–rye group had a significantly higher BWG than the rye–rye group (P < 0.0001). Genetic line had a significant effect on BWG, where modern broilers had significantly higher BWG than the 1995 broiler (P < 0.0001) and Jungle Fowl (P < 0.0001). As well, 1995 broiler had significantly higher BWG than the Jungle Fowl (P < 0.0001).Table P < 0.0001). At day 0–10, modern broilers in the rye–corn treatment group had significantly lower BWG than the corn–corn (P = 0.0011) and corn–rye (P = 0.0033). BWG from day 1 to day 10 in modern broilers was significantly lower in the rye–rye treatment group when compared with the corn–corn (P = 0.0233) and corn–rye (P = 0.0205). There was no difference in BWG between treatment groups consuming the same diet , which was expected. There was also no difference in BWG between rye–corn treatment group and the corn–corn (P = 0.4095) and the corn–rye (P = 0.3831) treatment groups. However, when BWG of chicks consuming the same diets were combined for day 1–10 (corn–corn with corn–rye and rye–rye with rye–corn), corn-fed chicks had a significantly higher BWG than rye-fed chicks  and corn–rye (P = 0.0033). There was no difference in BWG between the corn–corn and corn–rye treatment groups (P = 1.000) nor was there any difference in BWG between the rye–rye and rye–corn (P = 0.844). However, there was also no difference in BWG between rye–rye treatment group and the corn–corn (P = 0.6934) and the corn–rye (P = 0.8645) treatment groups. When BWG of chicks consuming the same diets were combined (corn–corn with corn–rye and the rye–rye with rye–corn), corn-fed chicks weighed significantly more than rye-fed chicks  and Jungle Fowl (P < 0.0001). As well, 1995 broilers fed corn in the first phase of the experiment had significantly higher BWG from day 1 to day 10 than Jungle Fowl (P < 0.0001).Interaction between the age of the chicks, dietary treatment and genetic line had a significant effect on the BWG ( P = 0.003. BWG frP = 0.9999) nor was there a statistical difference between the rye–rye and corn–rye groups (P = 0.5103). Modern broilers in the corn–corn and rye–corn groups had significantly higher BWG than the rye–rye and corn–rye groups (P < 0.0001). BWG from day 10 to day 20 in the 1995 broiler line was statistically different between all treatment groups, where the corn–corn group had the highest BWG (P < 0.0001), followed by the rye–corn group (P < 0.0001), then the corn–rye group (P < 0.0001), and lastly the lowest BWG occurred in the rye–rye group (P < 0.0001). From day 10 to day 20, Jungle Fowl in the corn–corn group had significantly higher BWG than the rye–rye treatment group (P = 0.0059); however, there were no difference in BWG between any of the other treatment groups. There was significant differences in BWG between genetic lines in the corn–corn and rye–corn treatment groups from day 10 to day 20, where the modern broilers had the highest BWG (P < 0.0001), followed by the 1995 broiler (P < 0.0001), and Jungle Fowl had the lowest BWG (P < 0.0001). A similar trend was observed in the corn–rye groups, where modern broilers in the corn–rye group had the highest BWG (P < 0.0001), followed by the 1995 broiler (P < 0.0001), with the Jungle Fowl having the lowest BWG (P < 0.0001). In the rye–rye group, modern broilers had significantly higher BWG than both the 1995 broiler and the Jungle Fowl (P < 0.0001). 1995 Broiler in the rye–rye treatment group also had significantly higher BWG than the Jungle Fowl (P = 0.0039).From day 10 to day 20 in modern broilers, there was no significant difference in BWG between the corn–corn and rye–corn treatment groups (P < 0.001). Modern chickens fed corn in the second phase of the experiment had significantly higher tibia strength when compared with rye-fed chickens (P < 0.0001). In the 1995 chicks, the corn–corn group had significantly higher tibia strength than those chicks fed rye in any phase of the experiment (P < 0.0001). Chicks in the rye–corn group had significantly stronger tibias than the rye–rye (P < 0.0001) and the corn–rye (P = 0.0065) groups; however, there were no difference in tibia strength between the rye–rye and corn–rye groups (P = 0.9511). There were no significant differences in tibia strength between treatments in the Jungle Fowl. When comparing genetic lines within each treatment group, the modern broilers and 1995 broilers had significantly stronger tibias than the Jungle Fowl in the corn–corn group (P < 0.0001). Modern broilers had significantly stronger tibias than the 1995 broilers in the rye–rye (P = 0.0465) and corn–rye group (P = 0.0153). Finally, in the rye–corn group, modern broilers had significantly stronger tibias than the 1995 broilers and Jungle Fowl (P < 0.0001).Table P < 0.0001; Table P < 0.0001). In the Jungle Fowl, there was no significant difference in tibia ash between the treatments. Between genetic lines, modern broilers had significantly higher tibia ash than the 1995 broilers in the rye–rye (P < 0.0001) and corn–rye (P < 0.0001) groups. Similar results were observed in the Jungle Fowl where the rye–rye (P = 0.0083) and corn–rye (P = 0.0016) groups had significantly higher tibia ash than the 1995 broiler. Modern broiler in the rye–corn group had significantly higher tibia ash than the 1995 broiler (P = 0.0002) and the Jungle Fowl (P < 0.0001). In the corn–corn group, modern broilers had significantly higher ash content than the 1995 broilers (P = 0.0167) and the Jungle Fowl (P < 0.0001). As well, 1995 broiler had significantly higher tibia ash content than Jungle Fowl (P = 0.034).At day 20, treatment, genetic line, and interaction between the two variables had a significant effect on tibia ash (P = 0.0152) and corn–rye (P < 0.0001). Similar results were observed in rye–corn groups, where the modern broilers in the rye–corn group had significantly higher tibia calcium content than the rye–rye (P = 0.0004) and the corn–rye (P < 0.0001) groups. Modern broilers in the rye–corn group had higher phosphorus content than the corn–rye group (P = 0.0036); however, no differences were observed between the other treatment groups. In the 1995 broilers, the corn–corn group had significantly higher tibia calcium concentrations than the corn–rye group (P = 0.003). The corn–rye group had significantly lower tibia phosphorus content than the corn–corn (P = 0.0064) and rye–rye (P = 0.0493) groups. There was no difference in calcium and phosphorus content between the other treatment groups in the 1995 broilers. There was no difference in calcium or phosphorus levels between treatments in the Jungle Fowl. Calcium content in the corn–corn group was significantly higher in the modern broiler compared with the 1995 broiler (P = 0.0045) and the Jungle Fowl (P = 0.0499). For the rye–corn group, modern broilers had significantly higher tibia calcium content than the Jungle Fowl (P = 0.004) and the 1995 broiler line (P = < 0.0001). In the corn–rye group, Jungle Fowl had significantly higher calcium content than the 1995 line (P = 0.0024). Tibia phosphorus content in the corn–corn group was significantly higher in the modern broiler than the Jungle Fowl (P = 0.016). In the rye–corn group, the modern broiler had significantly higher phosphorus content in the tibia than the 1995 broiler (P = 0.0166) or the Jungle Fowl (P = 0.0115). In the corn–rye group, modern broilers had significantly higher tibia phosphorus content than the 1995 broiler (P = 0.0287). There was no difference in calcium or phosphorus content between genetic lines for those birds maintained on the rye treatment throughout the experiment  and genetic line (P < 0.0001); however, there was no significant interaction between the two variables (P = 0.059). Regardless of genetic line, the corn–corn group had significantly shorter villi than the rye–rye (P = 0.0032) and the corn–rye groups (P = 0.0004). Overall, Jungle Fowl had significantly shorter villi than the modern broiler (P < 0.0001) and the 1995 broiler (P < 0.0001); however, VH was similar between the modern and 1995 broiler (P = 0.9009). Duodenal CD was altered by treatment, genetic line, and the interaction between the two variables had a significant effect on duodenal CD (P < 0.0014). Modern broilers in the corn–corn and rye–corn treatment group had significantly shorter CD than those fed rye–rye (P < 0.05) or corn–rye (P < 0.05). 1995 Chickens in the corn–corn group had significantly shorter CD than those chicks in the rye–rye (P < 0.0001) and the corn–rye (P = 0.0001). The 1995 chicks in the rye–corn group had significantly lower CD than the rye–rye (P = 0.0006) fed chicks; however, there was no differences in CD between the corn–rye and rye–corn groups (P = 0.1236). In the Jungle Fowl, there was no significant difference between treatments for duodenal VH and CD. There were no significant differences in CD between genetic lines in chicks fed a corn-based diet in the second phase of the experiment. However, Jungle Fowl in the rye–rye and corn–rye group had significantly shorter CD than the modern broiler (P < 0.005) and 1995 broilers (P < 0.005). In the duodenum, there was no difference between treatments, genetic lines, or interaction between the treatment and genetic lines in the VH:CD (Table P = 0.0391) where modern broiler had a significantly thicker muscularis than the Jungle Fowl (P = 0.0480). Treatment (P = 0.203) and interaction between the treatments and genetic lines were similar for muscularis thickness (P = 0.0609).Duodenal VH was significantly affected by treatment (s P = 0.09. RegardP = 0.048); however, after multiple testing correction (Tukey multiple comparison test), no statistical differences were observed between treatments or genetic lines for ileal VH. Jungle Fowl villi were shorter than the modern broiler (P = 0.0024) and the 1995 broiler (P = 0.0301). Genetic line had a significant effect on VH, CD, and muscularis thickness (P < 0.01) where modern broilers had significantly taller villi, deeper CDs, and thicker muscularis compared to the 1995 broiler (P < 0.05) and Jungle Fowl (P < 0.05). The 1995 broilers was in intermediate between Jungle Fowl and the modern broiler for all three traits (P = 0.0003).The results of the evaluation of a nutritional rehabilitation model on morphometric analysis of the ileum in three genetic chicken lines are summarized in Table P = 0.0002) and corn–rye (P = 0.0003) groups. Similar results were observed in the rye–corn group, where the rye–rye (P = 0.0292) and the corn–rye (P = 0.0467) groups had a significantly lower VH:CD ration. All other effects were not altered and P-values can be found in Table Dietary treatment had a significant effect on VH:CD ration, where the corn–corn group had a significant higher VH:CD ratio than the rye–rye  alone or in combination with selective lactic acid-based probiotic, using this model in 1995 chickens, before we expand our trials in children to test the lessons learned from our preclinical model. This process would include identifying a site in sub-Saharan Africa where these interventions could be tested in children with EE, ensuring that all of the human subject’s protections are in place, and developing the best strategies for measuring clinical outcomes.All animal procedures were approved and in compliance with Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (protocol #15006).MB, JL, and GT contributed to the overall study design and supervised all research. DK, SD, EG, SB, NA, and RM-G carried out the experiments and acquisition of data. MB, JK, RM-G, XH-V, and GT drafted and revised the first version of the manuscript. JL and GT analyzed the data. NA, WB, BH, and GT drafted the article and revised it critically for important intellectual content. XH-V and GT were responsible for the final editing of the manuscript. All the authors reviewed and finally approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GC and handling editor declared their shared affiliation.


The processes underlying persistence and remission of attention-deficit/hyperactivity disorder (ADHD) are poorly understood. We examined whether cognitive and neurophysiological impairments on a performance-monitoring task distinguish between ADHD persisters and remitters.On average 6 years after initial assessment, 110 adolescents and young adults with childhood ADHD  and 169 age-matched control participants were compared on cognitive-performance measures and event-related potentials of conflict monitoring (N2) and error processing (error-related negativity and positivity) from an arrow flanker task with low-conflict and high-conflict conditions. ADHD outcome was examined with parent-reported symptoms and functional impairment measures using a categorical (DSM-IV) and a dimensional approach.p < .05). ADHD remitters differed from persisters and were indistinguishable from control participants on the number of congruent (low-conflict) errors, reaction time variability, error-related negativity, and error-related positivity . Remitters did not differ significantly from the other groups on incongruent (high-conflict) errors, mean reaction time, and N2. In dimensional analyses on all participants with childhood ADHD, ADHD symptoms and functional impairment at follow-up were significantly correlated with congruent errors, reaction time variability, and error-related positivity .ADHD persisters were impaired compared with controls on all cognitive-performance and event-related potential measures (all Cognitive and neurophysiological measures of attention-vigilance and error detection distinguished ADHD remitters from persisters. These results extend our previous findings with other tasks and indicate that such measures are markers of remission and candidates for the development of nonpharmacological interventions. The identification of cognitive and neural processes underlying the trajectories of persistence and recovery from childhood-onset disorders during the transition to adulthood has the potential to prevent negative long-term outcomes , 2. AtteIt has been proposed that the cognitive processes associated with persistence of ADHD across development may be separate from those linked to the remission of the disorder . HoweverThe assessment of neurocognitive processes using cognitive and brain activity data may allow a deeper understanding of the developmental trajectories of ADHD. Our recent investigation of adolescents and young adults with childhood ADHD assessed on a range of cognitive, event-related potential (ERP), and electroencephalography (EEG) measures found that ADHD remitters differed from persisters, but not from control participants, on preparation-vigilance measures  and actigraphic data on movement. Executive-function processes of inhibition and working memory , instead, were not sensitive to ADHD persistence or remission, as ADHD remitters showed an intermediate pattern between persisters and control participants, without significant differences from either group . FurtherNeurocognitive impairments in ADHD include deficits in performance monitoring, an essential cognitive ability in goal-directed behavior to monitor ongoing performance and to adjust response selection , 17, 18.In ADHD, N2 attenuation in the flanker task has been reported in children and adults with ADHD , 25, 26,In the present study, we aimed to extend our recent findings  by invesThe sample consists of 279 participants, who were followed up on average 5.8 years (SD = 1.1) after initial assessments: 110 had a diagnosis of DSM-IV combined-type ADHD in childhood (10 sibling pairs and 90 singletons) and 169 were control participants (76 sibling pairs and 17 singletons) , 31. ParF = 11.34, p < .01) and functional impairment  than those who were not taking medication. However, the proportion of participants on medication did not differ between ADHD persisters and remitters . A 48-hour ADHD medication-free period was required prior to assessments. Three ADHD persisters (3.4%) were also on antidepressant medication, but for ethical reasons they were not asked to stop taking them. These participants were included in all analyses as their exclusion did not alter the results. Parents of all participants gave informed consent following procedures approved by the London-Surrey Borders Research Ethics Committee (09/H0806/58).Among those with childhood ADHD, 87 (79%) continued to meet clinical (DSM-IV) levels of ADHD symptoms and impairment (ADHD “persisters”), whereas 23 (21%) were below the clinical cut-off (ADHD “remitters”) . Among AThe Diagnostic Interview for ADHD in adults (DIVA)  was condAn estimate of IQ was derived with the vocabulary and block design subtests of the Wechsler Abbreviated Scale of Intelligence .The task was an adaptation of the Eriksen Flanker paradigm designed to increase cognitive load as used in previous studies , 25, 36.The EEG was recorded from a 62-channel DC-coupled recording system (extended 10–20 montage), using a 500-Hz sampling rate, impedances under 10 kΩ, and the FCz electrode as the recording reference. The electro-oculograms were recorded from electrodes above and below the left eye and at the outer canthi. EEG data were analyzed using Brain Vision Analyzer 2.0 . Raw EEG recordings were down-sampled to 256 Hz, rereferenced to the average of all electrodes (turning FCz into an active channel), and filtered using Butterworth band-pass filters . All trials were visually inspected for electrical artifacts or obvious movement, and sections of data containing artifacts were removed manually. Ocular artifacts were identified using the InfoMax independent component analysis algorithm . SectionAnalyses of ERPs of performance monitoring were restricted to incongruent trials, as the task used in this study is known to elicit strong N2, ERN, and Pe components in high-conflict, but not in low-conflict, conditions , 25, 36.d effect sizes are presented along with means, SDs, and test statistics for the group analyses , condition , and group by condition interaction using random intercept models in Stata  to control for genetic relatedness of the sibling pairs in a repeated-measures design. A random intercept model was also run to test the effect of group, scalp site  and group by site interaction on the N2. ERN and Pe were analyzed with regression models with dummy variables to identify overall group effects, controlling for sibling relatedness with the “robust cluster” command in Stata. Age correlated significantly with several of the cognitive-ERP measures  and was analyses , where 0analyses . PearsonBecause ADHD persisters had lower IQs than remitters did  14), an, an14), An overall group effect emerged on all cognitive-performance and ERP measures . Post hon = 63) and remitters (n = 20) became a trend for the ERN and nonsignificant for the Pe. Given the small female sample sizes  and the discrepancy in the size of male and female groups, sex differences were not directly tested. However, the effect sizes in the male-only sample  were comparable or only slightly reduced compared with those of the full sample. Remitters significantly differed from control participants on incongruent MRT, congruent RTV, and incongruent RTV, but not on incongruent errors. All other results remained unchanged. For further details, see the Controlling for IQ, group effects on MRT in both conditions and N2 at FCz were nonsignificant . Differen = 110), both ADHD symptoms and impairment at follow-up significantly correlated with the Pe  and neurophysiological error processing (ERN and Pe) compared with persisters. Attention-vigilance measures and conscious error processing were also associated with the continuum of ADHD symptoms and impairment at follow-up. Conversely, measures of executive control (incongruent errors), speed of processing (MRT), and neurophysiological conflict monitoring (N2) did not distinguish remitters from persisters, and thus they were not sensitive to remission or persistence of the disorder. Processes of attention-vigilance and neurophysiological error processing can be markers of remission from ADHD and may be sensitive to the effects of training or compensatory mechanisms.RTV, measuring intraindividual variability in reaction time, and number of congruent errors in the low-conflict condition distinguished ADHD remitters from persisters, but not from control participants, and were also correlated with continuous ratings of ADHD symptoms and impairment. Impairments in such measures in the congruent condition of the flanker task may result from lapses in attention and index attention-vigilance processes. Neurophysiological measures of error processing (ERN and Pe) showed the same association with ADHD remission. Conscious error processing (Pe) also correlated with the continuous ADHD symptoms and functional impairments at follow-up. Of note, the group differences observed on this peak-to-peak ERN were likely explained by the voltage change from the PNe to the negative ERN peak see the . This mePrimary analyses did not control for IQ, as lower-mean IQ in ADHD samples represents one of multiple cognitive processes underlying ADHD pathophysiology , 44, andThe present study extends the findings in our previous investigation that used a cued continuous performance test (CPT-OX), a four-choice reaction time task, and Wechsler Abbreviated Scale of Intelligence measures of IQ and digit span . Attentid = 0.44–0.75) between persisters and remitters in measures representing markers of remission, but small or negligible effect sizes (d = 0.02–0.28) in measures not sensitive to ADHD outcome at follow-up, suggesting this study had sufficient power to detect the major correlates of remission with the current sample sizes. Furthermore, when we repeated the analyses for male participants only, differences between remitters and persisters in the ERN and Pe were reduced. However, the small sample of female participants did not allow a direct examination of sex differences. Future studies that include a higher number of female participants are needed to further investigate these processes also in females. Finally, our sample included young adults as well as adolescents, who are still undergoing rapid cortical maturation. Although we controlled for age in all analyses, future follow-up assessments with participants having reached adulthood and when more ADHD participants may have remitted could clarify matters further.A limitation of this study is that, despite the large sample size, the low ADHD remission rate at follow-up resulted in a relatively small group of remitters. Therefore, we could not rule out the possibility that some nonsignificant differences between remitters and other groups could be due to low power. However, we observed medium-to-large effect sizes (Overall, we report that attention-vigilance and neurophysiological error processes were impaired in ADHD persisters but not in remitters and may be sensitive to compensatory mechanisms in those who remit from the disorder. These processes may be targets for nonpharmacological interventions or behavioral training aimed at alleviating some of the long-term outcomes of ADHD. Conversely, cognitive measures of executive control, speed of processing, and conflict monitoring were not sensitive to ADHD remission/persistence. Considering the importance of using a broad range of cognitive and neural measures in investigating the mechanisms underlying neurodevelopmental disorders , our cog


STDT) is the shortest interval at which an individual recognizes paired stimuli as separate in time. We investigated whether and how voluntary movement modulates STDT in healthy subjects. In 17 healthy participants, we tested STDT during voluntary index‐finger abductions at several time‐points after movement onset and during motor preparation. We then tested whether voluntary movement‐induced STDT changes were specific for the body segment moved, depended on movement kinematics, on the type of movement or on the intensity for delivering paired electrical stimuli for STDT. To understand the mechanisms underlying STDT modulation, we also tested STDT during motor imagery and after delivering repetitive transcranial magnetic stimulation to elicit excitability changes in the primary somatosensory cortex (S1). When tested on the moving hand at movement onset and up to 200 msec thereafter, STDT values increased from baseline, but during motor preparation remained unchanged. STDT values changed significantly during fast and slow index‐finger movements and also, though less, during passive index‐finger abductions, whereas during tonic index‐finger abductions they remained unchanged. STDT also remained unchanged when tested in body parts other than those engaged in movement and during imagined movement. Nor did testing STDT at increased intensity influence movement‐induced STDT changes. The cTBS‐induced S1 cortical changes left movement‐induced STDT changes unaffected. Our findings suggest that movement execution in healthy subjects may alter STDT processing.The somatosensory temporal discrimination threshold ( Ample evidence describes changes in brain processing of tactile sensory information while healthy subjects prepare and execute voluntary movements . The STDT is the shortest interval at which an individual recognizes paired stimuli as separate in time .For the study, we enrolled 17 healthy subjects (aged 24–43 years) at the Department of Neurology and Psychiatry.All participants gave written informed consent. The experimental procedure was approved by the institutional review board at Sapienza University Rome (CE REF# 4041) and conducted in accordance with the Declaration of Helsinki.During the study, participants were comfortably seated in an armchair beside a table. STDT was investigated according to the experimental procedures used in previous studies , equipped with three infrared cameras , was used to record index finger abductions and proximal arm movements. For index finger movements, the right arm was firmly secured and the arm position was kept constant throughout the experiment by visually inspecting the joint angles and by keeping the distance between the armchair and the table stable. Care was taken to secure the right forearm firmly in the same position on the table. The right arm was abducted at the shoulder by about 45–50° and the elbow joint was flexed at about 90°  cup electrodes placed over the FDI muscle or the deltoid muscle, in a belly‐tendon configuration. EMG signals were recorded and filtered with a Digitimer D360  (bandwidth 20 Hz–1 kHz), then analyzed off‐line with a personal computer through a 1401 plus A/D laboratory interface .Data were stored on a laboratory computer for on‐line visual display and further off‐line analysis .All 17 healthy subjects underwent the main experimental protocol first and some took part in the control experiments (applied in random order).Paired stimuli for STDT were triggered by movement execution at various intervals after movement onset. The movement consisted in right index finger abductions and the STDT was tested on the volar surface of the right index finger. Subjects were asked to abduct the index finger as widely and as fast as possible and were continuously encouraged to do so throughout the motor task. To define the time course of movement‐induced STDT changes, paired stimuli were delivered at five intervals: as soon as the EMG signals reached 100 μV in amplitude (defined as “0 msec” for simplicity), 100, 200, 500 msec, and 5 sec after movement onset, and STDT values were calculated for each interval. Specifically, paired stimuli for STDT testing at the 0‐msec interval were delivered concomitantly with movement onset. The subject was asked to abduct the right index finger while paired electrical stimuli were delivered at 0 msec after each movement onset. The threshold to identify movement onset for fast as well as slow index finger abductions was set at 100 μV of EMG activity. At each index finger abduction, the interval for STDT testing was progressively increased in 10‐msec steps, until the subject recognized the two stimuli as sequential  while we tested STDT on the volar surface of the right index finger; and third, we asked them to abduct the right index finger and tested STDT on the volar surface of the right digiti minimi. Paired stimuli for STDT were delivered 0, 100, and 200 msec after movement onset.To find out whether movement‐induced changes in STDT depended on movement velocity, in the same subgroup of 13 participants, we asked subjects first to abduct the index finger as slowly as possible. Paired stimuli for testing right‐finger STDT were then delivered at 0, 100 and 200 msec thereafter. In the second experiment, the investigator passively moved the index finger and paired stimuli for STDT were delivered concomitantly with movement onset (“0 msec”).To find out whether the STDT changes depended on the muscle contraction itself or on the type of movement , we asked 10 subjects to abduct the right index finger and keep the first dorsal interosseous muscle in a maximal tonic contraction with the aid of visual EMG feedback, while we delivered paired electrical stimuli for STDT to the right index finger.In this experiment, in 13 participants, electrical stimuli for STDT were delivered at twice the intensity used in the main experiment at 0, 100, and 200 msec after the right index finger abductions.To gain further information on the cortical areas responsible for STDT modulation, we investigated STDT values during motor imagery while participants mentally executed index finger abductions. Paired electrical stimuli for STDT testing were delivered to the right index finger while the subjects imagined they were abducting the index finger. A “go” signal was used to trigger the paired electrical stimuli for STDT testing and to tell subjects when to begin the imagined right index finger abductions.To investigate whether movement‐induced changes in STDT take place in the S1 cortex, in 10 subjects, we applied repetitive transcranial magnetic stimulation over the primary sensory cortex (S1), according to the experimental neurophysiological technique continuous theta‐burst stimulation (cTBS)  and index finger abduction (peak acceleration and peak velocity) kinematics. P values <0.05 were considered to indicate statistical significance.Unless otherwise stated, all values are means ± standard error (SE). To analyze changes in STDT values during index finger abductions in the main experiment, we used a repeated measures analysis of variance (ANOVA) with factor ISI . To analyze whether STDT values changed before the index finger movement (motor preparation), we used a repeated measures ANOVA with factor ISI . To analyze changes in STDT values in control experiments, we ran a repeated measures ANOVA with factors EXPERIMENTAL SESSION (five levels) and ISI . To analyze changes in movement variables for right index finger abductions within and across different experimental sessions, we used a further ANOVA with factor EXPERIMENTAL SESSION (two levels: when STDT was tested on the right and left hands) and ISI . The Greenhouse‐Geisser correction was applied when needed. Tukey's test was used for post hoc analysis. To analyze whether STDT values changed during motor imagery, during passive index finger abductions, during muscle tonic contraction, and after S1 cTBS (STDT percentage change at 0 msec pre‐S1 cTBS vs. STDT percentage change at 0 msec post‐S1 cTBS), we used a paired sample F5,80 = 19.36, P < 0.000001). Post hoc tests showed that STDT values increased significantly when paired stimuli were delivered after the onset of right index finger abductions: at 0 msec (P = 0.0001), 100 msec (P = 0.002) and 200 msec (P = 0.04). STDT values returned to baseline values at 500 msec (P = 0.6) and 5 sec (P = 0.15) after movement onset . Peak velocity of index finger‐abduction remained significantly unchanged across the various conditions testing different intervals elapsing after movement onset and paired stimuli for STDT . Nor did movement duration and kinematics of right index finger abductions vary when the STDT was tested on the left hand .Repeated measures ANOVA showed a significant factor ISI . STDT values therefore remained statistically unchanged during motor preparation at both 250 msec and 150 msec before movement onset . Mean movement onset latency was 219 ± 12 msec.Repeated‐measures ANOVA for STDT changes during motor preparation showed no significant factor ISI , ISI  and a significant EXPERIMENTAL SESSION × ISI interaction . Post hoc analysis showed that in the main experiment, changes in STDT values were significant after the onset of right index finger abductions  , and in the control experiments when STDT was tested on the right digiti minimi, after the onset of right index finger abductions  . Conversely, when STDT was tested on the left‐index finger during right index finger abductions STDT values remained unchanged  and also when tested on the right index finger during proximal arm movements   STDT values changed significantly at movement onset and within 200 msec thereafter   although it increased less than it did during voluntary index finger abductions.Paired sample T test showed no significant changes in the STDT values tested during first dorsal interosseous muscle tonic contractions .Paired F3,36 = 27.28, P < 0.0001) at 0 msec (P = 0.000164), 100 msec (P = 0.000166) and 200 msec (P = 0.002) after right index finger abductions .Paired sample P < 0.0001; percentage change at 0 pre‐S1 cTBS vs. STDT percentage change at 0 msec post‐S1 cTBS: 149 ± 9% vs. 146 ± 11%; T test: P = 0.48).Primary somatosensory cortex cTBS significantly increased baseline STDT values but left percentage STDT change at movement onset unmodified  at movement onset and up to 200 msec thereafter, STDT values increased from baseline. Conversely, during motor preparation, STDT remained unchanged. We also provide new observations showing how movement execution modulates STDT values: in all our healthy subjects, STDT values changed significantly during fast as well as slow index finger movements and, although to a lesser extent, also during passive index finger abductions, whereas during tonic index finger abduction they remained statistically unchanged. No differences were found in STDT when tested in body parts other than those involved in movement execution and during imagined movement. Nor did increasing the intensity used for STDT influence movement‐induced changes in STDT. Changes in S1 cortical activity with cTBS increased significantly STDT values but left the movement‐induced STDT changes unaffected. Overall, we show that movement‐related STDT values become evident only when STDT is tested on the same body part engaged in the motor task thus giving further insight into altered STDT in movement disorders.data not shown). Further evidence confirming participants’ performance reliability came from our observation that baseline STDT values remained statistically unchanged across the various experimental sessions. To make sure that fatigue or distraction did not distort the trial results, we gave experiments in randomized order. Our experiments excluded aspecific movement‐induced STDT changes attributable to “dual task effects” (a reduced discriminative ability owing to the two contemporary tasks), because the STDT tested on the hand contralateral to that involved in movement remained unchanged during and after the movement. This conclusion receives support also from our experiments showing that the three tasks testing movement preparation caused no significant STDT changes. Because movement‐induced STDT changes also when we delivered stimuli at an intensity twice higher than that used in the main experimental procedure, we exclude STDT changes depending entirely on sensory attenuation mechanisms  decrease in amplitude during movement execution (Chapman et al. Conversely, as a more likely explanation and one meriting future research, we suggest that STDT modulation during movement execution relies mainly on basal ganglia–thalamus interplay. A recent study in monkeys (Song and Francis In conclusion, our findings show that movement execution in healthy subjects interferes with STDT processing in subcortical areas, possibly through basal ganglia–thalamus connectivity thus linking STDT and motor circuits. Future studies in patients with movement disorders will provide insight into the STDT abnormalities reported in Parkinson's disease and dystonia (Conte et al. None declared.


Porphyromonas gingivalis, a periodontopathic gram-negative anaerobic bacterium, generally expresses two types of fimbriae, FimA and Mfa1. However, a novel potential fimbrilin, PGN_1808, in P. gingivalis strain ATCC 33277 was recently identified by an in silico structural homology search. In this study, we experimentally examined whether the protein formed a fimbrial structure. Anion-exchange chromatography showed that the elution peak of the protein was not identical to those of the major fimbrilins of FimA and Mfa1, indicating that PGN_1808 is not a component of these fimbriae. Electrophoretic analyses showed that PGN_1808 formed a polymer, although it was detergent and heat labile compared to FimA and Mfa1. Transmission electron microscopy showed filamentous structures (2‒3 nm × 200‒400 nm) on the cell surfaces of a PGN_1808-overexpressing P. gingivalis mutant (deficient in both FimA and Mfa1 fimbriae) and in the PGN_1808 fraction. PGN_1808 was detected in 81 of 84 wild-type strains of P. gingivalis by western blotting, suggesting that the protein is generally present in P. gingivalis. Porphyromonas gingivalis, a gram-negative anaerobic coccobacillus, is a member of the “red complex” of bacteria, which are pathogens primarily responsible for human periodontal diseases , and disrupted using a French press at 7.3 MPa. Unbroken cells and large debris were removed by centrifugation. The supernatant was subjected to precipitation with 50% ammonium sulfate saturation. The precipitate was dialyzed with the Tris buffer, and then applied to DEAE Sepharose Fast Flow chromatography  with 50 ml of bed volume. After washing thoroughly with the buffer, sample was fractionated by a linear gradient elution with 400 ml of NaCl (0 to 0.5 M) in the buffer.Fractionation were performed largely according to a previously described method . BrieflyIntact bacterial cells or the PGN_1808 fraction as described above was placed on a grid with an elastic carbon supporting film , negatively stained with 10 mM ammonium molybdate, pH 7.0, and observed by transmission electron microscopy .P. gingivalis strains were washed with PBS, pH 7.4, and the optical density at 600 nm was adjusted to 0.5, 1.0 and 2.0. The bacterial suspensions were mixed with the antisera to PGN_1808 or whole cells of P. gingivalis cells [The slide agglutination assay was performed following a standard protocol. is cells  on a glaSamples were mixed with a loading buffer consisting of 50 mM Tris/HCl, pH 6.8, 1% (w/v) SDS, 0.5 M 2-mercaptoethanol, 10% (w/v) glycerol, and 0.01% (w/v) bromophenol blue , and denatured by heating at the indicated temperatures for 10 min. Then, the samples were loaded onto an SDS-PAGE gel consisting of 11% or 5‒20% gradient polyacrylamide. After electrophoresis, protein bands were visualized by staining with Coomassie Brilliant Blue (CBB) R-250. For the western blot analysis, proteins separated in the SDS-PAGE gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in 20 mM Tris/HCl, pH 7.5, supplemented with 300 mM NaCl and 0.05% (w/v) Tween 20, and incubated with the primary anti-FimA , anti-MfFractionated PGN_1808 was applied to blue native-PAGE . BrieflyXL  at a flow rate of 1.0 ml/min in PBS, pH 7.4, supplemented with 0.02% NaN3. The protein elution was monitored by optical absorbance at 280 nm.The molecular size of the fractionated PGN_1808 was examined by using HPLC with a gel filtration chromatography column TSKgel G3000SWMass spectrometry analysis was performed as described previously . N-termipgn_1808 was examined by reverse transcription (RT)-PCR. Total RNA was isolated using ZR Fungal/Bacterial RNA MiniPrep  and treated with DNase I (Zymo Research Corp.) to remove residual DNA strands. No genomic DNA contaminants were detected in the total RNA samples (data not shown). The pure RNA was used to generate cDNA with a PrimeScript RT-PCR Kit and random 6 mers . Transcription unit was examined by a standard PCR for intergenic regions using the cDNA as a template and primers listed in Transcription unit comprising http://www.cbs.dtu.dk/services/LipoP/) was used for predictions of lipoprotein signal peptides [In silico structure homology-modeling of PGN_1808 was performed using the SWISS-MODEL online program (https://swissmodel.expasy.org/) [The LipoP 1.0 online program (peptides . In silisy.org/) .P. gingivalis strain TDC60 for the fractionation assay because TDC60 normally expresses both FimA and Mfa1 fimbriae [P. gingivalis ATCC 33277 expresses aberrant FimA fimbriae due to a nonsense mutation in fimB [Previously, we detected PGN_1808 concomitantly with FimA and Mfa1 fimbriae when the fimbriae were fractionated by ammonium sulfate precipitation and anion-exchange chromatography [ and unpufimbriae , whereasThe elution diagram showed a major peak at fractions 36 and 37 , which wDenaturation of FimA and Mfa1 fimbriae by heating at low temperatures  resulted in a smear and/or ladder-like pattern, due to partial dissociation of the FimA and Mfa1 polymers, respectively, in the SDS-PAGE, whereas denaturation by heating at 100°C resulted in a single band at the sizes corresponding to their monomers (FimA at 40 kDa and Mfa1 at 70 kDa), although degraded products of these proteins were also detected , as prevN-terminal sequencing of the PGN_1808 monomer band shown in  revealedP. gingivalis mutant deficient in both FimA and Mfa1 fimbriae , indicating that there might be variants of PGN_1808.PGN_1808 was detected in 81 of 84 wild-type strains of blotting , suggestBacteroides ovatus (PDB ID: 4rfj), that was resolved by Xu et al [We submitted PGN_1808, excluding the predicted signal peptide in the N-terminal 20 amino acids, to structure homology-modeling in the SWISS-MODEL program. The program found that PGN_1808 shared significant homology with a putative cell adhesion protein BACOVA_01548) from 48 from BXu et al . The PGNWe have not yet identified any accessory components of PGN_1808 fimbriae, because no proteins were concomitantly fractionated with PGN_1808. However, this possibility should be addressed in future research using a wild-type strain, rather than the PGN_1808-overexpressing mutant.pgn_1808 was examined in P. gingivalis wild-type strains ATCC 33277 and TDC60 by RT-PCR. We found that pgn_1808 was co-transcribed with the upstream genes including pgn_1805, pgn_1806, and pgn_1807, but not with pgn_1804 and the downstream gene pgn_1811 (note that pgn_1809 and pgn_1810 are absent). The pgn_1805, pgn_1806, and pgn_1807 genes are annotated as cysteine-tRNA ligase, patatin (a lipase), and glycosyl transferase, respectively [Transcription unit comprising ectively . FurtherP. gingivalis. However, PGN_1808 was detergent and heat labile compared to FimA and Mfa1. The biological function of PGN_1808 fimbriae should be examined in the future.We showed that PGN_1808 polymerized to form a fimbria and that it was generally expressed in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The goal of immunosuppression in transplantation has shifted to improving long-term outcomes, reducing drug-induced toxicities while preserving the already excellent short-term outcomes. Long-term gains in solid organ transplantation have been limited at least partly due to the nephrotoxicity and metabolic side effects of calcineurin inhibitors (CNIs). The alloimmune response requires activation of the costimulatory pathway for T cell proliferation and amplification. Belatacept is a molecule that selectively blocks T cell costimulation. In June 2011, the U.S. Food and Drug Administration approved it for maintenance immunosuppression in kidney transplantation based on two open-label, randomized, phase III trials. Since its introduction, belatacept has shown promise in both short- and long-term renal transplant outcomes in several other trials. It exhibits a superior side effect profile compared to CNIs with a comparable efficacy. Across all solid organ transplants, the burden of chronic kidney disease, its associated cardiovascular morbidity, mortality, and inferior patient/allograft survival is a well-documented problem. In this review, we aim to discuss the evidence behind the use of belatacept in solid organ transplants as an effective alternative to CNIs for renal rescue in patients with acute and/or chronic kidney injury. Transplantation is the treatment of choice for end stage organ failure. In non-renal solid organ transplants, with no replacement therapies, transplantation is a lifesaving procedure. When compared to dialysis, kidney transplantation provides patients with a better quality of life and decreased cardiovascular morbidity and mortality . The intp < 0.001)  . At 3 ye< 0.001) . There i< 0.001) .In renal transplantation, the leading causes of death and graft loss are cardiovascular disease and chronic allograft nephropathy . These dGraft survival in the long run is impaired by the nephrotoxicity of CNIs. Five years after transplantation histological evidence of CNI induced nephrotoxicity is seen in greater than 50% of allografts, and by 10 years, this finding is universal .Many alternative regimens such as CNI minimization, withdrawal and avoidance with use of mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) have been tried but rejection risks are higher, and sustained improvement in kidney function has not been seen consistently across different studies –15.The overall goal of immunosuppression in transplantation includes minimization of acute and chronic rejection while managing the adverse cardiovascular, nephrotoxic, infectious, and malignant sequelae of chronic immunosuppression.Belatacept , a molecule that blocks T cell costimulation, has shown promise in both short- and long-term renal transplant outcomes with a superior side-effect profile compared to CNIs .In this review, we aim to discuss the mechanism of action of belatacept, its role in kidney transplantation as an effective alternative to CNIs, and its future in non-kidney solid organ transplantation.Many immunosuppressive agents in use today interfere with specific steps that lead to T cell activation. It was theorized that a CTLA4-Ig protein with high affinity for CD80/86 could attenuate the T cell response by blocking “Signal 2” or costimulation. Abatacept was the first such molecule that was developed. Even though it proved successful in treating diseases mediated by an autoimmune T cell response (rheumatoid arthritis and psoriasis) it had an inadequate response in non-human primate transplant models –19.Using a rational mutagenesis and screening strategy, a daughter molecule LEA29Y (belatacept) with two amino acid substitutions (L104 → E and A29 → Y) was developed . BelatacPreclinical trials in non-human primate transplant models by Kirk et al. and Montgomery et al. revealed that treatment with anti-CD80 and anti-CD86 monoclonal antibodies delayed allograft rejection but did not induce tolerance , 22. Then = 221), less intensive (LI) belatacept (n = 226), and more intensive (MI) belatacept (n = 219). Induction regimen included basiliximab and maintenance immunosuppression included mycophenolate mofetil (MMF) and corticosteroids. At 12 months, the mean measured estimated glomerular filtration rate (eGFR) was higher in the belatacept arms as compared to the CsA group by 13–15 ml/min. Episodes of acute rejection at 12 months were higher in the belatacept groups compared with CsA . The majority of acute rejection episodes occurred in the first 3 months, and all of the rejection occurred within 6 months of transplantation. Despite higher rejection rates in the belatacept groups, the mean measured GFR at month 12 was higher than in the CsA group without acute rejection. No cases of antibody-mediated rejection were reported, and development of de novo anti-HLA donor-specific antibody (DSA) was lower in the belatacept groups . The prevalence of biopsy-proven chronic allograft nephropathy at month 12 was lower in the belatacept groups compared with CsA . There was no statistical difference in patient and graft survival at the end of 1 year between all three groups .The use of belatacept as first-line immunosuppression was first published in a phase III study by Vincenti et al. (BENEFIT study) . This stp = 0.001 MI vs CSA, p < 0.0001 LI vs CsA). Mean non-HDL cholesterol increased by 8.1, 8.0, and 18.3 mg/dl in the belatacept MI, belatacept LI, and CsA arms, respectively . Patients in either belatacept arm had a significant reduction in triglycerides; −23.6 mg/dl , −27.8 mg/dl  compared to an increase in the CsA group, +6.6 mg/dl. There was no statistically significant difference in the incidence of new onset diabetes after transplant (NODAT) between the belatacept treatment arms and the CsA treatment arm.The belatacept treated group had better cardiovascular and metabolic outcomes. Mean blood pressure (systolic and diastolic) were statistically lower in patients treated with either belatacept regimen when compared to the CsA group. Mean systolic blood pressure was 133, 131, and 139 mmHg in the belatacept MI, belatacept LI, and CsA group, respectively (An extended criteria donor (ECD) is any donor who at the time of death is over the age of 60, or over the age of 50 with two of the following three criteria: (1) history of high blood pressure, (2) a terminal creatinine of >1.5 mg/dl, and (3) death by cerebrovascular accident. ECD kidney allografts are associated with delayed graft function (DGF) and poorer long-term outcomes . In reciDe novo use of belatacept in ECD kidneys was studied in the BENEFIT-EXT study. The mean measured GFR at 12 months was significantly different in the MI belatacept (52.1 ml/min/1.73 m2) vs the CsA group (45.2 ml/min/1.73 m2) p = 0.008. There was no difference between the belatacept LI group (49.5 ml/min/1.73 m2) and CsA group, p = 0.1. Similar to the BENEFIT study, the belatacept-treated groups had non-inferior patient and graft survival compared to the CsA group . The rates of DGF were also similar between the groups .p = 0.0016 MI vs CSA, p = 0.0006 LI vs CsA). Mean triglycerides decreased by 1 mg/dl (belatacept MI) and 18.2 mg/dl (belatacept LI) compared to an increase of 34.5 mg/dl in the CsA arm . Incidence of NODAT at 12 months was lower in the belatacept MI group (2%) compared to the CsA group 5% (p = 0.03 MI vs CsA)  .p = 0.11) in the MI belatacept group and by 45%  in the LI belatacept group when compared with the CsA group. Similarly, the likelihood of graft loss at 7 years was reduced by 44%  in the MI belatacept group and by 41%  in the LI belatacept group.The 7-year outcomes from the BENEFIT study have yielded encouraging results. One hundred fifty-three of the 219 patients (70%) treated with the MI belatacept regimen; 163 of the 226 (72%) treated with the LI belatacept regimen; and 131 of the 215 (61%) treated with CsA were followed for the full 7-year period. While the results were not conclusive (likely due to inadequate power), there was a trend toward a decrease in the likelihood of death at 7 years by 38%  or belatacept LI group  and the CsA group. Similarly, there was no significant difference in death censored graft lost at 7 years between the belatacept MI regimen , or belatacept LI regimen  and the CsA group (19.3%).Seven-year outcomes from the BENEFIT-EXT study were also reported recently. Of the original 543 patients who comprised the intent-to-treat population, 372 (69%) had data available for analysis at 7 years; 128 of the 184 (70%) treated with the MI belatacept regimen, 138 of the 175 (79%) treated with the LI belatacept regimen, and 108 of the 184 (59%) treated with CsA. At 7 years, there was no difference in the likelihood of death between the belatacept MI , belatacept–sirolimus (n = 26), or Tac–MMF (n = 30). All patients received induction with thymoglobulin, 6 mg/kg total dose. At 6 months, four patients in the belatacept–MMF group (12%), one in the belatacept–sirolimus (4%), and one in the Tac–MMF (3%) had acute rejection. At 12 months, one additional patient in the belatacept–MMF group had acute rejection. Patient survival with a functioning graft was similar at 12 months; 91, 92, and 100% in the belatacept–MMF, belatacept–sirolimus, and Tac–MMF groups, respectively. Renal function was better in both belatacept groups compared to the Tac–MMF group. Mean calculated GFR was 63.6 ml/min/1.73 m2 in the belatacept–MMF group, 61.8 ml/min/1.73 m2 in the belatacept–sirolimus group, and 54.0 ml/min/1.73 m2 in the Tac–MMF group. No difference in cardiovascular risk factors including blood pressure, cholesterol, or new onset diabetes was noted  and less risk of chronic kidney scarring  as compared to the CNI-treated transplant recipients. There was no statistically significant difference in risk of death, loss of allograft, and acute rejection. Cardiovascular and metabolic outcomes also were congruent with the BENEFIT study with lower blood pressures, better lipid profiles, and reduced incidence of NODAT  in the belatacept-treated groups  or remained on their CNI-based regimen (n = 89). At 1 year, the mean GFR improved by 7 ml/min/1.73 m2 in the belatacept group as compared to 2.1 ml/min/1.73 m2 in the CNI group (p = 0.0058). There were six (7%) cases of acute rejection in the belatacept group as compared to none in the CNI group. No grafts were lost in the belatacept group, and one patient died with a functioning graft in the CNI group due to myocardial infarction. Cardiovascular and metabolic outcomes did not differ clinically between the two groups . At 3 years 8.4% of patients converted to belatacept had experienced an acute rejection compared to 3.6% of patients who remained on a CNI . There was no difference in patient or graft survival between groups. A trend in improvement of renal function may have been blunted by the addition of 16 patients who were switched from a CNI-based immunosuppression regimen to belatacept after 24 months post-randomization , 34.9 ± 14.5 ml/min at 1 year (p = 0.001), and 35.6 ± 13.6 ml/min at 2 years (p = 0.001)  underwent pre-conversion biopsies. The majority of these biopsies revealed histological patterns of acute tubular injury due to ischemia reperfusion and/or donor-derived vascular lesions, which are associated with renal dysfunction and CNI nephrotoxicity. Having failed CNI minimization, the patients were converted to belatacept-based regimen within 6 months of transplantation. Six months after conversion 22 (88%), at 12 months 20 (80%), and at 24 months 14 (56%), patients had functioning grafts. Three patients did not recover kidney function. Of the remaining 22 patients, renal function improved from a nadir eGFR of 18.28 ± 12.3 ml/min before conversion to a sustained long-term improvement of 33.9 ± 10.1 ml/min at 6 months (2 and increased to 29.7 ± 15.0 ml/min/1.73 m2 (p = 0.002), 31.6 ± 14.5 ml/min/1.73 m2 (p < 0.005), and 34.0 ± 15.2 ml/min/1.73 m2 (p < 0.005) at 3, 6, and 12 months post-conversion, respectively. Proteinuria improved at all time points’ post-conversion but did not differ significantly from baseline. Nine (11.4%) patients experienced biopsy-proven acute rejection; of these two suffered graft loss shortly after conversion to belatacept . Seventeen of the 22 patients who were switched from an mTOR inhibitor had previously been switched from a CNI due to biopsy confirmed CNI toxicity. Mean eGFR at the time of conversion was 26.1 ± 15 ml/min/1.73 mBased on data from these trials conversion from CNI-based immunosuppression regimen to a belatacept-based regimen appears safe with a slight increase in acute rejection soon after conversion. Patients converted to belatacept have been shown to have stabilization of renal function and some experience improvement in eGFR. Long-term data are need to assess whether short-term improvements in renal function lead to improvements in graft survival.2 prior to the conversion to an eGFR of 42 ± 12.5 ml/min/1.73 m2 (p = 0.028) at the end of a mean follow-up of 16.5 months. Surveillance biopsies after conversion were performed on five of six patients. Histologically, no new episodes of rejection were observed, and there was no worsening of chronicity scores. Immunologically, there was no emergence of new DSA. One patient who had DSA prior to conversion had declining DSA post-conversion without histological evidence of rejection  relative to the standard-of-care tacrolimus and MMF (15%) control group , serious infection rates (11 vs 13 events per 100 person-years of treatment), and cancer incidence rates (1.8–2.1 vs 2.6 events per 100 person-years of treatment) were equivalent to the CNI-based immunosuppression . Also, iThere was a higher incidence of posttransplant lymphoproliferative disease (PTLD) with belatacept in the first year after transplantation. Epstein–Barr virus (EBV) seronegative status was the strongest associated risk factor and represented a ninefold increase in risk . Owing tProgressive chronic kidney disease is a well-known complication of all solid organ transplants. Prior disease, peri-operative insults, and long-term exposure of vasoconstrictive CNIs can all contribute to this. In addition, chronic kidney disease worsens cardiovascular morbidity and mortality and has been shown to be an independent risk factor for adverse transplant outcomes.de novo belatacept as compared to CsA showed that belatacept was associated with higher rates of early low-grade steroid responsive acute rejection. This was offset by higher GFR, lower blood pressures, and lower incidence of NODAT. A few important limitations of these studies included the use of CsA as the comparator of choice rather than the more potent tacrolimus. In addition, only low immunologic risk patients were enrolled. Long-term outcomes have now been reported to show a clear trend toward improved patient and graft survival. Nevertheless, it remains to be seen whether the improved metabolic profile leads to a reduction in cardiovascular morbidity and mortality.In kidney transplantation, the BENEFIT studies that investigated the use of From an immunological standpoint, the observed reduced rate of development of donor-specific antibodies in belatacept-treated groups has encouraging long-term implications. Inadequate immunosuppression either due to non-compliance or reduction in immunosuppression is the major cause of late DSA formation and chronic antibody-mediated rejection . Developde novo studies, a higher rate of early acute rejection post-belatacept conversion was reported. Despite this, belatacept was shown to improve or stabilize intermediate-term kidney allograft function at up till 3 years follow-up. Thus, based on the data available so far, a careful conversion strategy might be safe in allografts with stable function and even those with dysfunction and established intolerance to CNIs. The findings of belatacept conversion studies that are ongoing are eagerly awaited.In randomized conversion trials, as with the The use of belatacept should be carefully individualized in kidney transplant recipients. Factors to be taken into account are the cardiovascular and hemodynamic complications associated with poor allograft function along with the immunological risk of the recipient. Future studies are underway to assess the safety and efficacy of belatacept in the setting of immunological sensitization and in combination with lymphocyte depleting agents. The results of these studies may help broaden the scope of this well-tolerated drug and make CNI free immunosuppression a real possibility for the vast majority of kidney transplant patients.As outlined in this review, the non-kidney solid organ transplant experience with belatacept is sorely lacking. The role of belatacept is not yet established in this group of transplant patients. The scarce data that are available currently do not lend itself to generalizability. More studies are urgently needed in these end organ failure transplant groups where renal dysfunction remains a widely prevalent and important contributor to patient mortality.DK, GG, and SL made substantial contributions to acquisition of data, and/or analysis and interpretation of data; participated in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be submitted and any revised version; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.GG has served on the Scientific Advisory Board of Bristol-Myers Squibb. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.