Circoviridae family, including PiCV. In this study, three groups of pigeons acquired from separate lofts were screened for PiCV, and their genome sequence was determined. Following this, they were housed in a single loft for 22 days, during which blood and cloacal swab samples were taken. From these blood and cloacal swabs, PiCV genomes were determined with the aim to study the spread and recombination dynamics of PiCV in the birds. Genome sequences of PiCV were determined from seven pigeons (seven tested PiCV positive) before they were housed together in a loft (n = 58 sequences) and thereafter from the ten pigeons from blood and cloacal swabs (n = 120). These 178 PiCV genome sequences represent seven genotypes (98% pairwise identity genotype demarcation), and they share >88% genome-wide pairwise identity. Recombination analysis revealed 13 recombination events, and a recombination hotspot spanning the 3′ prime region, the replication-associated protein (rep) gene and the intergenic region. A cold spot in the capsid protein-coding region of the genome was also identified. The majority of the recombinant regions were identified in the rep coding region. This study provides insights into the evolutionary dynamics of PiCV in pigeons kept under closed rearing systems.Pigeon circovirus (PiCV) infects pigeon populations worldwide and has been associated with immunosuppression in younger pigeons. Recombination is a common mechanism of evolution that has previously been shown in various members of the  Columba livia f. domestica), a species in the Columbidae family, is strongly associated with larger human populations [Columba livia) can flock in groups of up to 400 individuals, and their range can span several kilometers. Similar behavior of living in groups is also observed in the feral domestic pigeon population inhabiting cities around the world, which are referred to as urban pigeons [Adenoviridae, Anelloviridae, Circoviridae, Herpesviridae, Paramyxoviruses, Parvoviridae, Picornaviridae and Reoviridae [The domestic pigeon , also referred to as columbid circovirus, has a single-stranded circular DNA genome of ~2 kb that encodes two bidirectionally transcribed genes, a replication-associated protein (rep) and capsid protein (cp) genes [Pigeon circovirus  genes ,20,21. Pp) genes ,27,28,29p) genes ,31,32. Ap) genes ,34.cp and rep genes and near the origin of replication [The phylogeny of PiCV sequences does not show any clear geographic structuring . Howeverlication ,35.In recent years, a popular pigeon sport called “one loft races” (OLR) involving young pigeons has become popular. In this type of competition, all young birds supplied by various breeders are identically housed, fed and trained before the racing season in a single loft. This procedure usually takes 2–3 months with an aim to exclude additional factors affecting the result of the race, such as the location of the loft of origin, care and nutrition, which means that the race can be won by the best individual. However, OLR likely violates all biosecurity measures and can lead to the transmission of various pathogens. Thus, the morbidity of pigeons in OLR can be very high, and if proper veterinary care is not provided, the mortality rates can also be very high. The prevalence of PiCV in young racing pigeons is close to ~80% . Taking In this pilot study, we investigate the recombination dynamics of PiCV in young domesticated pigeons sourced from different facilities and then housed in a common loft.The research protocol was approved by the Local Ethics Committee on Animal Experimentation of the University of Warmia and Mazury in Olsztyn . The researchers made every effort to minimize the suffering of birds.n = 10) were randomly selected and bought from three different pigeon lofts of ca. 50 birds located in Warmińsko-Mazurskie voivodeship in Poland. The lofts where the pigeons were sourced from were not connected in any way, so the passive transfer of viruses via equipment, humans or food was unlikely. In all of the pigeon lofts, PiCV was detected in the past during routine checks, but there were no clinical symptoms of any disease in the animals on the day of purchase. The young birds purchased had not been vaccinated against common viral and bacterial pathogens.Eight-week-old pigeons (Myoviridae) [Birds TSP1-4 originated from loft 1 located in Olsztyn (racing pigeons), birds TSP5-8 originated from loft 2 located in Bartoszyce , and birds TSP9-10 originated from the loft 3 located in Ruś (racing pigeons). Blood samples were collected on 4 May 2020 from each of these purchased pigeons. Following this, the ten pigeons (TSP1-10) were housed in a single loft. Four of the birds died during the experiment because of severe dehydration and fibrinous aerosacculitis, probably as a result of stress and mixed infection with bacteria and immunosuppressive PiCV. However, no detailed laboratory examination of deceased pigeons was performed. For the deceased pigeons, the final blood collection date was taken when they died, whereas the second date for the rest of the birds is 26 May 2020 . Cloacalviridae) .DNA was extracted from 10 µL of blood samples using DNeasy Blood & Tissue Kit  in accordance with the manufacturer’s instructions. For cloacal swabs, viral DNA was isolated from 200 µL of UTM  using the High Pure viral nucleic acid kit  according to the manufacturer’s instructions.Escherichia coli XL blue competent cells. Ten clones were selected from the blood, and 5 from each swab positive sample for Sanger sequencing of the PiCV-cloned genomes. The recombinant plasmids were purified using Fast DNA-spin Plasmid DNA Purification Kit  and Sanger sequenced at Macrogen Inc.  by primer walking. The sequences were assembled and annotated using Geneious v11.0.3. 178 . Poorly sequenced clones were not included in downstream analysis. The assembled sequences from high-quality Sanger-sequenced clones were deposited in GenBank  with TempliPhi 2000 kit . Abutting primers  were designed in a conserved region of PiCV based on an alignment of all PiCV genome sequences available in GenBank (downloaded 14 December 2020). This primer pair was used to amplify the PiCV genomes from the RCA products using polymerase chain reaction (PCR) with Kapa HiFi DNA polymerase  with the following thermal cycling condition: initial denaturation 95 °C for 3 min followed by 30 cycles of 98 °C for 20 s, 55 °C for 15 s, 72 °C for 2 min and a final extension at 72 °C for 2 min followed by cooling to 4 °C for 10 min. The PCR amplicons were resolved on a 0.7% agarose gel, and ~2 kb amplicons were excised from the agarose gel and purified. The purified amplicons were ligated into the pJET 1.2 vector , and the recombinant plasmids were transformed into Complete PiCV genomes determined in this study together with those available in GenBank (downloaded on 15 February 2021) and beak and feather disease virus sequence as an outgroup were aligned using MUSCLE . This alp-value < 0.05 were accepted as credible.Recombination analysis was performed using RDP5  with defThe alignment of the sequences, with recombinant regions removed, was used to infer a Maximum Likelihood phylogenetic tree using PhyML  with K2 Ten birds were purchased from three different lofts, and blood samples were collected from each animal. Thereafter, they were all housed in a single loft for 22 days, and blood and cloacal swab samples were collected as summarized in n = 127). Pairwise identity analyses showed the all PiCVs (those available in GenBank and from this study) share >83% genome-wide identity. Based on the genome-wide pairwise identities, we identified seven genotypes for PiCV genome sequences based on the criteria we set for the purpose of this study, which was that viral sequences that share >98% genome-wide identity are part of the same genotype  of the PiCV sequences in five pigeons in the three lofts prior to being housed in a single loft. Once all the animals were housed in the same loft, this genotype was found in seven pigeons, which accounts for 47.5% (57/120) .n = 13), Belgium (n = 6), Brazil (n = 7), China (n = 63), France (n = 1), Japan (n = 1), Nigeria (n = 1), Poland (n = 30), United Kingdom (n = 2) and United States of America (n = 3). Global phylogenetic analysis of the PiCV recovered from 10 pigeons in this study (n = 178) together with those PiCV genomes available in GenBank was undertaken (PiCV genomes available in GenBank (download 15 February 2021) are those identified in pigeons from Australia (dertaken . The phydertaken .rep. Twelve of the recombination events detected are within the rep gene (rep and the intergenic region, whereas a cold spot was identified near the 5′ region of the cp (rep and cp, as well as the intergenic region. The differences in recombination patterns could be due to the variation in samples, i.e., global datasets with “circulating” recombinants as opposed to the ones in this study that address recombination in a short time frame within a small set of animals in a loft. The cold spot located in the cp, along with low amounts of recombination events, shows a higher region of conservation and importance of the cp likely for transmission and infectivity. Phylogenetic analysis of the genomes without recombination shows five clades present in the population (To investigate PiCV recombination dynamics, a pilot simulation of “housing together” naturally infected and non-infected animals originated from different maternal lofts was undertaken. The recombination analysis of the PiCV sequences from this study revealed 13 detectable recombination events. The recombination event detected in a large portion of the sequences was event 2, occurring in 109 genomes , and thirep gene . We idenf the cp . These rpulation  in contrpulation .There is no evidence of recombination in sequences present in Clade II and IV . Clade IOnly three recombination events were identified in sequences derived from the animals while in the three separate lofts of origin, whereas ten recombination events were detected in sequences derived from animals once they were housed in the same loft.In this study, we undertook a pilot experiment to simulate the viral dynamics at play when infected and non-infected pigeons are acquired from different sources and then housed together in a single loft. We show that genotypes of PiCV spread to non-infected birds and also to already infected birds, either resulting in co-infection or displacement of the previous genotype, e.g., the genotype 4 in animal TSP7 that appears to have “out competed” genotypes 1, 2 and 7 . We alsoThis study demonstrates the PiCV spreads in animals within a loft in a short time frame, in which the animals can harbor multiple genotypes and dominant genotypes possibly outcompete previous variants. Pigeon circovirus is known as an immunosuppressive factor in pigeons. A previous study performed on 30 PiCV full genomes recovered from pigeons of various breeds and different health statuses did not find any correlation between genotype, sequence mutation of circovirus strain and health status of its host . There i


Epidermolysis bullosa (EB) is a severe hereditary disease characterized by defective epithelial adhesion causing mucocutaneous fragility. The major types are EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and more than 35 EB subtypes. Another very rare type of EB is Kindler EB (KEB). Clinically, it is a very heterogeneous disease which ranges from localized to extensive skin lesions with frequent multisystem extra cutaneous involvement. The role of a pediatrician-dermatologist cooperation within a multidisciplinary team is fundamental for both the diagnosis and management contributing to these patients’ better life expectancy. Aim of this study is to describe clinical and laboratory characteristics of the main EB subtypes focusing on nutritional and gastrointestinal aspects, providing information to aid the paediatric management of children with EB. This retrospective study reviewed the cases of 160 pediatric EB patients : 31 patients affected by EBS (mean age ± SD: 4.37 ± 7.14), 21 patients affected by JEB (mean age ± SD: 9.26± 17.30) and 108 with DEB (mean age ± SD: 11.61 ± 13.48). All patients were admitted at the Bambino Gesù Children’s Hospital in Rome, between June 2005 to June 2020. The reduced gastrointestinal absorption, chronic losses, esophageal stenosis and chronic inflammatory state, represent the basis of nutritional problems of EB patients. In particular, anemia represents one of the most important complications of DEB patients which could require transfusion-dependent patterns. Malnutrition, vitamin deficiencies and anemia have been related to growth delay in EB patients. A specific diet with a balance of all macronutrients is required and improving caloric intake with sugar limitations is fundamental to prevent dental caries and tooth decay typical of EB patients. While sepsis proved to be the major cause of morbidity and mortality in younger patients, squamous cell carcinoma was mostly observed in older patients, especially those affected by DEB. Patients with EB require regular monitoring for complications and sequelae with a frequency of evaluations which varies based on age and EB subtypes. Cooperation among medical teams involving paediatricians, dermatologists, specialist clinicians including nutritionists such as families and patient’s association is fundamental to approach the disease and improve the quality of life of these patients.The online version contains supplementary material available at 10.1186/s13023-021-02144-1. Inherited epidermolysis bullosa (EB) is a heterogeneous group of genodermatoses characterized by defective epithelial adhesion causing mucocutaneous fragility with blister formation after minimal trauma , 2. EB cIncidence and prevalence of EB have been widely and variously defined through epidemiological studies considering clinical or molecular characteristics. Variegated data have been reported reflecting variations in patient’s recruitment from US to Europe , 4; in mEB clinical spectrum ranges from localized to extensive skin lesions with frequent extensive multisystem extra cutaneous involvement , 3, 4. LEB recognition and early diagnosis is necessary: the role of the pediatricians, especially those involved in pediatric primary healthcare evaluation, is fundamental for both the diagnosis and management of EB children to contribute to these patients’ better life expectancy. In this scenario, pediatrician and dermatologist cooperation is required. This aspect is under consideration in all European Countries. Indeed, the European Reference Network for Rare Skin Disorders (ERN-Skin) is focusing a major part of the activities to spread the knowledge on EB in non-reference centers .The aim of this study is to describe the clinical and laboratory characteristics of the three main EB subtypes and to provide recommendations to aid the pediatric management of children with EB. This would help define the role of the pediatrician-dermatologist cooperation within the multidisciplinary team composed of professionals such as dentists, pain therapist, physiotherapist, psychologists, endocrinologist, endoscopist and nutritionists as well as families and patients associations.We retrospectively reviewed the cases of 160 patients of EB in children admitted at the Bambino Gesù Children’s Hospital in Rome, between June 2005 to June 2020. The Dermatology Unit in Bambino Gesù Children’s Hospital is a reference center for rare skin disorders including EB and the dermatologists are the case manager of these patients. All patients are followed by a multidisciplinary team upon the patients’ needs. However, the dermatologists and the pediatricians are regularly consulting together all patients.This retrospective study has been carried out within the framework of the European Reference Network for rare skin disorders (ERN-Skin) .The diagnosis of EB was initially clinical and subsequently confirmed by immunofluorescence antigen mapping and ultrastructural examination of skin biopsies . In the All data were deposited in a web dataset upon the first evaluation of the patient, either during hospitalization or day hospital recovery, depending on the severity of the disease. In particular, follow-up evaluation has been performed for all the duration of the disease along life.For each patient, we reported the following clinical and laboratory variables (when available), classified both per age group  and main EB subtypes : age; age group; gender; percentile of BMI or W/H ratio; gene mutations; EB complications, including cutaneous, ocular, gastrointestinal, neuropsychiatric and other complications; laboratory workup, including indices of anemia, inflammation, nutritional state, renal function, liver function, coagulation, hormones, and others (see Tables Anemia was defined considering age related differences and percentile curves for hemoglobin (Hb) as following: Hb <14.5 g/dl in new-borns; Hb <10 g/dl in patients from 1 to 2 months; Hb <9 g/dl in patients from 2 to 3 months; Hb <9.5 g/dl in patients from 3 to 6 months; Hb <11 g/dl in patients from 6 to 12 months; Hb <11.5 g/dl in patients from 2 years to 12 years; Hb <12 g/dl in female older than 12 years 86 old and Hb <13 g/dl in males older than 12 years old . Sepsis is one of the most frequent extra cutaneous complications in EB patients. This was detected in a small percentage of our population, mainly in patients younger than 1-year-old (data not shown). This could be related to the multidisciplinary management of our third level referral hospital for EB patients where paediatricians, dermatologists, haematologists and EB expert nurses and clinicians work together preventing and managing EB patients’ complications. In accordance with the literature, the most frequent pathogen found in EB infections was S.aureus . In moreS.aureus –19. WhilS.aureus . MonitorS.aureus . Indeed,S.aureus , 21. MorS.aureus . In our Other common problems affecting EB patients involved the gastrointestinal system and nutritional aspects. Among these complications, esophageal stenosis, constipation, dental caries, and malnutrition were the most frequently reported. A percentage as high as 81.4% of DEB patients underwent esophageal dilatation (data not shown), in accordance with literature reports . FrequenAccording to literature, GERD prevalence may be underestimated as its assessment is often based on patient recall .MoreoverAnemia represents one of the most important complications of DEB patients [23 ex 21], as also shown by our findings (66%). Although hemoglobin was found to be below normal ranges for age, the mean ferritin value found was 81.5 ng/mL , with values sometimes above the normal limit , reflecting the chronic inflammation typical of EB patients. Therefore, regular serum iron dosing could be particularly useful in these patients. Indeed, mean serum iron values below the normal range limit 35-130 mcg/dl) were observed in patients older than 20 years 27.6 mcg/dl). In this context, adequate evaluation of anemia should include a complete iron asset examination with serum iron values. Oral iron supplementation is widely used to treat anemia even if it is poorly tolerated and associated with gastrointestinal disorders such as: heartburn, constipation or diarrhea, with a consequent reduction in compliance . Moreove.6 mcg/dl mcg/dl wNone of our patients performed tube feeding because our methodology is to perform a prompt diagnosis and adequate treatment of esophageal stenosis in addition with nutritional support.Moreover, patients require higher protein because of protein loss through skin lesions and inflammatory processes, as discussed in literature .In particular, according to nutritional experts, protein intake should be up to 200% of the recommended intake .To better define EB patient’s growth, specific growth charts have been recently proposed . For exaIn contrast to literature findings, we found out that the majority of our patients had 271 normal values of vitamin A and high levels of vitamin E. These findings are rarely observed in both the general population and EB patients. Our findings could be probably explained by the overuse of topic agents containing vitamin E in this population, where the lack of an adequate hydrolipidic film is connected to the absence of skin protective function and to a more percutaneous absorption. Moreover, the general nutritional deficit detected in our cohort demonstrates that nutritional supplementation should always be recommended as the risk of over dosage is low . As repoReports on quality of life (QoL) of EB patients show the heavy burden of this disease on families, which requires psychological individualised support and close monitoring following psychological changes and specific physical requirements , 39. SpeIn our population, a low rate of depression was detected, probably underestimating the real psychological consequences of the disease. Further studies are necessary to evaluate EB patient’s QoL, thus helping paediatricians-dermatologists cooperation and the multidisciplinary equipe to manage with psychological consequences of EB for both patients and families. Moreover, pediatricians should refer EB patients and families to patients’ associations in addition with social workers which play an important role in counselling families of affected children.Laboratory, imaging and clinical monitoring are important aspects of EB management. Patients with EB require regular monitoring for complications and sequelae. The frequency of the evaluations varies depending on age and EB subtypes. All EB patients should perform vaccinations as recommended in the specific Country Vaccines Plan. Tables EB is a rare disease with a wide range of clinical presentations, from mild 300 to devastating severe forms. Diagnosis and management are both challenging: specialised hospitals should be the referral centre for this complex role guaranteeing a multidisciplinary evaluation and follow-up. However, the role of the family pediatrician is regularly connected with the reference center in order to reduce difficulties related to the management and to prevent/delay severe complications.Considering EB rarity, the family pediatrician should be informed about EB peculiarity and specifically skin fragility. However, the pediatrician should be involved in the follow-up of EB patients reducing the frequency of moving to the reference center.This study highlights the most frequent complications in EB severe subtypes focusing on nutritional and gastrointestinal aspects. Multidisciplinary care involving pediatrician-dermatologist cooperation within specialist clinicians, families and patients Association is fundamental, providing instruments to face the disease and improve EB QoL.Additional file 1.Supplementary Table 1. Laboratory workup of EBS patients per age group. Supplementary Table 2. Laboratory workup of JEB patients per age group.Supplementary Table 3. Laboratory workup of DEB patients per age group.


PDGFRA) and chromosome 19 (CDC6) have been found associated to trypanotolerance in West African taurines. Screening for FST outliers in trypanosome positive/negative animals we detected seven variants putatively under selection. Finally, we identified a minimum set of highly ancestry informative markers for routine admixture testing. The results of this study contribute to a better understanding of the genetic basis of trypanotolerance in Baoulé cattle and their crossbreeds. Furthermore, we provide a small informative marker set to monitor admixture in this valuable indigenous breed. As such, our results are important for conserving the genetic uniqueness and trypanotolerance of Baoulé cattle, as well as for the improvement of Baoulé and Zebu crossbreds in specific community-based breeding programs.The genomes of crossbred (admixed) individuals are a mosaic of ancestral haplotypes formed by recombination in each generation. The proportion of these ancestral haplotypes in certain genomic regions can be responsible for either susceptibility or tolerance against pathogens, and for performances in production traits. Using a medium-density genomic marker panel from the Illumina Bovine SNP50 BeadChip, we estimated individual admixture proportions for Baoulé x Zebu crossbred cattle in Burkina Faso, which were tested for trypanosome infection by direct ELISA from blood samples. Furthermore, we calculated local ancestry deviation from average for each SNP across 29 autosomes to identify potential regions under selection in the trypanotolerant Baoulé cattle and their crossbreds. We identified significant deviation from the local average ancestry (above 5 and 10% genome-wide thresholds) on chromosomes 8 and 19 in the positive animals, while the negative ones showed higher deviation on chromosomes 6, 19, 21, and 22. Some candidate genes on chromosome 6 ( Bos taurus taurus and Bos taurus indicus subspecies are the two most important cattle types in West Africa  genotyping, inferring selection signatures from differences in local admixture levels has received considerable attention in human genetics . SimilarThe genomic ancestry proportions between trypano-susceptible indicine Zebu and the trypano-tolerant taurine Baoulé cattle in Burkina Faso were assessed based on microsatellites and 155 SNPs in 23 candidate regions . In thisThis study was carried out in the province of Poni in the South-western administrative region of Burkina Faso. We selected three study sites with different management and breeding systems of Baoulé cattle and Baoulé x shorthorn Zebu crossbreds, including 27 villages from the Bouroum-Bouroum, Kampti and Loropéni departments . In the A total of 737 blood samples, including 387 Baoulé and Baoulé x Zebu crossbreds from Bouroum-Bouroum, and 350 crossbreeds from Kampti and Loropéni were collected in EDTA tubes during the health monitoring activities of the “Characterization and Sustainable Utilization of Local Cattle Breeds” (LoCaBreed) project. DNA extraction from EDTA blood samples was performed with the MasterPureTM DNA Purification Kit for Blood Version II  following the manufacturer’s protocol. The trypanosomosis status was recorded by indirect ELISA test to diagnose positive or negative trypanosome infection in the blood samples  resultinP-value 1 × 10 − 6. After quality control, 28,034 SNPs and 776 animals were available for subsequent analyses.The genotyping of the 737 DNA samples with the Illumina Bovine SNP50 BeadChip was performed at Neogen . Additional genotypes from 30 purebred Zebu and 35 crossbreds  were incK = 2). The admixture bar plots for ancestry proportions were created in R with the barplot function  is a program for estimating locus-specific ancestries in admixed individuals, using allele frequencies of the reference populations . We applFST outlier loci putatively under selection between the trypanosome positive (n = 244) and negative (n = 266) crossbred animals using a cut-off at p < 0.05 corrected for a false discovery rate . Significance of the Pearson’s r was assessed with the P-value from the Pearson Correlation Coefficient Calculator  in chromosomes 8 and 19 for trypanosome positive crossbreds , and in FST values and disregarded the pure-bred Baulé and Zebu. Among these crossbred animals we grouped them in trypanosome positive and negative animals to avoid detection of breed differences unrelated to trypanosome tolerance. We detected seven variants with a FDR corrected threshold of p < 0.05  and 0.990  (p < 0.001).To reliably detect hybrids even with a small set of SNPs applicable for routine genetic monitoring, we selected the most ancestry informative markers resulting in the highest differentiation between Baoulé and Zebu cattle. The 100 SNPs with the highest divergence presented and 0.79 . We estiand 0.79 . The Pea/top100) . All PeaThe high amounts of global admixture observed in the taurine cattle population in the three studied departments of Burkina Faso indicated mixed genetic backgrounds of the cattle in Bouroum-Bouroum, Kampti and Loropeni , 3. The The uncontrolled mating in extensive production systems, which are typically practiced in West Africa, can lead to the introgression of Zebu genes in the small taurine cattle population, which represents a threat to their genetic integrity , and migB. t. indicus), Eurasian taurine, and African taurine (both B. t. taurus) ancestry proportions.In a recently admixed population, ancestral populations have been mixing for a relatively small number of generations, resulting in a new population with different proportions of their genome derived from the original parental populations . Local aWe applied an approach of significance testing and performed a permutation test of circularizing the genome by concatenating the SNPs of all autosomes in a single string, cutting this string once and rearranging the two resulting segments, as proposed by VAV1, PIK3R5, RAC1, VAV2, GAB2, and INPP5D genes in chromosome 8 to be genes under selection in Muturu and N’Dama cattle breeds in response to trypanosomes infection. Surprisingly, we also found higher Baoulé ancestry in chromosome 8 (35–50 Mb) also in trypanosome positive cattle, which could indicate that these regions harbor beneficial Baoulé haplotypes, which are not connected to trypanosomosis tolerance. These regions might harbor genes of general importance for adaptation to the environment. Some canadidate genes on chromosome 6 at 71373513-71421283 (PDGFRA) and chromosome 19 at 41185975-41196948 (CDC6) for trypanotolerance in West African taurines have been found on these chromosomes  genotyping assay. Such an easy and fast genotyping array can be implemented at any laboratory equipped with Real-Time PCR machine and can be used for routine monitoring of hybridization in Baoulé cattle. FST values showing the highest levels of allele frequency differentiation between the two parental breeds similar to our methodology. These AIMs provided close estimation with pedigree information. Correlation coefficient between breed level based on admixture estimates from 25 SNP data obtained in this study .Quality controlled Bovine 50k SNP chip data, including 31,612 SNPs of the 802 animals included in this study, were uploaded to DRYAD. The dataset has been assigned a unique identifier , Burkina Faso.JS conceived the original idea of the study and together with GM, PB, BY, and NK further developed the idea and decided on the set of analysis. BY and PB did the statistical analysis and wrote the text. BY, DO, BZ, AS, SO-K, SM, HT, and ATr collected the data for the analysis and together with JS, GM, PB, NK, ATa, PO-W, MW, ATr, and OM critically reviewed the text. All authors approved the final version of the manuscript.NK was employed by company SUISAG, Switzerland. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-nitrosodimethylamine (NDMA), a probable human carcinogen, in simulated gastric fluid at physiologic pH levels and nitrite concentrations?Does ranitidine convert to In this in vitro study of 150-mg ranitidine tablets added to simulated gastric fluid, NDMA did not form when gastric nitrite concentrations were at the upper range of physiologic or at nitrite concentrations as much as 50-fold greater than the upper range.In this study, 150-mg ranitidine tablets did not convert to NDMA in simulated gastric fluid with physiologic nitrite concentrations. N-nitrosodimethylamine (NDMA) formation following the addition of ranitidine to simulated gastric fluid using combinations of fluid volume, pH levels, and nitrite concentrations, including physiologic levels.This study characterizes  N-nitrosodimethylamine (NDMA), a probable human carcinogen, was formed when ranitidine and nitrite were added to simulated gastric fluid. However, the nitrite concentrations used were greater than the range detected in acidic gastric fluid in prior clinical studies.A publication reported that To characterize NDMA formation following the addition of ranitidine to simulated gastric fluid using combinations of fluid volume, pH levels, and nitrite concentrations, including physiologic levels.One 150-mg ranitidine tablet was added to 50 or 250 mL of simulated gastric fluid with a range of nitrite concentrations from the upper range of physiologic (100 μmol/L) to higher concentrations (10 000 μmol/L) with a range of pH levels. NDMA amounts were assessed with a liquid chromatography–mass spectrometry method.NDMA detected in simulated gastric fluid 2 hours after adding ranitidine.At a supraphysiologic nitrite concentration , the mean (SD) amount of NDMA detected in 50 mL simulated gastric fluid 2 hours after adding ranitidine increased from 222 (12) ng at pH 5 to 11 822 (434) ng at pH 1.2. Subsequent experiments with 50 mL of simulated gastric fluid at pH 1.2 with no added nitrite detected a mean (SD) of 22 (2) ng of NDMA, which is the background amount present in the ranitidine tablets. Similarly, at the upper range of physiologic nitrite  or at nitrite concentrations as much as 50-fold greater (1000 or 5000 μmol/L) only background mean (SD) amounts of NDMA were observed . With 250 mL of simulated gastric fluid, no NDMA was detected at the upper physiologic range (100 μmol/L) or 10-fold physiologic (1000 μmol/L) nitrite concentrations, while NDMA was detected  at a 50-fold physiologic nitrite concentration (5000 μmol/L).In this in vitro study of ranitidine tablets added to simulated gastric fluid with different nitrite concentrations, ranitidine conversion to NDMA was not detected until nitrite was 5000 μmol/L, which is 50-fold greater than the upper range of physiologic gastric nitrite concentrations at acidic pH. These findings suggest that ranitidine is not converted to NDMA in gastric fluid at physiologic conditions. The proposed mechanism for ranitidine8 to form NDMA involves 2 separate nitrite-dependent steps, and the reaction rate for the second step is proportional to the concentration of the NDMA precursor dimethylamine and the square of the concentration of protonated nitrite.9 Thus, pH level, ranitidine concentration, and nitrite concentration are key reaction conditions that will affect whether NDMA will be formed.In addition, ranitidine was proposed to convert to NDMA in vivo, as described in an article by Braunstein et al titled “Analysis of Ranitidine-Associated 7) had been considered the upper range of normal in the acidic fasting stomach based on published clinical studies.10 However, that assessment was performed in 1985, and an updated evaluation of physiologic nitrite concentrations was warranted, with additional consideration of the potential effect of food; of medications, such as H2 blockers and proton pump inhibitors; and among specific patient groups. The current in vitro study characterized the potential formation of NDMA following the addition of ranitidine to simulated gastric fluid using different combinations of fluid volume, pH, and nitrite concentration, which included physiologic levels.Review of the literature revealed that 25 μmol/L of nitrite  undergoing upper endoscopy for various gastrointestinal conditions. 11 Nitrite concentration increased with pH. The 95th percentile nitrite concentration was less than 1 μmol/L at pH levels less than 4, less than 10 μmol/L at pH levels 4 to 5, less than 100 μmol/L at pH levels greater than 5 to 6.99, and less than 200 μmol/L at pH levels of 7 or greater. As the reaction to form NDMA depends on the nitrite being protonated, Ka, which is the negative log of the acid dissociation constant (Ka) value. The lower the pKa, the stronger the acid. For nitrite, the pKa value is 3.3, which means that at pH 3.3, 50% of nitrite will be protonated and 50% will be deprotonated. In Figure 1, with pH levels of less than 5, increases in total nitrite concentration are offset by the decreased amount of nitrite that is protonated, such that protonated nitrite remains approximately constant. With pH levels greater than 5, protonated nitrite concentration decreases rapidly as pH increases. eTable 1 and eFigure 1 in the In 1993, Xu and Reed13 reported the association of concentrated potassium nitrate solution boluses administered via nasogastric tube on gastric nitrite concentration. After nitrate is absorbed from the intestines, secretion into saliva occurs in a concentrated form that can then be converted to nitrite by nitrate-reducing bacteria on the back of the tongue. However, a bolus of nitrate solution is not reflective of physiology that occurs after consuming nitrate in food, as the meal will increase pH as well as the volume of food and liquids in the stomach, diluting the concentration of salivary nitrite that enters the stomach.eTable 2 in the 15 from 45 years ago reported the effect of an extremely high-nitrite meal including canned luncheon meat from a time prior to modern regulations and technology that reduced nitrite in cured meats.16 Four other studies20 assessed gastric pH and nitrite every 30 or 60 minutes for 24 hours, thus including data from the fasting and fed state. Milton-Thompson et al19 reported median and upper range data from healthy volunteers before, during, and after cimetidine administration combined and binned by 1-unit pH increases . While food remains in the stomach longer during the digestive period, a drug dissolved in gastric fluid and/or liquids empties throughout the digestive period.24 After the incubation, 1-mL sample aliquots were taken, and 0.1 mL of 1 mol/L NaOH was added to prevent further potential NDMA formation  2-hour acid stage, simulating the time that a drug sample may stay in the stomach. Notably, the actual residence time of ranitidine in the fasted stomach may be much shorter .To test conditions under which ranitidine would be converted to NDMA, experiments were performed at a supraphysiologic nitrite concentration (10 000 μmol/L). At 10 000 μmol/L, the mean (SD) amount of NDMA detected in 50 mL simulated gastric fluid 2 hours after adding 1 ranitidine tablet (150 mg) decreased with increasing pH values of 1.2, 2.0, 3.0, and 5.0 from 11 822 (434) ng to 3616 (301) ng, 1310 (113) ng, and 222 (12) ng, respectively . ConsistSubsequent experiments with varying nitrite concentrations  were perWith 250 mL of simulated gastric fluid, no NDMA was detected at the upper physiologic range of gastric nitrite concentration (100 μmol/L) or 10-fold physiologic (1000 μmol/L) nitrite concentrations, while additional NDMA was detected  at 50- and 100-fold physiologic concentrations . Because the nitrite concentration was matched between the prior 50 mL simulated gastric fluid experiments and these experiments with 250 mL, the total amount of nitrite in simulated gastric fluid was 5 times greater in the 250 mL experiments. As 1 ranitidine tablet was added to both the 50 mL and 250 mL experiments, the ratio of nitrite to ranitidine molecules was 5 times greater in the 250 mL experiments compared with the 50 mL experiments.11 When increasing the simulated gastric fluid volume to 250 mL, as may occur when consuming 200 mL of water, conversion of ranitidine to NDMA was detected when gastric nitrite was 5000 μmol/L instead of 10 000 μmol/L. However, this scenario is unlikely to be encountered in patients, as consuming water will increase pH and decrease the gastric nitrite concentration by dilution. Furthermore, 5000 µmol/L is approximately 600-fold greater than the 95th percentile of gastric nitrite at pH values of less than 2 from patients with 24-hour (combined fed and fasted) gastric fluid measurements  may be the result of background NDMA being present in the ranitidine tablets before being placed in simulated gastric fluid and not the result of ranitidine-to-NDMA conversion in simulated gastric fluid. Zeng and Mitch have recently retracted or corrected reported NDMA findings in 2 journals26 because of analytical artifacts associated with the use of gas chromatography–mass spectrometry technology for measurements where ranitidine is present. The retraction and correction highlight the need for using a fit-for-purpose method for the analytical measurements in conjunction with conditions that as closely as practical reflect human physiology to assess the potential for drugs to be converted in vivo to nitrosamines. Regardless, the lowest nitrite concentration they studied (1000 μmol/L) was more than 1800-fold greater than the 95th percentile of fasting nitrite concentrations , which represents a worst-case scenario of nitrite intake from a past era. Since then, additional regulations in different countries were implemented to reduce the amount of nitrite in cured meat, and the industry evolved to reduce nitrite even further.30 In the United States, as of 2009, the mean daily exposure to nitrites from cured meats among those who eat cured meats was 0.25 mg per day, and the 90th percentile was 0.56 mg per day.30 Thus, the amount of nitrite in the meal described in Walters et al15 was 64 times higher than the mean and 29 times higher than the 90th percentile of cured meat eaters. Despite the high amount of nitrite in the meal in Walters et al,15 gastric nitrite only reached 290 μmol/L and occurred at pH 4.7, which is associated with a protonated nitrite concentration nearly identical to the fasting level.As described in the Methods section and shown in eTable 2 in the 31 and if more nitrite is present in gastric juice at a higher pH, then as pH decreases, nitrite will also decrease. Interestingly, when study participants in Mowat et al,12 who were receiving 40 mg of omeprazole for 4 weeks , were administered 2 mmol of potassium nitrate by nasogastric tube, there was no increase in gastric nitrite immediately. Rather, nitrite reached the highest levels from 40 to 90 minutes after the potassium nitrate bolus.12 The lag time for elevated nitrite is consistent with the increase in gastric nitrite only occurring after the nitrate bolus was absorbed, secreted in concentrated form by salivary glands, converted to nitrite by nitrate-reducing bacteria in the oral cavity, and then nitrite entering the stomach with saliva.32Increased gastric nitrite concentrations have been proposed to be because of increased nitrate-to-nitrite reducing bacteria at higher pHs in the stomach. However, nitrite is known to be more stable at higher pH,33 As shown in this study and other work, because amine nitrosation reactions, which form nitrosamines, are very dependent on the chemical environment and the presence of a suitably reactive amine group, care should be taken in ascribing observed reactivity under nonphysiologic conditions as evidence for the potential of drugs such as ranitidine to form NDMA in humans. Like this study, use of available clinical data to assess test conditions that should be used for in vitro testing have been previously reported. For example, in 1985 Gillatt et al34 assessed the propensity of various amine-containing drugs to form nitrosamines with a very high nitrite concentration (40 000 μmol/L) and compared those results with what was determined as the upper bounds of simulated gastric nitrite concentration from available clinical data (25 μmol/L). Gillatt et al34 highlighted the importance of the nitrite concentration and noted that reactivity at the higher concentration was not always predictive of nitrosamine formation at the lower concentration. Furthermore, nonclinical mutagenicity studies with ranitidine from the early 1980s had used excessive nitrite concentrations, leading to questionable clinical relevance, as commented on by the authors of those studies.37Questions regarding the potential for nitrosamines to form in vivo from drugs or food were asked approximately 50 years ago.34 the supraphysiologic nitrite concentrations used by in vitro studies8 were compared with nitrite concentrations observed in physiological gastric fluid clinical studies. In addition, the interplay of reactive nitrite and pH was evaluated. Overall, to investigate the potential for pharmaceuticals to lead to in vivo formation of NDMA or other nitrosamines, the test conditions used in vitro should include physiologically relevant conditions. When that was done with ranitidine in this study, in vitro formation of NDMA did not occur.In this study, as well as in the study by Gillatt et al,38 which demonstrated that ranitidine did not increase urinary excretion of NDMA across 2 different diets with, 1 containing large quantities of cured meats.The in vitro model used in this study has inherent limitations and does not reflect all aspects of human physiology, but it does provide a method for assessing the potential for physiologic nitrite reactions with drugs in the gastric fluid to lead to NDMA formation. The in vitro studies performed here did not include gastric conditions in the presence of a meal aside from experiments at higher pH levels that occur following a meal, which led to decreased formation of NDMA. In addition, the lack of conversion of ranitidine to NDMA at simulated physiological conditions observed here was further supported by the accompanying randomized, placebo-controlled clinical trial,38In this in vitro study of ranitidine tablets added to simulated gastric fluid with different nitrite concentrations, ranitidine conversion to NDMA was not detected until nitrite was 5000 μmol/L, which is 50-fold greater than the upper range of physiologic gastric nitrite concentrations at acidic pH. These findings suggest that ranitidine is not converted to NDMA in gastric fluid at physiologic conditions and are further supported by the results of a recent clinical trial.


Atopic dermatitis (AD) is a remitting relapsing chronic eczematous pruritic disease. Several studies suggest that gut microbiota may influence AD by immune system regulation.We performed the first in‐human efficacy and safety assessment of fecal microbiota transplantation (FMT) for AD adult patients. All patients received 2 placebo transplantations followed by 4 FMTs each 2 weeks apart. AD severity and fecal microbiome profile were evaluated by the Scoring Atopic Dermatitis Score (SCORAD), the weekly frequency of topical corticosteroids usage, and gut microbiota metagenomic analysis, at the study beginning, before every FMT, and 1–8 months after the last FMT.p = .011), 50% and 75% decrease was achieved by 7 (77%) and 4 (44%) patients, respectively. At Week 18 (8 weeks after the last FMT) the average SCORAD score decreased from baseline at Week 4 , 50% and 75% decrease was achieved by 7 (77%) and 6 (66.7%) patients respectively. Weekly topical corticosteroids usage was diminished during the study and follow‐up period as well. Two patients had a quick relapse and were switched to a different treatment. Two patients developed exacerbations alleviated after an additional fifth FMT.Nine patients completed the study protocol. There was no significant change in the SCORAD score following the two placebo transplants. The average SCORAD score significantly decreased from baseline at Weeks 4–12 (before and 2 weeks after 4 times of FMT) (59.2 ± 34.9%, Wilcoxon Metagenomic analysis of the fecal microbiota of patients and donors showed bacterial strains transmission from donors to patients. No adverse events were recorded during the study and follow‐up period.FMT may be a safe and effective therapeutic intervention for AD patients, associated with transfer of specific microbial species from the donors to the patients. Further studies are required to reconfirm these results. Fecal microbiota transplantation may be a safe and effective therapeutic intervention for AD patients, associated with transfer of specific microbial species from the donors to the patients. Nearly 20% of all cases are considered as moderate or severe, causing quality of life impairment, psychological, social, as well as financial burdens.Atopic dermatitis (AD), a remitting‐relapsing chronic eczematous pruritic skin disease, affects up to 20% of children and up to 10% of adults.Until recently, the therapeutic ladder of AD consisted of topical treatments, phototherapy, and immunosuppressant agents.Several studies addressed the role of microbiota in AD, with much of the emphasis being put on the skin microbiota, in particular Staphylococcus aureus.In this study, we evaluated the effect of fecal microbiome transplantation in AD patients.22.1AD patients treated at the Dermatologic department of the Tel Aviv Medical Center, who were ≥18 years of age, with moderate‐to‐severe AD, as defined by a Scoring Atopic Dermatitis (SCORAD) score ≥25, with minimum disease duration of 3 years, inadequately controlled by topical and systemic therapy. Key exclusion criteria comprised of another concomitant active dermatologic disease, pregnancy, and systemic therapy including antibiotics and phototherapy within 4 weeks before the beginning of the study.2.2This was a proof of concept, single‐blinded, placebo‐controlled cross‐over pilot study addressed to assess the safety and efficacy of FMT for the treatment of mild‐moderate AD, and to assess the change in the fecal microbiota following FMT. All patients received 2 doses of placebo FMTs, followed by 4 treatment FMTs  each 2 weeks apart Figure . Each paDuring the study period, patients could use only topical therapy including emollients and glucocorticoids or calcineurin inhibitors.The protocol was approved by the ethics review board of the Tel Aviv Medical Center. All patients provided written informed consent in accordance with the principles of the Declaration of Helsinki. There was no commercial support for the trial. All the authors vouch for the accuracy and completeness of the data, for the adherence of the trial to the protocol, and for the reporting of adverse events.2.32, excluded for any significant medical history or for any use of antibiotics in the preceding 3 months. They were eligible according to the Israeli Ministry of Health guidelines which include a physical examination and elaborative laboratory screening tests, including fecal enteric pathogens , serum antibodies to hepatitis A, B, and C; human immunodeficiency virus; HTLV, and Treponema pallidum as well as celiac, CBC . Preparation of capsulized FMT was previously described.The three volunteer stool donors were healthy, nonpregnant adults aged 18–50 years, with a normal body mass index of 18.5–24.9 kg/m2.4Two doses of 15 FMT capsules were administered on 2 consecutive days. Patients were asked to fast overnight before capsule intake.2.5The study primary endpoint was the overall mean change from baseline of the SCORAD score.2.6Stool samples collected from the donors, and the patients during the study period were sequenced into metagenomics reads using Illumina NextSeq. We used bowtie2The CP cut‐off means that dissimilarities below 0.00005  changes per base pair can differ due to a different number of CP. We therefore define the minimal dissimilarity detection threshold to be 0.00005 changes per base pair.NS is the number of shared strains between samples X and Y and NC is the number of comparable species between samples X and Y.Dissimilarities below the defined detection threshold are referred as “transmission events” or “strain sharing”. An aggregated dissimilarity score was defined for pair of samples as:2.7Statistical analysis was performed with Python scipy.stats package. The Wilcoxon signed‐rank test for comparison between two matched samples was used for calculation of significance levels between different groups. Correlations were calculated according to Pearson correlation method.3A total of 15 AD patients were enrolled to the trial. Five did not enter the study due to inability to comply with the trial regimen. Another patient (patient 6) breached the study protocol by using enemas, thus, possibly effecting the gut microbiome composition. Nine patients , with average age of 44.6 years (range: 24–68 years), completed the study. One of these patients (patient 8) failed to deliver stool samples and is, therefore, not included in the fecal microbiome analysis.Before treatment, the average SCORAD score of all 9 patients was 51.2 ± 16.2, with 4 patients classified as moderate (SCORAD 25–50), and 5 as severe (SCORAD >50). The average usage of topical corticosteroids was 10.4 ± 3.5 applications per week. Before enrolment all patients received topical therapy as well as one or more systemic treatment (Table p = .859), and the number of weekly applications of topical corticosteroids . One patient exhibited improvement of the SCORAD score of 31%, four patients exhibited improvement in the range of 1%–11%, and the remaining four patients experienced worsening by an average SCORAD elevation of 19% (range: 3%–51%). All 9 patients who started the study completed the FMTs protocol. Following each FMT there was a significant reduction in the SCORAD score compared to the score at Week 4, (after 2 placebo transplantations and before the FMT treatment)  . At Week 12 (2 weeks after the last FMT), 50% and 75% decrease of the score, compared to the score before FMT (Week 4) was achieved by 7 (77%) and 4 (44%) out of the 9 patients, respectively. Two participants had a quick relapse and were switched to a different treatment. Seven patients continued follow up and at Week 18 (8 weeks after the last FMT) the average SCORAD score continued to decrease  and 50% and 75% SCORAD decrease was achieved by 7 (77%) and 6 (66.7%), respectively. The frequency of the weekly topical corticosteroid usage was diminished during the follow‐up period as well .After the placebo part, there was no significant change in AD severity  had an exacerbation, reflected by an increase of the SCORAD score from 11 to 49, and from 8 to 36, 32 and 21 weeks after the last FMT, respectively, still a reduction from their baseline score. Additional single FMT led to SCORAD score decrease of 80% and 40% respectively  standing out as it was transmitted in 7 of the 8 patients.For most patients, we found high similarity between strains at baseline and after placebo treatment, indicative of high microbiome stability. In contrast, following the FMTs, we found high dissimilarities to strains at baseline, indicative of transmission events and changes in the microbiome strain pool. Comparing patients to their donors, we found a mirror picture, whereby there was high dissimilarity between the donor and both baseline and postplacebo samples of each patient, and low dissimilarity after the FMT. Together, these results suggest that without any treatment (including placebo) bacterial strains of patients are stable over time but following FMT some strains are transmitted from donors and replace previous strain that exist in patients.3.2We performed a t‐distributed stochastic neighbor embedding analysis on the pairwise dissimilarity matrix of all samples Figure . This gl3.3R = .68, p < .001),  and the sample dissimilarity between donor and their patients (4In this study we examined the effect of FMT treatment on moderate to severe AD. Four FMT sessions resulted in significant improvement in signs and symptoms as compared to baseline. Despite our small cohort these results provide the first clinical evidence for the importance of the gut microbiota in AD patients and demonstrate that FMT may be an effective therapeutic intervention. There were no side effects to the treatment during the study and the follow up period.We further found that FMTs resulted in strain transmission events to patients and that the higher the similarity was between the microbiome of donors and the patients after the FMT, the higher the clinical improvement was. Some strains were transmitted to multiple patients, with SGB_1626—Prevotella copri being a notable case as it was transmitted to 7 of the 8 eligible patients for whom stool samples were available. These results suggest that the gut microbiota may have a causal effect in AD and may be a viable therapeutic target for AD.There was a variability in the response to the FMT treatment among the patients. Some reacted immediately after the first FMT while others improved substantially only few weeks following the fourth FMT.Five patients retained the improvement during the follow up period. Four patients relapsed. Two had an early relapse, on Weeks 10 and 12. The other 2 patients, experienced late relapse and responded well to a fifth FMT, revealing that microbiota change after the FMT can be temporary.Two patients had a substantial improvement after placebo transplantation, not associated with microbial composition alteration, reflecting the placebo effect or natural course of the disease.This study demonstrates for the first time, that FMT can be an effective and safe procedure for moderate to severe AD patients. The results of the dupilumab studies, demonstrated a mean change of the SCORAD score at Week 16 of 57%–62% from baseline,The association between microbial transfer and clinical outcome was most strongly observed in two patients who responded to the FMT, experienced a relapse, and regained response after additional FMT, suggesting FMT can be therapeutically used for the induction of initial disease control but also maintenance of disease control of AD. Although this is a pilot study, its unique design enabled us to demonstrate that these changes were not apparent in the control arm. Nevertheless, this study has limitations, including the small number of participants, the lack of double blinded design and lack of information regarding additional factors that may impact the gut microbial composition, such as diet.FMTs have been shown to be the most effective therapy for recurrent Clostridium difficile infection.The skin and the intestine both have an important role as immunological barriers and immune regulation. Intestinal microbiota influence the skin through modulatory effect of the gut on systemic immunity.Probiotic‐fed mice exhibited increased dermal thickness, improved folliculogenesis, and increased sebocyte production resulting in thicker and shinier fur.In humans, local application of Roseomonas mucosa was effective for AD.Further studies, randomized double blind placebo controlled, should be performed to confirm the clinical efficacy and determine the best regimen of FMT for AD. Additional multiomics analysis should be performed to provide insight towards the exact bacteria, metabolites and immune mediators involved.Funding information is not available.The authors declare that there are no conflict of interests.Conceptualization: Jacob Mashiah, Nitsan Maharshak, Eran Segal; Data Curation: Anastasia Godneva, Tal Karady; Formal analysis: Tal Karady; Investigation: Nitsan Maharshak, Jacob Mashiah, Naomi Fliss‐Isakov, Dan Slodownik, OA, Liat Samuelov, Eran Ellenbogen, DH; Methodology: Jacob Mashiah, Nitsan Maharshak, Eran Segal; Project administration: Jacob Mashiah, Nitsan Maharshak, Eran Segal, ESp, Naomi Fliss‐Isakov; Resources: Eran Segal, Nitsan Maharshak, ESp; Supervision: Jacob Mashiah, Nitsan Maharshak, Eran Segal; Visualization: Tal Karady; Writing—original draft: Jacob Mashiah, Tal Karady, Nitsan Maharshak, Eran Segal; Writing—review and editing: Jacob Mashiah, Tal Karady, Nitsan Maharshak, Eran Segal, Naomi Fliss‐Isakov.


In recent years, many articles have demonstrated that magnetic resonance imaging (MRI) may be performed successfully in the study of the chest. The aim of this study was to evaluate the potential role of MRI in the differentiation of benign from malignant pleural disease with a special focus on malignant pleural mesothelioma and on MRI protocols. A systematic literature search was performed to find original articles about chest MRI in patients with either benign or malignant pleural disease. We retrieved 1246 papers and 17 studies were finally identified as being in accordance with our purpose. For a morphologic assessment, T1-weighted and T2-weighted sequences were usually performed, eventually associated with T1 post-contrast sequences for better detection of pleural lesions. Functional sequences such as Diffusion Weighting Imaging (DWI), associated with the evaluation of Apparent Diffusion Coefficient (ADC) maps, were lately and gradually introduced in chest MRI protocols and their potentiality in differentiating benign from malignant disease has been investigated by many authors. Many progresses have been performed to improve quality images and diagnostic performances of MRI. A better and early identification of pleural disease may be obtained, providing MRI as a possible tool that can differentiate malignant from benign pleural disease without using invasive procedures. Malignant Pleural Mesothelioma (MPM) is a rare aggressive tumor that derives from mesothelial cells found in the pleural and peritoneal surface . MPM is Patients with MPM are often symptomatic with dyspnea and chest pain; other symptoms may be cough, malaise and weight loss . The preMPM has a poor prognosis with a median survival of 4 to 18 months . NowadayMPM diagnosis is still based on histological assessment through thoracoscopy or percutaneous biopsies with imaging guidance by computed tomography (CT) or ultrasound (US) . In the Imaging examinations are crucial in MPM diagnosis and staging; in particular, chest radiography is the first-line test to assess pleural lesion, although it has a low sensitivity and specificity. Pleural effusion, pleural masses or plaques may be detectable on an X-ray but CT results as the gold standard for pleural lesions evaluation . CT alloThe role of MRI in pleural disease evaluation and in the diagnosis of MPM has long been discussed over the past 20 years. In particular, in the last 10 years, the number of reviews and articles regarding the role of imaging in MPM assessment increased. Moreover, international conferences, such as the International Conference of the International Mesothelioma Interest Group (iMig), founded in 1991, analyzed the role of imaging in the management, diagnosis, prognosis and response to therapies of MPM ,13,14.MRI was found to be superior to CT in the evaluation of pleural lesions, in the presence of small foci, and in the differentiation of malignant from benign disease ,16. Due MRI limits are well known, such as the higher cost and the reduced availability of scans. Moreover, MRI acquisition takes at least 20–25 min, and this could be a limit in patients with low performance status and breathing difficulties . RegardlThe aim of this study was to evaluate the potential role of MRI in the differentiation of benign from malignant pleural disease with a special focus on malignant pleural mesothelioma and on MRI protocols.A systematic literature research was performed to identify all relevant data on the potential role of MRI in differentiating benign from malignant pleural disease.The databases utilized for the research of relevant publications were Scopus, Web of Science and PubMed. The last search was run on 20 November 2021. The keywords used to identify articles included benign pleural disease; malignant pleural disease; malignant pleural mesothelioma; magnetic resonance imaging; MRI. Filters were applied to include only articles published in English, described as original research and published after the year 2000. Only articles with a sample size greater than 10 patients were included.Two authors (AM and FV) independently performed a first selection of the articles based on the title. Subsequently, the abstracts of the identified studies were screened and the full text of studies that passed the title and abstract screening was read. Any disagreement was overcome by discussion and reaching a mutual agreement.A total of 1246 studies were obtained from the research conducted. In total, 1090 records were removed for lack of inherence, while there were 112 duplicates. Titles and abstracts of the remaining 44 records were screened and after this process 35 articles were assessed for eligibility. Each article was fully read and after this process 18 papers were excluded because they did not meet all eligibility criteria. Finally, 17 studies were included in this review , Table .When evaluating pleural lesions on MRI, a morphological assessment should be performed primarily. To achieve proper anatomical imaging and to reduce susceptibility artifacts, the Turbo Spin Echo (TSE) T2w sequence with a short echo spacing sequence, on axial, coronal and sagittal plans is recommended ,15,17,22Malignant pleural mesothelioma shows an inhomogeneous hypointense to isointense signal on T1w and hyperintense signal on T2w MR sequences  and enhaIn 2021, Sabri et al., highlighted MRI morphological features such as contour and thickness as potential tools to differentiate malignant from benign lesions with a sensitivity of 89.29%, specificity of 76%, positive predictive value of 89%, negative predictive value of 76.92% and accuracy of 85.37% .In 2004, Weber et al. evaluated the role of a very short T2* gradient echo pulse sequence with fat suppression and radial K-space acquisition. The MRI protocol also included high-resolution breath-hold T1w TSE, a respiratory-gated T2w TSE, half-Fourier single-shot turbo spin-echo (HASTE) sequences and finally a contrast-enhanced T1w acquisition with fat suppression. Radial MRI application provides a shorter acquisition time because of its ultrashort echo time of 0.5 milliseconds. Due to the repeated sampling of the k-space, it is helpful in reducing the effect of motion artifact due to cardiac and breath activity .In a study by Plathow et al., HASTE sequences were defined as optimal for tumor delineation and pleural fluid evaluation . In thisCT is known to be a feasible and fast imaging modality for the assessment of structural changes in many chest pathologies, including pleural disease. The development of new MRI techniques, such as functional ones, has also led MRI to gain more importance in the study of the chest, where the use of contrast agent has proven to be useful for a better pleural evaluation ,22, therDiffusion weighted imaging may be useful for a better detection of pleural disease, helping in the differentiation of a benign origin of the disease from a malignant one and to distinguish malignant lesions from each other, also thanks to the involvement of post-processing analysis (ADC maps) ,28.A concordance on a threshold value for mean ADC able to differentiate lesions is still missing, although different technique of region of interest (ROI) positioning has been investigated to reduce errors in the collection of these values ,30.An easy, fast way of recognizing of malignant pleural lesions was also assessed and proposed, as to avoid the use of ADC .In 2006, Plathow et al., combined morphological and functional evaluation comparing standard 2D MRI and novel 3D dMRI (dynamic MRI) techniques. Functional MRI is of great importance not only in surgical/therapeutical planning but even in the assessment of tumor response. Two-dimensional dMRI was performed using true-FISP (time-resolved true fast imaging with steady-state precession) sequence measuring the displacement of chest wall and diaphragm on a workstation . Three-dimensional dMRI was obtained recording lung movement during breathing cycles in three dimensions (3D) using an isotropic time-resolved 3D gradient echo pulse sequence associated with the view-sharing implementation TRICKS (time-resolved interpolated contrast kinetics). Three-dimensional dMRI was found to provide functional parameters indicative of therapeutic response not available with simple spirometry. Regardless, 3D dMRI had a low temporal resolution and a long postprocessing time compared to 2D dMRI .For pleural lesion evaluation, intravenous contrast administration is mandatory. For this purpose, a 3D gradient echo with fat suppression sequence is suggested with an initial basal acquisition and subsequent post-contrast study ,22. PleuTo differentiate between malignant and benign pleural lesions, a novel, semi-objective biomarker, the early contrast enhancement (ECE), was proposed in a study by Tsim et al. . Sixty-sTumor response assessment is another critical issue. Modified Response Evaluation Criteria in Solid Tumors (mRECIST) are usually applied, measuring lesion thickness. CT is still the first line imaging method for MPM mRECIST application , but MRIThe evaluation of tumor vascularization, especially with the development of antiangiogenic drugs, has an important prognostic role. DCE analysis may provide information of lesion response to therapy when morphological evaluation is failing. A quantitative analysis of tumor enhancement was proposed by Tomšič et al., in 2019. DCE parameters were evaluated using two different kinetic models: the extended Tofts model and the adiabatic approximation of tissue homogeneity model .DWI is a type of functional imaging based on the random movements of water molecules in tissues. Many pathologies, such as malignancies and inflammatory disease, can alter water diffusion causing a restriction of molecules movements because of molecular and structural changes . ThroughMany studies have been conducted to confirm that both DWI and ADC maps can be used in the evaluation of MPM or malignant pleural disease (MPD) and in differentiating benign from malignant disease. Moreover, a mean ADC value that could reliably be used for distinguishing malignant from benign lesions has been investigated too.2), followed by DCE imaging acquisitions, used retrospectively for the evaluation of misclassified lesions. A statistically significant difference between ADC values of MPD and that of benign lesion was obtained; a mean ADC value of 1.52 × 10−3 mm2/s was suggested as an optimal cut off . Using an ADC range between 1.52 and 2.00 × 10−3 mm2/s a misclassification of lesions may occur; the authors hypothesized as the presence of necrosis or inflammation in the tumor as a probable explanation. The use of DCE in addition to information gained through ADCs improved the sensitivity to 92.8% with, regardless, a decrease in specificity to 94.1%. Accuracy, PPV and NPV all changed to 93.5%, 92.8% and 94.1%, respectively [In 2012, Coolen et al. prospectively evaluated the use of DWI in the differentiation of MPD from benign lesions . With a ectively .2) and multiple ADC maps were obtained . A statistically significant difference of ADC1 and ADC2 between metastatic pleural thickening and benign lesions was found (p < 0.05). The authors found that a mean ADC value ≤ 1.5 × 10−3 mm2/s suggests a malignant etiology, despite low sensitivity and specificity [In accordance with these results are those of İnan et al., (2016); in this work the authors tried to differentiate metastatic malignant lesions from benign pleural thickening through DWI. Multiple b values were used to acquire diffusion-weighted images  [The results obtained by Sabri et al., in 2021 are similar . A 1.5T a et al. ,31. Auth< 0.001) .A different result was reported by Jiang et al. in 2021; retrospectively analyzing the diagnostic performance of DWI using a 1.5T superconducting magnet in patients suspected of having pleural malignancies, the author found out that although hyperintense pleural areas on DWI are suggestive of malignancy, especially with high b values, the mean ADC value in the group of pleural malignancies was not statistically different from that of the benign group, nor they could find a cutoff value that was able to discriminate malignant lesions from benign ones. The authors supposed that these results may be the consequence of the impossibility to obtain the ADC value in a large group of benign lesions and that a subjective position of the ROIs chosen for ADC evaluation may have led to bias .Thus, a mean ADC value able to discriminate reliably MPD from benign lesions is still debated and more studies are needed to assess a robust cutoff value .The problem of different ROIs positioning in the ADC measurements of pleural abnormalities related to intra- and interobserver variability was assessed by Priola et al. in 2017 . Five diThe capability to differentiate histologic subtypes of MPM without using an invasive approach was proposed by Gill et al., in 2010 .2). The final group consisted of 50 investigated patients affected with histologically proven MPM . Each different histologic subtype showed different average ADC values: for the epithelioid it was 1.31 ± 0.15 × 10−3 mm2/s, for the biphasic it was 1.01 ± 0.11 × 10−3 mm2/s, and for the sarcomatoid it was 0.99 ± 0.07 × 10−3 mm2/s. No statistically significant difference was found between ADC values of biphasic and sarcomatoid tumors, while the ADC of epithelioid MPM appeared to be significantly higher than those of biphasic and sarcomatoid MPM. ADC value of the epithelioid subtype showed a sensitivity of 60%, a specificity of 94% and an accuracy of 84% in the differentiation from the sarcomatoid subtype [Through a 3T magnetic resonance, free-breathing DWI was acquired with different b values  led them to coin the term “pleural pointillism” to report the appearance of multiple hyperintense spots mostly at high values of b parameter (1000 s/mm2) as result of MPD in visual imaging examination. This diagnostic marker presented higher values of sensitivity, specificity, PPV, NPV and accuracy  compared to those obtained at the CT evaluation of mediastinal thickening and shrinking lung appearance. The authors suggested that this pattern of visualization may help in pleural biopsy and limit samples [For a more clinical and rapid assessment of pleural lesions, in 2014, Coolen et al., suggested a new visual evaluation of MPD through DWI in a group of patients suspected of being affected by malignant pleural mesothelioma . The ana samples .2 and resembling those of pleural pointillism, in 94% of patients with malignant pleural lesions [In accordance with these results are those reported in 2021 by Jiang et al. The authors found areas of signal hyperintensity, with b values of 800 s/mm lesions .A different type of appearance of MPM in DWI was reported by Usuda et al. . The autOn the basis of the papers found in literature, an MR protocol for patients with MPM was hypothesized and elaborated. The protocol was developed using a 1.5T MRI system . An eight-channel phased array coil was used, and the patient was in supine position.We elaborated an MRI scan protocol with pre-contrast acquisitions, including functional scans (DWI) and post-contrast acquisitions, performed at different acquisition times. We used commercially available sequences, while experimental MRI techniques were not considered, letting their use remain limited to a research setting. The proposed MRI protocol for MPM patients is summarized in For a morphological analysis, we proposed a 2D T2-weighted acquisition  on an axial, coronal and sagittal plan. Sequences were acquired at end expiration during multiple short breath-hold. A T1-weighted turbo spin echo (TSE) sequence was made to complete the morphological assessment.2) was performed to assess the presence of areas of signal restriction , acquisitions occurred at 40 s, 80 s, 3 min, 4:30 min and at 5 min, to assess lesions enhancement.Finally, T1-weighted fat-saturated 3D gradient echo (VIBE) sequences before and after contrast injection were made. After the administration of contrast injection (0.2 mL/kg of gadoteridol (ProhancePost-processing analysis of the data was also performed with a commercially available software, generating ADC maps.MRI application for chest disease evaluation has long been discussed. In this study we revised the potential role of MRI in the assessment of MPD and in the differential diagnosis between malignant and benign lesions. An MRI protocol in order to evaluate MPD with a special focus on MPM was proposed.Further studies are needed to confirm and improve MRI application for pleural and general chest disease assessment.


In our study, we analyzed 30years of climatological data revealing the bean production risks for Western Amazonia. Climatological profiling showed high daytime and nighttime temperatures combined with high relative humidity and low vapor pressure deficit. Our understanding of the target environment allows us to select trait combinations for reaching higher yields in Amazonian acid soils. Our research was conducted using 64 bean lines with different genetic backgrounds. In high temperatures, we identified three water use efficiency typologies in beans based on detailed data analysis on gasometric exchange. Profligate water spenders and not water conservative accessions showed leaf cooling, and effective photosynthate partitioning to seeds, and these attributes were found to be related to higher photosynthetic efficiency. Thus, water spenders and not savers were recognized as heat resistant in acid soil conditions in Western Amazonia. Genotypes such as BFS 10, SEN 52, SER 323, different SEFs , SCR 56, SMR 173, and SMN 99 presented less negative effects of heat stress on yield. These genotypes could be suitable as parental lines for improving dry seed production. The improved knowledge on water-use efficiency typologies can be used for bean crop improvement efforts as well as further studies aimed at a better understanding of the intrinsic mechanisms of heat resistance in legumes. Phaseolus vulgaris L.) is the most important legume in people’s diet in the tropics of Latin America and East Africa , maximum carboxylation rate (Vcmax), , , 2008, A(Vcmax), .sg), they sustain the diffusion of CO2 in the leaves, improving cooling from transpiration (WUE) by maintaining or increasing photosynthesis  has been established as an important attribute that describes the efficiency of plants to use available water for carbon sequestration  by maximizing water capture for more production with better gas exchange. However, these results were generated in lysimeter-like tubes, not in the field conditions.Water use efficiency  of common beans with better heat resistance that contributes to better physiological performance and grain yield under heat stress. The study provides novel information on the physiological behavior and response mechanisms to high temperatures in different common bean genotypes in Western Amazonia. Results and suggestions can be used for crop improvement efforts and for studies aimed at a better understanding of the mechanisms of heat stress resistance in beans.Throughout its evolution and domestication, the common bean eventually formed two cultivated gene pools, the Mesoamerican gene pool and the Andean gene pool , origina−1, an average temperature of 25.5°C, and a relative humidity of 84%. Under greenhouse conditions the mean maximum and minimum temperatures were 33.8°C and 24.6°C, respectively, the mean daily temperature was 28.5°C and the mean relative air humidity was 56% , located in Florencia, Caquetá (Colombia). Located in the moist climate of the tropical rainforest ecosystem, it exhibits an average annual precipitation of 3,800mm, 1700h of sunshine year was 56% . These ePhaseolus vulgaris L. , four Mesoamerican interspecific lines of P. vulgaris × P. coccineus , eleven Andean interspecific lines of P. vulgaris × , one interspecific line from P. vulgaris × P. acutifolius (SER 212), 11 interspecific lines from P. vulgaris × P. acutifolius × P. coccineus , one Mesoamerican type with interspecific genes, generated through crossing [(ALB 74 × INB 841)F1× RCB 593)], and four interspecific lines from P. vulgaris × P. coccineus which resulted in a group of Mesoamerican gene pool lines . The YR serves as a first standardization of obtained genotype yield toward the geometric yield from the whole individual experiment (year repetition). The second step was to calculate general stress intensity index (SII) as a second data standardization using ratio of geometric means from all (in our study two) years of experiments . The third step was to calculate GSI for each particular genotype used in the study. The GSI was calculated using the following equation: GSI={1−[Ÿ ]}/SII, where Ÿ is the geometric mean, YRx1 or x2 is the yield change per genotype X in first (1) or second (2) year, and SII is general stress intensity index.To evaluate the agronomic yield upon high temperature environment, destructive sampling (85–90 DAS) was carried out in the central row of each plot, the pods of the harvested plants were threshed, and the grains were cleaned and oven-dried to determine their yield , or Transpiration (E) were plotted on the X-axis. As the dependent variable, grain yield was plotted on the Y-axis. Four quadrants were visualized by plotting the corresponding averages on each axis.The Michaelis–Menten hyperbolic constant was used to adjust the age in R . PearsonA the model adjustment was carried out using linear segment models with two segments given by the following function sg and A as response variables was prepared. This point corresponds to the maximum ambient temperature value at which the A took a linear segment direction. For each genotype we calculated the mean expected value for responses (sg and A) when temperature was 27.74°C. With these estimated values we performed a scatter plot of A against gs and included a label for each genotype.Due to the non-linear behavior of WUE values as well as those that had higher heat tolerance allowing to maintain bean yield in advanced common bean lines. With the conformed bean genotypes typologies, a principal component analysis (PCA) was also performed and the effect of the leaf temperature depression on the different physiological mechanisms to maintain a higher photosynthetic efficiency was tested by means of a Monte Carlo permutation test. The ANOVA using LMM was adjusted to analyze the effect of the fixed factor (Genotype). Plots associated with genotypes within the monitoring period (repeated measurements) were included as random effects. The assumptions of normality and homogeneity of variance were evaluated using an exploratory residual analysis. Differences between genotypes were analyzed with Fisher’s post hoc LSD test with a significance of α=0.05. The LMM were made using the lme function in the nlme package, the cluster analysis, PCA and the graphic outputs were made in the packages “ade4,” “ggplot2,” “factoextra” and “corrplot” in the R language software, version 3.4.4  due to the reduction in stomatal conductance  and substomatal CO2 concentration  which resulted in lower grain yield , generating processes of stomatal limitation to photosynthesis (r=0.60 between LTD and lim pg<0.001). For an adequate grain yield, carbon fixation must be high , increasing gs, resulting in a transpiration cooling mechanism . Furthermore, this characteristic negatively affects the efficient use of water (r=−0.88 between E and WUE p<0.001). There were other characteristics such as LSP which was positively related to both GY and A, as well as to E.The high air temperature had a negative effect on common bean genotypes by decreasing carbon assimilation  of the 64 bean genotypes ranged from 336 to 1,614kgha−1 with an average value of 957kgha−1 , contrary to what was found in the different SEFs lines , SMR 173, SER 323, and SEN 52. Some genotypes found in the lower left quadrant  have low E and positive LTD which negatively affected A and GY. However, sometimes this mechanism was not efficient enough in the process of grain formation  probably because of additional effects from soil constraints.In general, grain yield  showed opposite behavior characterized by low gs, high limg, and a high GY. We found that for low left quadrant genotypes that have low sg, the glim was higher, and therefore, the leaf temperature was higher than the air temperature (positive LTD) with negative effect on GY. Other genotypes with similarly high GY values presented a lower E and high WUE that caused higher leaf temperature than air temperature  presented specifically for SEF 40  SMN 99, SMN 95, SEF 14, ALB 121 and SMR 123, and (2) ALB 352, SMR 174, and SEN 52] which with similar Ci, achieved high carbon assimilation  under very different leaf temperatures. These two responses suggest different physiological behavior as mechanisms of leaf-lead air temperature dissipation and reveal a physiological or biochemical player in the background  and a maximum rate of electron transport driving regeneration of ribulose-1, 5-bisphosphate . These genotypes were able to achieve very high GY  despite leaf temperatures more than +1°C above ambient temperatures . From the historical climate information, we found 85days of the year that have values higher than 27.74°C, distributed in two periods of the year that correspond to Julian days 1 to 53 (between the months of January to February) and Julian days 243 to 284 . EUW: Effective use of water, and (3). OEUW: Opportunistically effective use of water (p<0.0001) using leaf temperature differential (LTD), instantaneous water use (WUE), the transpiration (E), gas exchange (gs), gasometric limitation caused by air temperature increase (glim), and carbon use and fixation .According to the cluster analysis of different variables taken from the 64 bean genotypes, three statistically different groups with contrasting physiological mechanisms to tolerate high air temperature were found. According to their physiological traits, we divided the bean genotypes into: (1). of water . These tgs, glim), water use , intracellular carbon (Ci) and leaf temperature differential (LTD) is clearly opposed along axis 1 (32.8% of variance can be explained), separating genotypes with contrasting differences in heat dissipation strategy and water use. Axis 2 (20.5% variance can be explained) opposed genotypes with characteristics related to ΦPAR and LSP. The separation of bean genotype into three typologies according to physiological traits was significant and explained 30% of the total variance according to the Monte Carlo test  and SEF 10, SEF 46 and SEF 73 . The two important mechanisms involved  reduced glim, increasing sg and Ci, and therefore carbon fixation , should be involved as a heat resistant genotype trait  and SCR 56 (Phaseolus vulgaris) with GY of 1,493 and 1,479kgha−1, respectively. The average GY for this typology is 835kgha−1. This group represented the highest values of WUE, hence its name. This high efficiency had a notable impact on the capacity to carry out gas exchange processes, reducing Ci and E, which increased glim. In turn, the decrease in E was followed by increased LTD (Leaf temperatures were sometimes higher than air temperatures). Despite this, SEF 70 and SCR 56 showed higher capacity of translocation of photoassimilates (enhanced seed filling) likely because of their high LSP which translated into an increase in the maximum rate of electron transport driving regeneration of ribulose-1, 5-bisphosphate . This resulted in an increase in the maximum rate of RuBisCO carboxylation (Vcmax). The efficiency of the PSII is also demonstrated as it was able to regulate the excess energy and to carry it along the electron transport chain. The correlations of this typology were largely related to those variables related to carbon use , as well as reduced Ci, since this typology has a high glim . Generally, in some terminal drought scenarios, these traits can increase final GY. However, the lowest A, Rd and LCP which altogether surely lead to lowest GY proved that conservative crop behavior can significantly decrease the yield potential upon heat in Western Amazonia  we can postulate this group as conservative in water use despite water availability.This group demonstrates typical conservative traits of the water savers group  with yields of 1,608, 1,346 and 1,205kgha−1, respectively, as well as SEF 40  with a yield of 1,557kgha−1. The main characteristic of this typology is related to higher values of Rd and LCP and efficiency in carbon fixation (Vcmax). At the same time, with its medium capacity in the instantaneous photosynthetic use of water (WUE), middle E, middle Ci, glim and gs, these genotypes regulated leaf temperature less effectively than EUW group. However, this finely regulated water usage allowed efficient carbon fixation, achieving statistically similar performance that achieved heat-resistant ones through transpiration cooling (EUW typology). SER 16 was previously characterized as a typical high-yielding water saver in terminal drought. However, in our study, Western Amazonian soil allowed only very low yield performance of SER 16. We can characterize this third group OEUW as heat resistant genotypes with opportunistic water spender strategy and photosynthetic heat acclimation (This group of bean genotypes had an average limation  with hybGSI) we found differences between genotypes , SCR 56, SMR 173 and SMN 99 (mainly from EUW group) showed GSI values higher than three due to their ability to adapt.By analyzing heat stress resistance in beans under the western Amazonian conditions using the genotype stress index , i.e., an increase of the substrate (Ci) for carbon fixation (A) at the cellular level by increasing also E. Therefore, when E increased, some bean genotypes—through latent heat transfer—maintained a lower leaf temperature relative to that of the air (LSP with A and GY. To work this mechanism properly (LTD), water must be exchanged for carbon and carbon then incorporated into biomass, which relates to effective use of water , reducing latent heat flow, thus increasing leaf temperature . Another group made up of SEF 40 and SEN 52 which, in addition to having very similar characteristics to the WUE typology (anisohydric) presented a higher maximum carboxylation rate (Vcmax), and higher Rd and LCP compared to the other typologies. Interestingly, in some cases the rate of photosynthesis was not limited by the ability of the enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase to carboxylate RuBP (Vcmax) since the electron transport rate for the regeneration of RuBP was also not affected. The mechanism of this typology is to save soil water, dissipate to some degree the negative effect of air temperature but still remain efficient in carbon fixation and assimilate translocation leading to higher grain yield. It has been recently mentioned that a thermal acclimatization has allowed a coupling between Vcmax and Rd  with increased E generating an efficient canopy cooling  hypothetically allowed a translocation of photosynthates for grain formation, thus increasing the GY. Behavior of two genotypes with highest GSI  showed the pragmatic base for the above-mentioned statement: although they both had negative LTDs differences were found in carbon fixation efficiency that allowed high GY performance for both genotypes. However, the connection between E, gs, and overall water uptake and GY need to be further studied.According to the mechanisms described by the different typologies of beans, we found that some of them related to the use of water and others to the ability to increase carboxylation rate. We found that in each typology there were genotypic variations that allowed grain yields over 1,200kgha cooling . High E OEUW typology). Generally, under hot conditions the limitation of A is presented by a decrease in both the maximum rate of carboxylation (Vcmax), the maximum rate of RuBP regeneration (Jmax) and the maximum photochemical efficiency of PSII  and higher carbon fixation ×SEN 34], which has been reported as heat resistant . This ge of PSII . Howeverfixation .1×RCB 593, where ALB 74 provides genes from P. coccineus L. and INB 841 provides genes from P. acutifolius A. Gray. This allowed them to obtain genetic gain by inheriting its heat resistance (OEUW type) was more efficient in fixing carbon (Vcmax) and showed highest photosynthetic values observed in this study. SEF 70 was the most water efficient genotype compared to the others, with a high light saturation point (LSP) where the functionality of the PSII in relation to electron transfer was efficient. A common feature of SEF 10 and SEF 73 was high instantaneous water use.Other bean genotypes that showed heat resistance traits were SEFs  that were generated by crossing (ALB 74×INB 841) Fsistance . When weGY upon heat: BFS 10, SCR 56, SEF 10, SEF 40, SEF 70, SEF 73, SEN 52, SER 323, SMN 99 and SMR 177). The increase in temperature affected the adjustment of the thermal tolerance of PSII . However, we found another successful mechanism that some genotypes besides having a moderate use of water have developed a high carbon fixation efficiency (described for the genotype SEN 52).In this study, we elucidated the physiological mechanisms of ten heat-resistant genotypes  showed that approximately a third of growing seasons experienced at least 1day in which maximum daily temperatures exceeded 30°C and average daily minimum temperatures of greater than 20°C were experienced in all growing seasons. This suggests that high nighttime temperatures are more of a production risk than high daytime temperatures in Florencia of western Amazonia region. Furthermore, climatological profiling for the years 1984–2014 showed that the biggest challenges of growing beans in Western Amazonia are high maximum and minimum temperatures, high rainfall in the first half of the season, high E and therefore more negative LTD, a mechanism that allowed higher A due to the maximum rate of RuBisCO carboxylation (Vcmax) and high rate of electron transport driving regeneration of ribulose-1, 5-bisphosphate . As the air temperature increases, glim increases, decreasing Ci and consequently A. According to the analysis of different variables taken from the 64 bean genotypes, three statistically different groups were found with contrasting physiological mechanisms: (1) WUE: Water Use Efficiency, (2) EUW: Effective use of water, and (3). OEUW: Opportunistically effective use of water. However, we also found outliers from this rule showing photosynthetically effective genotypes, surprisingly on the background of positive LTD.By analyzing the effect of air temperature on carbon assimilation we found that some genotypes had higher LTD with reduced glim increasing sg and Ci as the most important traits therefore resulting in high GY. Heat sensitive water use efficient genotypes  represent the highest values of WUE with a notable impact on the capacity to carry out gas exchange processes, reducing Ci and E, which increased glim and reduced LTD. The value of conservative genotypes need to be verified, especially in regions where terminal drought and high temperatures occur together.Heat-resistant genotypes with effective use of water (EUW) showed profligate spender traits involving the transpiration and thermal dissipation by GSI values and these genotypes could be suitable for use as parental lines for improving dry seed production.By analyzing heat stress resistance in beans under western Amazonian soil and climatic conditions we found that conditions in the Amazonia make beans susceptible to heat and acidic soil resulting in reduced grain yield. However, genotypes such BFS 10, SEN 52, SER 323, different SEFs , SCR 56, SMR 173, and SMN 99 presented high WUE) can crystallize into higher grain yield in the extreme weather and soil conditions of Western Amazonia.Further study is needed to verify if mentioned trait combinations are stable over similar environments and quantify their effect on grain yield in heat areas, as water conservative behavior is not expected to increase bean heat resistance in general. Research focused on lysimetric-related water usage under heat stress  of the above-mentioned three groups is recommended to verify the hypothesis that profligate water-spender and not water conservative traits  funded project named Bean Breeding for Adaptation to a Changing Climate and Post-Conflict Colombia (BBACO). Grant number BB/S018964/1.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.