By applying a multidisciplinary approach, we morphologically and molecularly analysed the cat remains found in a canid faecal sample collected in a forested area of central Italy. Individual Canis lupus italicus) and the European wildcat (Felis silvestris silvestris), which can live in overlapping ecological contexts and sometimes share their habitats with their domestic free-ranging relatives, increasing the risk of anthropogenic hybridisation. In this case study, we exploited all the ecological and genetic information contained in a single biological canid faecal sample, collected in a forested area of central Italy, to detect any sign of trophic interactions between wolves and European wildcats or their domestic counterparts. Firstly, the faecal finding was morphologically examined, showing the presence of felid hair and claw fragment remains. Subsequently, total genomic DNA contained in the hair and claw samples was extracted and genotyped, through a multiple-tube approach, at canid and felid diagnostic panels of microsatellite loci. Finally, the obtained individual multilocus genotypes were analysed with reference wild and domestic canid and felid populations to assess their correct taxonomic status using Bayesian clustering procedures. Assignment analyses classified the genotype obtained from the endothelial cells present on the hair sample as a wolf with slight signals of dog ancestry, showing a qi = 0.954 (C.I. 0.780–1.000) to the wolf cluster, and the genotype obtained from the claw as a domestic cat, showing a qi = 0.996 (95% C.I. = 0.982–1.000) to the domestic cat cluster. Our results clearly show how a non-invasive multidisciplinary approach allows the cost-effective identification of both prey and predator genetic profiles and their taxonomic status, contributing to the improvement of our knowledge about feeding habits, predatory dynamics, and anthropogenic hybridisation risk in threatened species.Non-invasive genetic sampling is a practical tool to monitor pivotal ecological parameters and population dynamic patterns of endangered species. It can be particularly suitable when applied to elusive carnivores such as the Apennine wolf ( Trophic relationships within ecosystems are key parameters in wildlife ecological studies . Feedingloci, permits reconstructing the multilocus genetic profiles of both predator and prey when analysing faecal DNA for diet studies. Later on, unknown multilocus genotypes can be assigned to well-representative parental populations through statistical procedures to assess species, native populations or signs of admixture. Such data can also provide key information on differences in food dynamics, hunting behaviours and ecological relationships among taxa or between pure and admixed individuals of the same species [Canis lupus italicus) and the European wildcat (Felis silvestris silvestris), which often share their natural habitats and individual territories across the Italian Peninsula, and that can successfully mate with their domestic counterparts, the domestic dog (C. l. familiaris) and the domestic cat (Felis catus) [During the last decades, diet analyses of vertebrate predators have been extensively conducted through multidisciplinary approaches, mainly based on morphological and molecular identifications of their prey remains contained in non-invasively collected faecal materials ,5. In pa species . This wos catus) ,12.The wolf and the European wildcat experienced very similar demographic scenarios in Italy, with protracted isolation south of the Alps and recurrent bottlenecks that made them sharply genetically differentiated from any other wolf or wildcat population ,14. NowaEven though distribution ranges and anthropogenic hybridisation, rates in Italy are continuously studied in wolves and European wildcats, especially through non-invasive genetic projects ,17. Intemultilocus genetic profiles of both the predator and the prey. In particular, we exploited the availability of reliable forensic genetic protocols [In this case study, we analysed the DNA contained in the remains of a canid faecal deposition collected in a forested area of central Italy to determine the individual rotocols , well-perotocols  and felirotocols  unlinkedrotocols  to genotOn 9 February 2020, we collected a canid faecal sample containing the consumption remains of a cat, consisting of hairs and claw fragments, in a wooded area of Tuscany in central Italy. The sample was initially preserved at −20 °C, subsequently stored in an oven at 80 °C for four hours to deactivate possible pathogens, and then frozen again at −20 °C until downstream morphological and molecular analyses.After separating hairs from claw fragments with water and a 1 mm sieve, a preliminary morphological identification of the hair sample was conducted by observing cuticle and medulla patterns with a 100–400× zoom microscope .n > 10) and the claw fragment, respectively, in a paper envelope to search for possible canid intestinal endothelial cells and in 40 mL of 96% ethanol to be genetically analysed, thus clarifying the taxonomic status of both predator and prey. Total DNA contained in the hair or on its surface  and in the claw samples was individually extracted using the Blood & Tissue Kit® (Qiagen), following the manufacturer’s instructions.Additionally, since stray dogs and cats, as well as wolf-dog and wildcat-domestic cat hybrids, were recorded in the study area, we stored a tuft of hairs (loci (STRs), discriminating among wolves, dogs, and their first three generations of hybrids [Amelogenin marker, to molecularly sex the extracted DNA; (c) 4 Y-chromosome STRs  determining the paternal haplotype in male individuals; and (d) a dominant 3-bp deletion at the β-defensin CBD103 gene (the K-locus) associated with black coat colour. The diagnostic cat marker panel included 29 domestic cat-derived dinucleotide STRs, discriminating among European wildcats, domestic cats, and their first two generations of hybrids [Each DNA sample was amplified by Polymerase Chain Reaction (PCR) and genotyped through a multiple-tube approach  at diagn hybrids ,17; (b)  hybrids ,29,30.All PCR reactions were performed in a total volume of 10 µL containing the following: 2 µL of DNA, 5 µL of MasterMix (Qiagen Multiplex Kit), 3 µL of Q-solution (Qiagen Multiplex Kit), 0.15–0.30 µL of primers adjusted to the volume with RNAse-free water. PCR products were analysed in an ABI 3130XL automated sequencer and allele sizes were estimated using Genemapper v.5.0 (Applied Biosystems).Hair and claw DNA samples were extracted, amplified, and genotyped in three separate rooms dedicated to low-template DNA samples under sterile UV laminar flood hoods. Negative  and positive  controls were included in each step to check for possible contaminations and correct allelic weights, respectively.locus performed during the multiple-tube approach, using Gimlet v.1.3.3 [Consensus genotypes, amplification success (AS) and error  rates were assessed from the four replicates per  v.1.3.3 .R ≥ 95%.Genotype reliability was calculated by RelioType , consideReliable genotypes were then assigned to their populations of origin  through a first explorative Principal Coordinate Analysis (PCoA) using GenAlEx v.6  and Baye5 iterations following a burn-in period of 5 × 104 iterations, using the Admixture and Independent Allele Frequencies models [K = 2. As reference parental populations, we selected—from the ISPRA Canis database—the 39-STR genotypes of 190 unrelated wild individuals belonging to the Italian wolf population and 89 wolf-sized dogs living in rural areas [qi ≥ 0.955, to the dog population if qi < 0.2, otherwise it was considered as admixed for intermediate (0.2 < qi < 0.954) values  values [A similar approach was used for the felid genotype, using as reference parental populations the 29-STR genotypes of 48 unrelated European wildcats, representative of the species distribution range in the central Apennines, and 65 free-ranging domestic cats, selected from the ISPRA database . Howeverdatabase ,29,30, s) values .Felis sp., suggesting it was a domestic cat, though genetic analyses on the same specimen returned a reliable multilocus genotype (R > 95%) only at the wolf microsatellite panel with an average rate of missing data of 0.154. Conversely, the claw DNA sample yielded a reliable genotype (R > 95%) only at the felid microsatellite panel with an average rate of missing data of 0.315. Mean rates of AS, ADO, and FA across loci were 0.846, 0, and 0  for the hair sample and 0.638, 0.049 and 0.009  for the claw sample, respectively. In the PCoA including canid reference populations, the hair genotype plotted marginally to reference wolves  individual with a Y-chromosome haplotype typical of the Italian wolf population [locus deletion theoretically indicated an animal with a wild-type coat colour. The claw genotype was unambiguously assigned to the domestic cat group with a proportion of posterior probability qi = 0.996 (95% C.I. = 0.982–1, The analysis of cuticle and medulla patterns morphologically classified the hair sample as belonging to e wolves a, whereae wolves b. Assignpulation , whereasNon-invasive genetic sampling can provide useful insights into trophic relationships within ecosystems, clarifying fundamental ecological aspects such as feeding habits, predation behaviour, and dynamics of animal species, thus contributing to designing sound and long-term conservation strategies ,37,38. Ttaxa below the specific level [In this case study, we used the ecological, morphological, and genetic information contained in a biological canid faecal sample to detect any sign of trophic interactions between wolf and European wildcat individuals or their domestic counterparts. Though traditional molecular diet analyses are not able to identify with certainty the taxonomic status of the involved ic level , we overFirstly, we conducted traditional morphological analyses, which revealed the presence of felid remains in the collected canid faecal sample. Then, we used reliable molecular tools and highly discriminant canid and felid STR panels ,26, whicloci. Such a profile resulted in a wolf with traces of dog ancestry at autosomal microsatellite loci, consistently with the presence of wolf-dog admixture cases, repeatedly documented in this region using both video-trapping and genetic tools [Interestingly, despite the initial water treatment of the faecal sample to separate its content, the few remaining canid intestinal cells on the hair sample allowed us to fully reconstruct the predator genetic profile with very high amplification success rates and no sign of ADO or FA across ic tools ,42. We wic tools ,20,43, ppost-mortem consumption of a cat carcass. Although our results were obtained from a single case study, future genetic identifications from faecal samples combined with accurate detections of the consumed prey, if extensively applied, could provide additional useful data about possible shifts in food habits and habitat use between wild predators and their domestic counterparts. This is particularly useful in areas with a documented presence of wild and domestic overlapping [Sporadic cat consumption by wolves has been reported  and our rlapping ,45, thourlapping , and no Our multidisciplinary approach could also be applied to support future studies on possible behavioural alterations between wolves and wolf-dog hybrids in predatory strategies, contributing to detect the presence of admixture on one of their main preys, the wild boar, which is also facing anthropogenic hybridisation with the domestic pig ,47. HoweOur methodological procedures could be further improved by using highly performing markers to genotype non-invasively collected samples such as metabarcoding or highly informative and easily inter-lab comparable SNP panels, in cases of canid or felid predation on wildlife or other domestic pets to establish the real taxonomic status of the predators through a finer scale admixture identification and to provide a more reliable assessment of their potential impact on other threatened species ,47,48,49multilocus genetic profiles and the accurate detection of their taxonomic status from non-invasive DNA. This will contribute to improve our knowledge about feeding habits, predatory dynamics, and anthropogenic hybridisation risk in threatened species, thus collecting information which is key to plan adequate conservation management actions.Our results, although limited to the analysis of a single biological sample, clearly show how a multidisciplinary approach allows the cost-effective identification of both predator and prey


Brucella spp., Neospora caninum, and Bovine Leukemia Virus frequencies were significantly different (p < 0.05) between clusters. The differences between the occurrences of diseases were not sufficient to indicate a genetic subpopulation with increased resistance to infections.Curraleiro Pé-Duro (CPD) cattle, a Brazilian local breed, are animals that are highly resistant to infectious and parasitic diseases. Strategies for the conservation of the breed and the genetic resistance to diseases should also consider the characteristics of the breed. The objective of this study was to analyze the diversity and population structure of the CPD breed using microsatellite markers and to correlate the serological profiles for causative agents of brucellosis, leptospirosis, neosporosis, leukosis, infectious bovine rhinotracheitis, and bovine viral diarrhea. DNA samples of 608 bovines were amplified and genotyped using 28 microsatellite markers for breed characterization. The genotypes were assigned to three clusters, indicating a substructure population related to the geographic distance. The observed heterozygosity was lower than that expected in most loci, and fixation index (Fst) in the subpopulation values ranged from 0.03073 (ETH3) to 0.402 (INRA63) on cluster 1, from 0.00 (INRA32) to 0.39359 (INRA63) on cluster 2, and from 0.00 (INRA32) to 0.28483 (TGLA53) on cluster 3. The  Local breeds have become a target of interest owing to their ability to live and reproduce in extreme climatic conditions and their adaptation characteristics to local conditions, characterizing them as resilient breeds. These characteristics mean that these breeds have a genetic heritage that should be preserved, especially considering the need for food production in the face of global climate change .During the colonization period, the first cattle were introduced in Brazil from the Iberian Peninsula, which underwent natural selection. They eventually adapted to local conditions and became rustic cattle, tolerant to heat, water, and food stress, with high prolificacy and resistance to diseases and parasites . Such anin situ conservation centers in the central-west and northeast regions of the country and ex-situ , companies and research institutions had initiated plans for the conservation of animal genetic resources. Such initiatives aim to identify populations at risk, characterize them phenotypically and genetically, and evaluate their productive potential, both at  ex-situ .There has been concern about the genetic diversity of local breeds, which could be seriously affected, putting these breeds at risk of extinction. The study of this diversity through geographic genetics aids our understanding of the gene flow and the adaptation of species within heterogeneous and fragmented spaces by understanding the geographical distances and how the transmission of genes occurs, making it possible to detect environmental factors that stimulate the adaptation process .Animals separated by geographic distances may present different genetic structures, to the extent of leading to subdivisions between breeds and within the same breed . GeograpGenetic characterization is a criterion used to decide which population should be conserved and is especially important to optimize the choice of samples when resources are scarce, ensuring genetic variability. The selection of breeds and individuals that should participate in the conservation program considers economic interests, adaptability, and the presence of unique alleles .The adaptive characteristics of animal genetic resources can be transmitted to other generations. Therefore, the most adapted genotypes need to be identified and managed. The purpose of identifying genotypes and preserving local breeds is for the possible future use of genetic material as a way to introduce their unique characteristics of adaptability in crossbreeding with commercial breeds, making the offspring more efficient conditions . In addiGiven that drug use and vector controls are difficult strategies to implement, and the environmental and food security impacts of these strategies are negative, one of the most valuable features of disease control is exploiting local breeds that are tolerant to infection. Examples are West African N'Dama cattle, which are tolerant to trypanosomosis, and East African Red Maasai sheep, which are resistant to gastrointestinal worms. Adding to the problem are the lack of resources of low-income cattle breeders, which hamper access to treatment and veterinary services, as well as the resistance of pathogens against the inappropriate use of antimicrobials. In this scenario, genetic management of animals is an alternative to increase resistance to diseases by using the most appropriate breeds for each type of production and improving herds by choosing animals with an increased level of resistance, in addition to crossbreeding to incorporate resistance genes in breeds lacking them .The aim of this study was to analyze the diversity and population structure of CPD cattle by evaluating microsatellite markers (STR) of breed characterization. Additionally, STR were associated to animals serological profile of brucellosis, leptospirosis, leucosis, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), and neosporosis.A total of 1,100 blood samples collected in 2011 from male and female CPD cattle of different age groups were used. The samples were obtained from the project “Central-West Pro Network—Characterization, Conservation, and Use of Local Brazilian Cattle Races: Curraleiro and Pantaneiro,” which also maintains data on the serological profile of herds in the states of Piauí, Tocantins, and Goiás.® Brazil) to detect anti-Brucella abortus antibodies  and responses to epidemiological questionnaires applied at the time of sample collection. The serological tests and places where they were performed as follows: serum agglutination test with buffered acidified antigen  and the International Society for Animal Genetics (ISAG) for studying genetic diversity in cattle. ®, United States).Polymerase chain reaction (PCR) was prepared and the PCR was performed as recommended with 2 µL of DNA, 3 µL of primer mix (0.2–0.4 µM of each primer), and 5 µL of commercial mix , one with an annealing temperature of 55°C and the other with an annealing temperature of 62°C, chosen based on the best amplification signal in each reaction.Primer amplification was optimized The first and second multiplex PCRs of the first electropherogram and the second multiplex PCR of the second electropherogram  followed the amplification protocol of an initial cycle at 95°C for 5 min; 34 amplification cycles ; final extension at 72°C for 30 min, followed by an infinite temperature of 12°C. PCR of the Gel2M1 primers was performed with an initial cycle at 95°C for 5 min; 34 cycles (95°C for 30 s and 62°C for 4 min), final extension at 60°C for 20 min, and infinite temperature of 12°C.®) at the Replicon/LaGene Research Center of the Pontifical Catholic University of Goiás and the ABI 3130 automatic sequencer (Applied Biosystems®) at the University of Córdoba, Spain. The data were analyzed using the GeneMapper program . The size of the amplified fragments was defined in comparison with the standard Gene Scan LIZ 600® (Life Technologies) and two control samples in each electropherogram. The controls were samples previously genotyped by the company Animal Breeding Consulting, Spain, with known and standardized genotypes.The amplification products of microsatellite loci were genotyped in capillary electrophoresis systems using the ABI3500 automatic sequencer (Applied Biosystemshttps://figshare.com/account/projects/98876/articles/14069039).Filtering of the genotypes was performed to remove samples that failed to amplify half or more of the markers because more genotyping failures mean that more assignment errors can occur. The P6 property was excluded as it had no DNA samples for the genetic analyses. At the end of the quality control, 608 remaining samples were evaluated together, without prior population definition . The dathttps://figshare.com/account/projects/98876/articles/14069039).The data obtained were treated in order to eliminate samples with low genotyping quality and monomorphic markers, leaving 608 Curraleiro Pé Duro samples that showed more than 50% of the genotyped loci and, therefore, were selected for the association analyses . The datThe genotyping data were entered into Structure 2.3.4 software , and theTo estimate population structure in Structure software, runs were conducted using a parameter set of 10,000 burn-in generations followed by 500,000 MCMC iterations. One to 22 populations (k = 1 to k = 22) and five iterations for each k value were assumed in the admixture model . The resThe genetic diversity indicators were calculated in the Microsatellite Toolkit for Excel program . The allThe deviations in Hardy–Weinberg equilibrium (HWE) were estimated with the software Genepop 4.0.5.3  by applyThe F coefficients, namely, Wright’s fixation index (F), which includes Fis (inbreeding coefficient/heterozygosity deficiency coefficient), Fit , and Fst (fixation index in the subpopulation or estimate of genetic differentiation between subpopulations)  were calGenetic divergence among populations was assessed based on analysis of molecular variance (AMOVA)  in ArleqBreed characterization markers were associated with the frequency of animals seropositive against infections by pathogens causing brucellosis, leptospirosis, neosporosis, leukosis, IBR, and BVD.odds ratio), which indicates how frequently a disease occurs in an individual carrying a certain marker.A population study was carried out adopting each genetic cluster as a population. The allele frequencies were observed and compared between groups of sick (positive) and healthy individuals (negative) using Fisher’s exact test. The strength of association was measured using the relative risk . In clusters 1 and 2, some markers were in equilibrium, i.e., no decrease in heterozygotes relative to homozygotes was observed at these loci. The PIC values per marker are compiled in Higher presence of alleles was observed in breed clusters 2 and 3  than in cluster 1 . The markers used were tested using HWE, and all loci in cluster 3 were in disequilibrium  per marker, considering all animals independently of clusters, was similar for all loci. Fis values ranged between −0.70401 (INRA63) and 0.6136 (TGLA53) in cluster 1; 0.46182 (INRA63) and 0.47398 (HAUT24) in cluster 2; and −0.03575 (BM8125) and 0.51811 (HAUT24) in cluster 3. The negative Fis values in some loci indicated that the observed heterozygosity was greater than expected, with no reduction of heterozygotes in comparison with homozygotes.Fst values ranged between 0.03073 (ETH3) and 0.402 (INRA63) in cluster 1, between 0.00 (INRA32) and 0.39359 (INRA63) in cluster 2, and between 0.00 (INRA32) and 0.28483 (TGLA53) in cluster 3, indicating varying levels of differentiation from markers with little genetic differentiation (Fst 0.25). Fst levels close to zero indicated less genetic differentiation.CFA  graphicap < 2.2e-16), with 53.45% (325/608) of concordant samples. Moreover, the properties grouped according to environmental characteristics were also grouped according to genetic characteristics  was found between genetic and environmental clusters, with 53.45% of samples in agreement . The percentages of individuals allocated to each cluster in the positive, negative, and suspect groups for each infection were tabulated , meaning that the total variation found among populations was 24.49%. Of this, 5.91% corresponded to the variation in populations between states and 18.57% to the variation between populations of the same state.AMOVA considered three groups referring to Goiás, Piauí, and Tocantins states to estimate the amount of total diversity due to variation in populations between and within states . The AMOThe 19 herds were tested as independent groups by AMOVA. This test indicated that 24.72% of the total variation (Fit) was between herds and 75.51% within individuals. The variation between herds was lower than the variation between individuals of the same herd .The differentiation of the same breed into three clusters may be explained by the role of the environment in selection. The physical separation of the populations of Goiás, Tocantins, and Piauí caused reproductive isolation, and adaptation to local environmental conditions may have affected the emergence of new phenotypes . The genExpected heterozygosity was higher than observed except for loci BM8125 and INRA63 (cluster 1), which may indicate increased inbreeding and reduced genetic variability. The loss of diversity in breed characteristics may also indicate an increase in inbreeding coefficient.All loci were polymorphic, with a total of 295 alleles in cluster 1, 233 in cluster 2, and 303 in cluster 3. Mean values of 10.54, 8.32, and 10.82 alleles per locus were observed in clusters 1, 2, and 3, respectively, a value lower than the average of 13 alleles per loci previously described in the breed. Although polymorphic, the loci were in HWE imbalance, which was in agreement with the results of The increase in the number of homozygotes, observed by the deviation in the HWE, can be explained by the high level of inbreeding due to the small effective number of animals in reproductive age and mating between related individuals. Other factors that can also explain the deviation in the HWE are subdivisions within the population, natural selection, migration, and null alleles .The Fst values were lower than 0.15, indicating that the two subpopulations share alleles and, therefore, the genetic differentiation is low to moderate. Owing to the geographical isolation of the populations of the states of Piauí, Tocantins, and Goiás, high differentiation between the populations of the states and increased of inbreeding within each state would be expected because the populations do not mate at random . HoweverFis ranged from 0.16441 (TGLA53) to 0.19427 (INRA63) and Fit from 0.27191 (TGLA53) to 0.29383 (BM8125). Fis values indicated inbreeding. The loss of genetic variability in CPD herds is a consequence of inbreeding due to the replacement of sires with animals from their own herd. The CPD breed showed higher F indices than other native Brazilian breeds such as Caracu, Pantaneiro, and Mocho Nacional .Leptospira infection, in cluster 3. The high rate of seropositivity for viral infections in CPD was reported by The number of positive animals was lower than that of negative animals, except for IBR virus infection in all clusters and for It is possible to infer that past environmental changes have triggered phenotypic diversification events that fixed or eliminated alleles  in populIn CPD animals, the most advantageous alleles for adaptation to the environmental conditions of Piauí and Tocantins showed increased frequency in each environment and were fixed in the populations, subdividing the breed into three distinct populations. AMOVA findings showed that most of the total genetic variance was due to the difference between alleles within individuals (75.28%), which is a similar result to that described for the local Brazilian breed Crioulo Lageano . CPD herBrucella spp., N. caninum, and BLV between clusters. The greatest variation in genotypes related to the BoLA genes occurred at the individual level, and has a high number of alleles that provide varied phenotypes to CPD.Diversity and a high number of alleles were detected in the three genetic clusters; however, the deviation from the HWE and the inbreeding coefficient indicated the occurrence of inbreeding in the populations. The difference in antibody detection was significant for We conclude that the populations are in the process of genetic differentiation. However, this differentiation is more pronounced in the Piauí herds compared to Tocantins and Goias. The results were not sufficient to indicate a genetic subpopulation with increased resistance or susceptibility to infection.


BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation,  Stat3 is a member of the signal transducer and activator of transcription (Stat) family of transcription factors and exerts different and sometimes contrasting functions in normal and transformed cells –3. In thDespite the observations for oncogenic roles, immunohistochemical studies of Stat3 give conflicting results for prognosis –15. ImpoThese findings suggest that Tyr705- and Ser727-Stat3 phosphorylation may be independent regulators of cancer cell phenotypes. Thus, we studied pTyr705- and pSer727-Stat3 in a set of well-characterized TNBCs and correlated the relative levels of each Stat3 phosphorylation with each other and with clinicopathological and phenotypic features. We show that pTyr705-Stat3 and pSer727-Stat3 are often independent of each other and influence the basal/luminal phenotype of TNBC cells in opposite directions. We also show that high levels of pStat3 are associated with good prognostic indicators but not with improved survival suggesting it may be a useful target for therapeutic intervention in these patients.Tissue microarrays (TMAs) were prepared from excess material from formalin-fixed and paraffin-embedded histological tissue blocks of TNBCs from patients before treatment, as described previously . The sam+ HRP reagents with DAB+  were used for visualization. Primary antibodies to pTyr705-Stat3  or pSer727-Stat3  were used. These antibodies have been independently characterized for specificity and performance in immunostaining previously (Sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked in hydrogen peroxide (3%) and antigen retrieval was performed by boiling for 20 min in EDTA . Primary antibodies were applied overnight at 4°C and EnVisioneviously –30. Slidhttps://qupath.github.io/) (QuPath (0.1.2) was used for quantification (hub.io/) . TMAs weAssociations between pTyr705- and pSer727-Stat3 and clinicopathological data were evaluated using chi-square tests or Student´s t-tests in the indicated cases. Kaplan-Meier and Mantel-Cox log-rank tests were used for survival data . We initImmunostaining data were obtained for pTyr705- and/or pSer727-Stat3 in 173 TNBCs. Data were available for both in 151 tumors. Uninterpretable spots were due to loss of tissue on the array or insufficient numbers of tumor cells.pTyr705-Stat3 localized to the nuclei of tumor cells and stromal cells including lymphocytes and fibroblasts, which served as convenient internal controls for staining. pTyr705-Stat3 was not seen in the cytoplasm. In contrast, pSer727-Stat3 was observed in the nucleus and the cytoplasm of tumor cells and stromal cells, compatible with previous data on the location of these two phosphorylated forms of Stat3 . To inveData were available for pTyr705-Stat3 in 4 lobular-pleiomorphic, 3 metaplastic, 3 micropapillary, 3 papillary, 5 pleiomorphic tumors, 33 tumors with medullary features, and 117 tumors of no-special type. Data for pSer727-Stat3 were available in 3 lobular-pleiomorphic, 5 metaplastic, 2 micropapillary, 3 papillary, 6 pleiomorphic tumors, 35 tumors with medullary features, and 111 no-special type cancers. There were no significant associations between pTyr705- or pSer727-Stat3 levels and histological subtype, although the numbers of each special histological subtype were very low .p < 0.05). Clinicopathological correlations with the combined levels of both pTyr705- and pSer727-Stat3 are shown in The clinicopathological characteristics in association with individual pTyr705- and pSer727-Stat3 groupings are shown in p = 0.029) and with combined ERβ and androgen receptor (AR) positivity (p = 0.022). Interestingly, dual ERβ and AR positivity correlated specifically with high levels of pTyr705- and low levels of pSer727-Stat3 (p = 0.036). Furthermore, tumors with high levels of pTyr705-Stat3 were more likely to be mucin-1  positive (p = 0.012). The proportion of patients carrying BRCA2 mutation was higher in the high pTyr705-Stat3 tumor group (p = 0.039). High pTyr705-Stat3 also correlated with less aggressive tumor characteristics such as smaller tumor size (p = 0.029), lower clinical stage (p = 0.002), and absence of lymph node metastases (p = 0.006).High levels of pTyr705-Stat3 were associated with ERβ positivity (p = 0.036) but negative for EGFR (p = 0.038). High pSer727-Stat3 was associated with smaller tumor size (p = 0.016) and lower clinical stage when high levels of pTyr705-Stat3 were also present (p = 0.002), but pSer727-Stat3 did not associate with lymph node metastasis. Patients with basal-like cancers showed high levels of pSer727- and low levels of pTyr705-Stat3 (p = 0.045).Tumors with high levels of pSer727-Stat3 were more likely to be positive for CK5/6 (Neither pTyr705- nor pSer727-Stat3 showed an association with TAp63 or ΔNp63 levels, or with p53 status. Furthermore, although Stat3 phosphorylation of Tyr705 and Ser727 was associated with less aggressive tumor characteristics such as tumor size or clinical stage, neither phosphorylation showed an association with survival .The role of Stat3 in cancer is controversial, with several studies demonstrating an oncogenic role in the development of various cancers, whereas others have indicated that Stat3 behaves as a tumor suppressor , 25. TheStat3 activation involves phosphorylation to allow dimerization, nuclear transport and binding to response elements in the promoters of target genes. In general, it is thought that Tyr705 phosphorylation is the initial event for Stat3 activation, followed by Ser727 phosphorylation to provide higher activity. Thus, most immunochemical studies evaluate total Stat3, or pTyr705-Stat3 as a general indicator of activity and only exceptionally pSer727-Stat3. However, data suggest that these two events have distinct functional effects during embryonic stem cell differentiation and in dictating epithelial cell identity in pancreatic and lung cancers , 26.BRCA2 mutation, which is more common in ER-positive luminal B tumors (In our study, we found that pTyr705- and pSer727-Stat3 are related events that are often associated with each other. On the other hand, individual tumors show discrepancies, where one phosphorylation event occurs in the absence of the other, suggesting that different signaling events are operating in these tumors. Most importantly, each phosphorylation is associated with a distinct tumor cell phenotype, indicating that pTyr705- and pSer727-Stat3 regulate distinct pathways within the tumor. In particular, high levels of pTyr705- but not pSer727-Stat3 were associated with ERβ alone and with ERβ and AR combined. Our results further showed that patients with high levels of pTyr705-Stat3 were more likely to be MUC1-positive. High-level AR is the defining feature of luminal-AR TNBCs  and BursB tumors . MoreoveB tumors .+/ERβ+/MUC1+ epithelial phenotype, pSer727-Stat3 is associated with a basal cell phenotype, again analogous to the mesenchymal phenotype induced by pSer727-Stat3 in other cancers (In contrast to the association of pTyr705-Stat3 with an AR cancers . In part cancers . On the  cancers , whereas cancers –45. In t cancers , 47.Stat3 is an important regulator of CSC phenotypes , 48. It High pTyr705-Stat3 is associated with less aggressive tumor characteristics such as smaller tumor size, lower clinical stage, and absence of lymph node metastasis, similar to some previous studies , 56–58, Our data show that although pStat3 phosphorylated at either site is associated with less aggressive tumor characteristics that are independently associated with better survival, such as tumor size or clinical stage, pStat3 itself is not associated with survival. Thus, these data imply that high pStat3 is an unfavorable indicator in tumors with a good prognosis according to their clinicopathological factors. This observation may relate to the ability of Stat3 to induce drug resistance , 61. FurIn conclusion, we show that pTyr705- and pSer727-Stat3 are associated with more favorable clinicopathological features but not with better survival, inferring its negative role in TNBC prognosis. Our results further indicate that Stat3 phosphorylation at Tyr705 and Ser727 have different effects on the phenotype of TNBCs, analogous to their distinct roles in embryonic stem cell self-renewal/lineage commitment  and cell


The Cambodian dairy sector collapsed due to the civil war. Thereby, the Cambodia dairy sector has not obtained basic information, such as cattle breed and milk production. To encourage the recovery of the Cambodian dairy sector, the present study aimed to reveal the genetic variation and the milk production in Cambodian crossbred dairy cattle. Initially, we conducted interviews to understand the breeding background and milk production of two local farms (Farm R and Farm M) in Cambodia. The percentage (%) of milk fat content in Farm R was higher than that in Farm M. A genome-wide analysis of the genetic characterization for 75 cows in the two dairy farms implies that some cows in Farm R are genetically far from other crossbred cattle and retain a higher proportion of genetic background derived from Cambodian-native cattle. The present study indicated genetic characteristics and milk composition in Cambodian crossbred cattle. Gene character in Cambodian local crossbred cattle could contribute to milk production in the Cambodian dairy system. Thus, our basic genetic study could provide a new breeding strategy by using local Cambodian crossbred dairy cattle to establish an adequate dairy strain.To improve the dairy sector in Cambodia in the future, we aimed to reveal the genetic variation and the milk production in Cambodian crossbred dairy cattle. We calculated the percent (%) milk fat content and the average milk yield per cow (L/day) for two farms (Farm R and M) based on the farmers’ records and interviews. The crossbred cows originated from Cambodian local farmers and Thailand breeders in Farm R, whereas the crossbred cows originated in Thailand breeders in Farm M. Then, we performed genetic characterization for 75 individuals from the two farms and an individual Japanese pure Holstein-Friesian cow based on 133,705 single nucleotide polymorphisms (SNPs) obtained by the GRAS-Di method. The milk fat contents in the bulk milk in the dry season and the average milk yield per cow on Farm R were 3.77 ± 0.98% and 7.81 ± 2.66 L/day, respectively, and were higher than those on Farm M (3.35 ± 0.54% and 6.5–7.5 L/day). Cattle originating in Cambodia in Farm R possessed a unique genetic character different from cattle from Thailand in Farm M. The present study suggests that the differences in milk fat content between the two farms might be explained by the genetic differences in crossbred cows. Historically, Cambodia produced enough milk for domestic consumption, and exported it to neighboring countries, such as Thailand and Vietnam, in Southeast Asia, before the civil war between the 1970s and 1990s. However, the livestock sector, including the dairy industry, was destroyed by the civil war , and theBos taurus (humpless cattle) and Bos indicus (zebu cattle), in tropical regions. There is no reproductive barrier between them [Bos indicus) is a domesticated breed, which has small hump and yellow hair, and originated from the wild Banteng (Bos javanicus) [Bos indicus) is an exotic breed, imported from India for draft power in 1956; and the Brahman (Bos indicus) were imported from the Philippines, as a donation from Lutheran Christians in 1984 . Since the 2000s, various breeds have been introduced to Cambodian cattle farmers, with the spread of artificial insemination techniques. For example, in 2002, Heifer International, a non-government organization in the United States , imported and introduced the Jersey breed semen to the farmers in Koh Krabei village, Prek Tmey Commune, Kandal Province, and thus, crossbred cattle were developed locally. In 2004, the Santa Gertrudis semen and Barbara semen were imported from the United States, under the EU project, and introduced in Takeo, Kampong Speu, and Pursat provinces. The Cambodian Center for Study and Development imported the Holstein-Friesian-Brahman, crossbred cattle from Thailand, in 2011. Cambodia had temporary opportunities to access several types of bull semen, but the genetic variations in imported semen were not diverse, because only a few institutes and communities were assigned to these breeding projects. Furthermore, cattle breeding was conducted without any integrated strategies, across the local communities. Therefore, the breeds for production, particularly dairy, have not been strategically managed by the Cambodian cattle industry so far. Elucidation of the genetic background of dairy cattle can help improve breeding practices, which can aid in the development of the dairy industry in Cambodia.Traditionally, two cattle species have been recognized, een them . Both spvanicus) , which iDuring the last decade, several cost-effective approaches have been developed for analyzing genome-wide single nucleotide polymorphisms (SNPs) in many individuals with high genetic diversity, including restriction site-associated DNA sequencing (RAD-seq and ddRAD-seq) ,7, and mAs a tentative assessment of the genetic background of Cambodian dairy cattle, we conducted a genome-wide survey of two dairy farm populations, using GRAS-Di analysis, in the suburb area of Phnom Penh city, Cambodia. We then performed STRUCTURE analyses of the two farm populations to understand their genetic characteristics, in comparison with a pure Japanese Holstein-Friesian cow, as a reference. In this study, we aimed to reveal the genetic variation and the milk production in Cambodian crossbred dairy cattle and provide information for improving the efficiency of future cattle crossbreeding strategies to establish an adequate dairy strainThis study was conducted with permission from the Committee of the Care and Use of Experimental Animals at the Graduate School of Bioagricultural Sciences, Nagoya University , General Directorate of Animal Health and Production (GDAHP), and MAFF in Cambodia .Initially, we conducted interviews to understand the breeding background, cattle supply, and milk production for two local farms (Farm R and Farm M), both of which are located in Cambodia. The herd sizes of Farm R and Farm M were 35 and 300 individuals, respectively. The yearly averages milk yield per cow (L/day) were obtained from the farmers’ records and interviews. The milk samples were collected randomly in dry season from November to March, and 92 and 34 samples were obtained from Farm R and Farm M, respectively. The percent (%) milk fat content was measured using FT-IR analytical equipment  in our laboratory in Cambodia.Hair roots were collected from 75 crossbred cows at the two farms, from all lactating cows in Farm R and randomly from lactating cows in Farm M; in addition, from one pure Japanese Holstein-Friesian cow at a Japanese dairy farm . At Farm R, a total of 25 crossbred dairy cows were examined, which consisted of 11 Cambodian and 14 Thai cow-derived individuals, and a total of 50 Thai cow-derived individuals were also examined at Farm M. All hair root samples were stored in a deep freezer at −80 °C until DNA extraction, and 40–50 follicles (around 4.5 mg) per animal were used for DNA extraction using the QIAGEN DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. DNA concentrations were measured using a spectrophotometer .The GRAS-Di and subsequent SNP call analyses were conducted by GeneBay in Yokohama, Japan. The random amplicons were produced with 64 primers and sequenced on a HiSeq4000 sequencer , with a paired-end (PE) read length of 150 bp. Low-quality bases and Illumina sequencing adapters were trimmed using the Cutadapt v 2.5 .http://www.R-project.org/, accessed on 30 April 2022), stats R packages. The sites were filtered using the criteria of minor allele frequency (--maf 0.02), missing data (--max-missing 0.2), minimum distance of 100 bp (--thin 100), and Hardy–Weinberg equilibrium (--hwe 0.05) using VCFtools [The fat content (%) in dry season in two farms were analyzed by the Mann–Whitney U test using algorithms in R version 3.5.1 .The Farm R cattle had different breeding backgrounds in the herd. This farm had two types of cattle: a Cambodian crossbreed between a Holstein-Friesian cow from Thailand and a Cambodian-native sire ; and an imported Thai crossbreed between a Thailand Holstein-Friesian and a Thailand Brahman sire. These crossbreeds were developed by local Cambodian farmers. In contrast, the Farm M cows were all crossbred between Holstein-Friesian  and Brahman breeds . There wWe first obtained a large number of SNPs, using the GRAS-Di analysis, from the genetic surveys of crossbred dairy cows in Cambodia. The present study implies that the difference in milk fat content between the two farms was explained by the genetic differences in each crossbred cow, which was originated from other regions. It might be explained that local Cambodian cattle  have different genetic characteristics from the Thai breeds (of the Brahman breed). The present study had limitations in the samples such as a low number of collection sites; therefore, further studies are needed.Bos taurus (humpless cattle) and Bos indicus (zebu cattle), respectively. This suggests that the local Cambodian dairy farmers cannot maintain their pedigrees by using pure semen from dairy breeds, leading to the restricted number of individuals with the high proportion of indicus-type (orange) genetic clusters at K = 2. The indicus-type genetic clusters at K = 2 were subdivided into two different clusters (orange and purple) in some crossbred individuals between the Holstein-Friesian and Cambodian native cattle at K = 3. The individuals that showed relatively higher proportions of the purple cluster at K = 3 were individuals that were placed to the left in the PCA plot, in addition, four individuals were crossbred between Cambodian-native and Holstein-Friesian. Therefore, it was implied that the genetic character included in the purple cluster is explained by Cambodian-native cattle. On the other hand, as to the two individuals that were placed above in the PCA plot, even though we could not find obvious differences to other individuals in the STRUCTURE plots, they tended to have a little bit higher proportions of the orange cluster than others. Further studies are needed to investigate whether the orange cluster was a genetic cluster of zebu cattle. A previous study suggested that several Bos species were involved in the history of Asian cattle [Bos javanicus). Cows that had a large portion of the purple cluster in Farm R were crossbred between local Cambodian cows and a purebred Holstein-Friesian sire. On the other hand, some cows on Farm M also had a large proportion of the purple cluster. Wild Banteng cattle have been domesticated in several regions in Southeast Asia [Bos taurus-derived populations, the genetic erosion of the Holstein-Friesian breed might have occurred locally, in dairy cattle populations from Cambodia. In this experiment, we could not obtain enough samples to conduct an association analysis; therefore, further experiments are needed. However, our genetic survey also revealed slight differences in the genetic characteristics between farms R and M. Farm R appeared to retain a higher proportion of genetic background derived from the local, Cambodian-specific cattle, than Farm M.Local farmers in Cambodia could face some limitations in purchasing cattle semen due to a lack of knowledge and skills for artificial insemination and difficulties in accessing semen markets. All semen from dairy bulls is imported from neighboring countries at a high price . Intervin cattle . Our resast Asia , the Braast Asia , suggestast Asia  and 117 ast Asia , revealeThe study indicated differences between the two farms in their relationship between milk yield and milk fat content. It is speculated that the local Cambodian cattle have effective genetic diversity for maintaining milk fat content high under tropical conditions. Generally, the production of milk, and its fat and protein contents, significantly decline under tropical conditions ,29; howeThis possibility could support future cattle breeding programs in Cambodia. Generally, the fat content of milk is one of the biggest concerns for the dairy industry in the tropics. Previous reports indicate that many genetic characteristics support milk yield and milk quality, under heat stress conditions, and many dairy sectors consider improving their dairy strain based on this genetic information in tropical areas . In CambThis report is the first to perform genetic characterizations of Cambodian crossbred dairy cows using a genome-wide analysis tool. Although further experiments are needed, the present study provides useful information for Cambodian cattle breeding stakeholders. In addition, our findings could be used to improve dairy breeding programs in Southeast Asian countries interested in increasing milk production in the tropics.


Circa situm conserves planted or remnant species in farmlands, where natural vegetation has been modified through anthropogenic intervention. It has been proposed that trees planted or retained under circa situm conditions may contribute to maintaining genetic diversity, however information on the role of this strategy in preserving genetic diversity is scarce. The aim of this study was to determine the levels of genetic diversity and structure, and mating patterns in planted and unmanaged stands of the tropical fruit tree Spondias purpurea L. in north western Costa Rica. In three localities, we used seven polymorphic microsatellite loci and genotyped 201 adults and 648 seeds from planted and wild stands. We found no differences in genetic diversity among planted and wild stands. Genetic structure analysis revealed that gene flow occurs among planted and wild stands within localities. Clones were present and their diversity and evenness were both high and similar between planted and wild stands. The number of pollen donors per progeny array was low (Nep = 1.01) which resulted in high levels of correlated paternity (rp = 0.9). Asexual seeds were found in 4.6% of the progeny arrays, which had multilocus genotypes that were identical to the maternal trees. Our results show that although planted stands under circa situm conditions can maintain similar levels of genetic diversity than wild stands, the low number of sires and asexual seed formation could threaten the long term persistence of populations.The term  Tropical dry forests (TDFs) constitute one of the most important reservoirs of biodiversity in the tropics, with high levels of species richness and endemism –5. TDFs circa situm conservation [In north western Costa Rica, TDFs have been reduced to a series of small forest patches surrounded by large cattle fields or cultivated areas . These forest patches shelter only 0.1% of the original TDF cover in Costa Rica , 17. Severvation .circa situm has been used to describe different conservation strategies within agricultural landscapes  outside natural habitats, but within the native range of the species [circa situm conserves planted or remnant trees in farmlands or forest patches where natural forests or woodlands containing the same trees were once found; but where natural vegetation has been lost or modified significantly through anthropogenic intervention [Circa situm plantations are not normally created for conservation [circa situm plantations need to be sufficiently close to wild populations, so that pollen flow  from wild individuals into planted stands is still possible, securing fruit production and reducing the loss of genetic diversity [The term  species . Generalrvention . Circa servation , but ratervation ; or moreervation . Indireciversity , 30–33.Spondias purpurea L. is a dioecious fruit tree domesticated in the Mesoamerican region [Spondias purpurea is pollinated by social bees and wasps and fruits are dispersed by small mammals and birds [S. purpurea fruits are easily distinguished by their fruits which are usually smaller, more acidic and less fleshy than cultivated fruits [S. purpurea like other perennial domesticated species is generally vegetatively propagated by stem cuttings in homegardens and living fences, frequently in close proximity to wild stands (unmanaged trees), that occur in secondary forests and TDF’s remnants [S. purpurea trees are more likely to be vegetatively propagated in circa situm and along live fences for fruit consumption. Fruit production can be achieved via sexual reproduction, if male trees from nearby populations act as pollen sources or via asexual reproduction  [S. purpurea flowers offer pollen and nectar, while female flowers only offer nectar as a reward, thus females are less likely to attract pollinators [n region , 35 known region , 36, 37.nd birds . During d fruits . In Mesoremnants , howeverremnants –32. Femalinators . This maS. purpurea in secondary forests and isolated trees are in close proximity to plantings along live fences, small farms or in homegardens. The objectives of our study were (1) to compare genetic diversity and structure among wild and planted stands of S. purpurea (2) to estimate mating patterns in terms of correlated paternity of wild and planted stands of S. purpurea, and (3) to determine the frequency of sexual and asexual seeds produced in wild and planted stands of S. purpurea. This will allow us to analyze the role of planted stands for circa situm conservation of genetic diversity in this tropical dioecious species.Our study took place in the north west pacific region of Costa Rica, where wild stands of S. purpurea individuals may be found in rustic plantations, propagated vegetatively for fruit consumption or found in natural habitats. Planted and wild stands of S. purpurea were located in three localities: Agua Caliente (AC), Murciélago (MU) and Horizontes Forestal Research Station (HO)  [stats library implemented in the R [The study was conducted in north western Costa Rica in the province of Guanacaste. The study region consists primarily of TDF with a mean annual rainfall of 1600 mm and a marked dry season that extends from December to May . The majion (HO) . The thrion (HO) . Along bion (HO) . HO is aion (HO) . Within + males) . To detein the R . We collS. purpurea [GeneMarker Software version 2.6.4 (SOFTGENETICS). To reduce genotyping error, all samples were independently scored by two different researchers to reach a consensus in the final data set. In addition the software MICROCHECKER [DNA from leaves and embryos was extracted using a modified Cetylmethylammonium Bromide (CTAB) protocol . Ten micpurpurea , were ampurpurea  and fragOCHECKER  was usedNa), allelic richness (Ar), observed (Ho) and expected heterozygosities (He), and inbreeding coefficients (F). Allelic richness was estimated by rarefaction of alleles as implemented in the hierfstat library [poppr [e, Ho and F) between planted and wild stands were analyzed using 10, 000 permutations to test significance in GenoDive v.3.04 [GenAlEx v.6.5 [genet. Genotypes with missing data were ignored [pi are allele frequencies. This index provides an estimation of the probability that two randomly selected genotypes are different and scales from 0 (no genotypes are different) to 1 . Genotypic evenness is a measure of the distribution of genotype abundances, which takes values from 0 to 1 [A total of 201 adult individuals (46 planted and 155 wild) and 600 seeds (281 planted and 319 wild) were sampled and genotyped in the three study sites. Genetic diversity was quantified for planted and wild stands using the following parameters: allele number averaged across loci  [GenoDive v.3.04. This coefficient is based on the relative probability of identity by descent of the alleles within the two compared individuals [To explore the overall genetic structure of planted and wild stands, we used the software STRUCTURE , 53 to dtatistic , with 10nt (FIJ) , estimatividuals .rpm) was estimated using MLTR [ep) as the reciprocal of the rpm [Multilocus correlation of paternity (ing MLTR . Correla the rpm .PI = 0.00003. The proportion of distinguishable genets based on multilocus genotypes in planted trees (R = 0.74) was similar to wild trees (R = 0.77)  . Clonal  = 0.77) . Genetic = 0.77) . Sex rat = 0.77)  (p>0.05)ST estimates show a lack of differences in allele frequencies between planted and wild stands within sites, with the exception of HO  and two wild stands  are predominantly grouped into cluster 1, with evidence of admixed individuals from cluster 2 . WHO indrp PLANTED = 0.99; rp WILD = 0.91) . We identified 30 seeds with multilocus genotypes that were identical to the maternal tree (MGM), which suggests asexual seeds production (FIJ = 0.012 (S7 Table in Paternity correlations were similar for both planted and wild progenies ( = 0.91) . Therefooduction . Our kinoduction  shows thoduction . Lower kSpondias [S. purpurea planted and wild stands showed no differences, suggesting that circa situm conditions may have conserved genetic diversity in adults. Our genetic diversity estimates were similar to those found in natural and fragmented Mexican populations of S. purpurea, and comparable to genetic diversity values found in other perennial edible plants  K. Koch., Malus domestica (Suckow) Borkh., Olea europea L., Pistacia vera L., Prunus avium L., Castanea dentata (Marsh.) Borkh., Vitis vinifera L., Leucaena esculenta (DC.) Benth., Polaskia chichipe (Rol-Goss.) A.C.Gibson & K.E.Horak, Euterpe edulis Mart.) [Genetic diversity estimates in this study were comparable to those found in other tropical trees –62 and sSpondias –65. The s Mart.) , 37. In s Mart.) .Ficus carica L., Dioscorea rotundata Poir., Olea europaea L., Theobroma cacao L.) [Musa paradisiaca L., Ficus carica L., Pyrus communis L., Pistacia vera L.) [circa situm stands may not be a viable option as propagule sources for regeneration or the creation of future orchards.Our results show the presence of trees with identical multilocus genotypes  both in planted and wild stands. In addition, a small proportion of progenies have multilocus genotypes that are identical to their maternal trees, which is an indirect evidence of apomictic seed formation , 68. Cloacao L.) –72. At overa L.) . This revera L.) , 73. Howvera L.) , 75. ThuS. purpurea may originate from a few genotypes that have become common in planted and wild stands in Guanacaste. Both our STRUCTURE and GST results suggest that gene flow is likely to occur between wild and planted stands within sites. Furthermore, individual STRUCTURE assignments present indirect evidence of gene flow among sites; however this occurs less frequently, as evidenced by significant pairwise GST estimates among populations. In the agro-ecosystems we studied, the source of the planted populations may have been nearby wild populations, which would explain the comparable levels of genetic diversity and the low levels of genetic differentiation observed between wild and cultivated plantings. In our study, planted stands are small orchards and live fences in close spatial proximity to wild populations, which inhabit nearby secondary forests or forest remnants. The fact that planted trees likely originated from nearby wild populations and the unconstrained gene flow among planted and wild trees, is congruent with the observed levels of genetic structure among wild and planted populations. There is evidence that gene flow can be sustained between planted plants and their wild relatives by pollinators inhabiting forest remnants [ST values that show that HO differs significantly from all other populations, supporting our previous suggestion that trees at AC and MU probably have different origins compared to HO. Therefore, population historical origin may have a greater influence on genetic diversity and better explain the structure than the simple comparison between planted and wild stands.The genetic admixture observed in individuals of wild and planted stands at AC and MU suggests that planted trees in these sites were likely selected from seedlings or cuttings of trees in nearby wild populations. In contrast, at HO the genetic differences observed between wild (WHO) and planted (PHO) stands reflect that planted trees (PHO) may have been propagated from other locations with allele frequencies similar to those observed at AC and MU. Planted trees are commonly selected for their larger fruits, and our results suggest that these trees may have been selected from a few sources or farmers have propagated similar genotypes across all planted stands. During the process of domestication trees that bear large, fleshy, sweet fruits have been selected, and also those that can be reproduced easily from cuttings . Our resremnants , 76. An remnants , 40. Theremnants . These dS. purpurea depends on pollinators for sexual reproduction and seed production [S. purpurea could also be related to pollinator behavior. In S. purpurea, a previous study showed that in disturbed habitats, floral displays are larger but pollinator visitation is negatively affected, reducing fruit-set and increasing paternity correlation [p = 0.63) as a result of a lower number of sires (Nep = 1.58). Our results show a limited number of sires both in planted and wild stands of S. purpurea in Costa Rica. Pollinator behavior may explain the high correlated paternity observed in the progenies at HO, where sex ratios did not statistically differ from a 1:1 ratio. These results are congruent with previous findings that habitat loss and fragmentation reduce the number of pollen donors and progeny fitness [circa situm and remnant wild populations may conserve adult genetic diversity, the highly correlated paternity of their progenies may compromise the regeneration ability of seeds produced by wild and planted trees alike [circa situm may be a viable conservation strategy for adult trees, it may limit the regeneration ability of populations in disturbed habitats.Previous studies have shown that oduction , 38. In oduction –82. Highoduction , increasrelation . Larger relation , 82–84. relation  showed t fitness –22, 83. S. purpurea. Our results indicated that planted trees harbor similar levels of genetic diversity as wild stands. The genetic structure is explained by differences among sites, possibly due to genetic admixture between planted and wild stands within sites, and fruit production is the result of sexual reproduction from pollen flow from a limited number of males and reproductive assurance mechanisms such as apomixis. Therefore, although circa situm conditions have maintained moderate levels of genetic diversity in this species, conservation of natural populations may be necessary to increase gene flow and the diversity of pollen donors, which may result in higher fitness of future generations [circa situm may only represent a subset of the original genetic diversity of the species and may falsely be considered a genetic reservoir, without considering its relationship with the overall fitness of the species. Therefore, to maximize the effectiveness of circa situm strategies in dioecious plants the vegetative propagation of trees should originate from spatially distant individuals to promote high genetic diversity and should strive to maintain equal sex ratios to guarantee sexual reproduction and more diverse progenies in the long term.In summary, we evaluated the differences in genetic diversity and structure and paternity correlation between planted and wild stands of the fruit tree erations , 83. ThiS1 Data(ZIP)Click here for additional data file.


There is no consensus on a single classification system for the obstructive findings in drug-induced sleep endoscopy. Previous classification systems have neglected to address the upper retropalatal obstruction, the segmental division of the lateral pharyngeal wall and the primary or secondary nature of laryngeal collapse.To propose, illustrate and evaluate a more comprehensive and yet simple classification for drug-induced sleep endoscopy findings.Cross sectional study in a tertiary sleep surgery unit. A total of 30 patients with obstructive sleep apnea underwent drug-induced sleep endoscopy according to a new classification system called LwPTL, and its findings were analyzed according to obstructive sleep apnea severity and body mass index. LwPTL incorporates the description of upper retropalatal collapse, distinguishes the lateral pharyngeal wall collapse into three levels and clarify when laryngeal collapses are primary or secondary.93.3% of the patients presented lateral pharyngeal wall collapse, usually at the level of the velum (73.3%). 80% presented multilevel collapse. Regarding the upper retropalatal region, LwPTL identified 10% of the cases presenting lateral salpingopharyngeal obstruction and 6.6% with high palatal collapse. 3.3% presented epiglottic collapse. Patients presenting lower levels of collapse, either from the lateral wall and/or tongue and/or larynx, represented 30% of the cases and had significantly more severe obstructive sleep apnea, without significant differences in body mass indexes.LwPTL seems a simple and straightforward staging system for classifying drug-induced sleep endoscopy, distinguishing the important upper retopalatal obstruction and the primary and secondary laryngeal collapses, providing more information for appropriate treatment selection. Obstructive sleep apnea (OSA) is a multifactorial disease. Multiple pathophysiological mechanisms contribute to airway collapse including anatomical narrowing of the upper airway, poor upper airway dilator muscle activity, low arousal threshold and ventilatory control instability.Considering the wide variety of surgical procedures currently available for treating chronic snoring and OSA, the assessment of the site(s) of upper airway obstruction is important for selecting the appropriate procedure. Multiple evaluation techniques have been developed to examine an individual's pattern of upper airway obstruction.Previous classification systems have neglected to address the upper retropalatal obstruction, the segmental division of the lateral pharyngeal wall and the detailed structural pattern of primary or secondary laryngeal collapse. In order to address these issues, we aim to propose, illustrate and evaluate a more comprehensive and yet simple classification for DISE findings, which we called LwPTL.After becoming experienced with both DISE technique and our proposed classification system, we have collected data on the last 30 consecutive OSA patients that underwent DISE and upper airway surgery at the Mansoura University Hospital, a tertiary referral sleep surgery unit in Egypt.All patients underwent level 1 polysomnography preoperatively . The severity of OSA is expressed in the apnea–hypopnea index (AHI). Obstructive apneas were defined as cessation of airflow for at least 10 s. Hypopneas were defined as periods of reduction of >30% in oronasal airflow for at least 10 s associated with a >4% decrease in oxygen saturation. The AHI was calculated as the sum of total events (apneas and hypopneas) per hour of sleep.All patients had preoperative DISE for assessment of obstruction levels. We applied our new staging system for DISE findings and correlated the data with the OSA severity and body mass index (BMI).Age from 18 to 50 years old;Available level one sleep study;OSA patients with AHI > 5 with CPAP failure or refusal.Previous sleep surgery;Pregnancy;Obvious craniomaxillofacial anomalies.For our DISE technique, the patient was in a supine position on the operating table. The patients had basic cardiorespiratory monitoring . The target depth of sedation is the transition from consciousness to unconsciousness , in practical terms, when the patient started to snore and choke. This description for the desired level of sedation is because we do not have the bispectral index monitoring in our department. Atropine was used for all patients once 30 min before anesthesia at a dose of (0.6 mg/kg) as it is anticholinergic drug which decreases saliva during evaluation. Sleep was induced using propofol in a dose of (1.5 mg/kg) as a bolus and then maintained with simply manual controlled infusion. Propofol is an ultra-short acting hypnotic that enables greater control of the depth of sedation during sleep endoscopy. So, slow stepwise induction was used to avoid oversedation. Deeper levels of sedation are associated with progressive decreases in both upper airway dilator muscle tone and neuromuscular reflex activation, which increase airway collapsibility. To avoid overestimation of airway collapse, collapses that occurred while oxygen saturation was less than the minimal saturation in the polysomnography were disregarded.Once the patient has reached a satisfactory level of sedation, a flexible endoscope lubricated with lidocaine 2% gel was introduced into the nasal cavity. The lateral pharyngeal wall, soft palate, tongue base and larynx were observed. The sites generating snoring and/or obstruction were assessed.Our experience with DISE examination suggested that this classification could guide the management protocol. According to data gained from DISE we classified airway collapse into four levels: Lateral pharyngeal Wall (LW), Palate (P), Tongue (T) and Larynx (L) . In all We subclassified it into collapse at the Level of the Salpingeopharyngeal folds (LS) , of the Palatal collapse refers to anteroposterior collapse only. We classified it according to Woodson classificationWe classified tongue base collapse into High Tongue base collapse (TH)  which meAny site of laryngeal collapse not caused by low tongue base collapse or lateral wall hypopharyngeal collapse is put under category Larynx (L) including epiglottic , aryepigSpecial tests were done when needed; we performed positional DISE by rotating the head to one side. In addition, transoral DISE was done to assess the tongue palate interaction  . During According to the staging system summarized in The rationale for using transpalatal advancement pharyngoplasty for treating the lateral pharyngeal wall collapse at the hypopharyx is to benefit from the anterior traction of the soft palate to increase the tension at the lateral pharyngeal walls more distally.t-test and Mann–Whitney test; p-value < 0.05 was considered to be statistically significant.Data was analyzed using  SPSS version 15. Quantitative normally distributed data was presented as mean ± SD. Comparison of continuous data like AHI and BMI as regard to each level of collapse being present or absent was done using Student 2).After analyzing the data gained from DISE, we could find the following in the 30 patients with median AHI (31.72 ± 20.39 events/h) and mean BMI of (32.97 ± 2.77 kg/mp = 0.003), without significant differences in BMI (p = 0.50) (Only 11 patients (36.6%) had one level of collapse and the other 19 patients (63.3%) had multilevel collapse. The presence of the multilevel pattern, compared to those with single level collapse, implied in a significant increase in AHI ( = 0.50) .Table 2Sp < 0.0001) and in BMI (p < 0.002)  was present in 28 patients (93.3%) and this emphasizes our look for the importance of the lateral wall collapse in OSA patients. Segmental classification of lateral wall collapse showed that of those 28 patients we could find 3 patients with LS, 22 patients with LV and 3 patients with LH. The presence of LH pattern, compared to those without LH collapse, implied in a significant increase in AHI (< 0.002) .Table 3Sp = 0.011)  . The pre= 0.011) .Table 4Sp = 0.0008), without significant differences in BMI (Low Tongue base collapse (TL) was found in 4 patients (13.3%) and High Tongue base collapse (TH) was found in 2 patients(6.6%). Laryngeal collapse was found in only one patient (3.3%), who had primary epiglottic collapse. Patients presenting the lower levels of collapse, either from the lateral wall and/or tongue and/or larynx, represented 30% of the cases and had significantly more severe OSA .Proper lateral wall splinting is still one of the limitations in sleep surgery. Improving outcomes may arise from a more accurate evaluation. In our view, it is an over simplification to describe the whole lateral pharyngeal wall as a single level of collapse. Lateral wall collapse should be thouroughly evaluated, segmentally classified and accordingly managed, because it does not collapse as one single unit.Recently we used transoral DISE to look at tongue palate interaction and to see if the oral tongue is pushing the soft palate or not. We could find that there is tongue palate contact in almost all patients. However, this simple contact did not seem to affect the collapsibility of the soft palate. In only one case out of the thirty patients we found a positive tongue palate interaction, meaning that we had the impression that this palate was being pushed by the tongue and, actually, this did not change our procedure selection because tongue palate interaction is not yet taken into account in our treatment protocols. Future follow-ups on cases presenting that feature are important for establishing whether tongue base management is required or not in those cases.The severity of upper airway collapse in patients with sleep apnea may decrease significantly when the head is rotated to the lateral side. However, there is no significant difference between the rotation of the head to the right or to the left side.In our classification, we intentionally did not quantify the degree of obstruction because there is no data showing any physiological meaning for partial visual collapses and usually a partial obstruction does not produce a recognizable decrease in flow,Woodson 2015 described 3 patterns for palatal morphology: the oblique palate in which the narrowing is at the velum, the intermediate palate in which the narrowing is at the velum and genu and the vertical palate at which the narrowing is at the velum, genu and the hard palate. These patterns were the rationale for selecting patients for the transpalatal advancement procedure.The classification of tongue base collapse into high or low would guide the procedure selection, respectively, toward bony framework surgery or tongue base reduction. To avoid overestimation of the laryngeal level of collapse, secondarily pushed epiglottis by low tongue base or lateral hypopharyngeal wall are described with the pushing level.The European position paper stated that: “hypopharynx has its superior limit at the level of the hyoid bone, where it is contiguous with the oropharynx and the major subsites of the hypopharynx are the pyriform sinuses, the post-cricoid region, and the pharyngeal wall. Therefore, this region is not involved in the collapse”.The small number of patients in our study is one limitation for a more robust statistical analysis. Another limitation is that we are not presenting the impact of this staging system on the outcomes of the surgical procedure selected, which is still under investigation and makes room for future studies.LwPTL seems a simple and straightforward staging system for classifying DISE, being the first to segmentally evaluate the lateral pharyngeal wall collapse and to distinguish the important upper retropalatal obstruction and to standardize primary and secondary laryngeal collapse. Application of this simplified detailed staging system may help with accurate surgical planning.The authors declare no conflicts of interest.


Ethiopia is recognized as a center of diversity for barley, and its landraces are known for the distinct genetic features compared to other barley collections. The genetic diversity of Ethiopian barley likely results from the highly diverse topography, altitude, climate conditions, soil types, and farming systems. To get detailed information on the genetic diversity a panel of 260 accessions, comprising 239 landraces and 21 barley breeding lines, obtained from the Ethiopian biodiversity institute (EBI) and the national barley improvement program, respectively were studied for their genetic diversity using the 50k iSelect single nucleotide polymorphism (SNP) array. A total of 983 highly informative SNP markers were used for structure and diversity analysis. Three genetically distinct clusters were obtained from the structure analysis comprising 80, 71, and 109 accessions, respectively. Analysis of molecular variance (AMOVA) revealed the presence of higher genetic variation (89%) within the clusters than between the clusters (11%), with moderate genetic differentiation (PhiPT = 0.11) and five accessions were detected as first-generation migrants using Monte Carlo resampling methods. The Mantel test revealed that the genetic distance between accessions is poorly associated with their geographical distance. Despite the observed weak correlation between geographic distance and genetic differentiation, for some regions like Gonder, Jimma, Gamo-Gofa, Shewa, and Welo, more than 50% of the landraces derived from these regions are assigned to one of the three clusters. Hordeum vulgare L.) ranks fifth in the acreage and production of cereals after tef, maize, wheat, and sorghum in Ethiopia. It accounts for 5.63% of the total cereal production ) with a productivity of 2.18 ton/ha in 2018/19 [‘meher’, which is the major rainy season (June to October) in which diverse genotypes are grown, and ‘belg’ with less amount of rain (late February to early July) in which most early maturing varieties are grown [‘meher’ is by far exceeding the one in ‘belg’, which covered 84.5% of the total area of production and 93.0% of the total yearly barley harvest in 2013/14 [Barley ( 2018/19 . It is cH. vulgare ssp. spontaneum) revealed that the wild barley might not be an ancestor for barleys of Ethiopia/ Eritrean and these landraces might have different domestication path [The origin and domestication of barley believed to be ‘Fertile Crescent’ . Ethiopiion path . The preion path , 13. Oneion path . Additioion path . Therefoion path .The genetic resources of Ethiopian landraces are still rich and well maintained, as a report indicated that 95% of the Ethiopian smallholder farmers use landraces as the major seed source , 18. AltTraditionally, farmers classified barley landraces based on kernel type as hulled, hull-less, and partially hulled barley . AdditioThe genetic structure of a population is influenced by variation in geographical collection distance, presence of geographical barriers like wetlands, mountains and gorges, as well as by the compatibility of genotypes to cross to each other. Besides this, the genetic structure is also due to the presence of barriers on the human local population over a long period of time .Application of molecular tools improved the efficiency and precision of analysis of genetic relatedness in different crop species, as they helped to decipher whether the morphological, chemical, traditional and geographical classifications are in consistence to molecular structural analysis . DiffereST) is widely used to investigate the genetic distance between populations [ST, were likely to be true. Therefore, Monte Carlo resampling methods [The presence of genetic divergence between populations can be studied using Nei’s genetic distance . Geneticulations , 44. As ulations  indirect methods  is bette methods . The nei methods .Several studies on the genetic distance of Ethiopian landraces using different molecular markers were conducted. Distinctive genetic features of Ethiopian landraces compared to other barley collections were reported, although a minimum genetic distance between different Ethiopian landraces was detected using RFPLs (restriction fragment length polymorphism) markers . AnotherTherefore, the aims of this study were, (i) to investigate the genetic diversity of Ethiopian barley landraces, and (ii) to analyses the role of the geographic origin, and defined agro-ecological zones in the formation of genetic structure using a highly informative 50k iSelect SNP array . The outhttps://www.arcgis.com) provided by Esri [A panel of 260 Ethiopian barley accessions was analyzed in this study . The 239 by Esri . Additio2O5 and 18% K2O in kg/m3 . When plants had grown to the two to three leaf stage, leaf samples with an approximate size of 300 mg were taken from a single plant for genotyping. The genomic DNA was extracted using a modified CTAB (cetyltrimethylammonium bromide) method [Three seeds from each of the 260 accessions were grown in the greenhouse at day (16h)/ night (8h) temperatures of 20–22°C/17-19°C as described by  in multi) method  and geno) method  at Traithttp://www.cog-genomics.org/plink/1.9/) [An initial set of 40,387 markers was successfully extracted from genotyping. 10,644 SNP markers were obtained, after removing all monomorphic markers and imputation using Beagle  followednk/1.9/) , which uhttp://taylor0.biology.ucla.edu/structureHarvester/) [The 983 highly informative SNP markers were used for population structure and genetic diversity analysis. The population structure was calculated using the Structure software v.2.3.4 . Computavester/)  was usedvester/) . From thvester/)  based onST)) were obtained from the analysis using the GenAlEX 6.5 software plugin for Excel [The 983 highly informative SNP markers were used for genetic diversity analysis. AMOVA was performed based on the number of genetically distinct clusters obtained from the structure analysis. Information about genetic variation within and between clusters based on PhiPT , expected heterozygosity (He), unbiased expected heterozygosity (uHe), and percentage of polymorphic loci (PPL) were also calculated using frequency based analysis in the GenAlEX software . AdditioFrom 43,461 scorable SNPs markers of the 50k iSelect SNP array ; 40,387 Analysis of the population structure based on 983 SNP markers identified the best probable number of the subpopulation based on k-value at K = 3, which therefore has been selected as an optimal number of inferred genetically defined clusters . AccordiAMOVA analysis was conducted based on the three genetically distinct clusters obtained through the analysis of population structure. The results revealed that variation within a cluster was accounting for higher variation (89%) than the variation among clusters (11%). The genetic differentiation was moderate (PhiPT = 0.11) with statistical significance at p < 0.001 . Based oThe study of the genetic diversity indices of the three genetically distinct clusters indicate, that cluster 3 is more diverse than the other two clusters with values of I = 0.47, He = 0.31, uHe = 0.31, PPL = 99.1%, followed by cluster 2  while cluster 1 is the least divers one  .Based on the results of pairwise PhiPT value, there is a moderate genetic differentiation between the subpopulations. The results indicate that the variation between genetically distinct cluster 1 and 2 is relatively larger (0.13) than between the other populations .The Mantel test, which is used to demonstrate the presence of spatial population structure indicated that the accessions were poorly structured, based on the GPS data of sampling with an R-squared value of 0.019 .From 43,461 scorable SNPs markers of the 50k iSelect SNP array ; the finThe distribution across the barley genome of the SNPs markers obtained after filtering was compared with the one of the 50k SNP array . The genAccording to the average membership coefficient, the predefined Welo and Shewa subpopulations were classified as genetically distinct in cluster 1 and 2, respectively . By the Estimation of the population structure along the geographical and agro-ecological arrangement gives an important view on the pattern of population structure. In Ethiopia, studies conducted on different cereal crops highlighted the presence of higher genetic variation within geographical locations and altitude ranges for barley , 67, 68,The influence of altitudinal gradient on population structure was reported as important by , 73, 74,Previous studies , 73, 74,To proof the impact of altitude gradient in specific province; Shewa and Welo subpopulations were selected, as they can be defined by cluster 2 and 1 respectively . The lanAs mentioned by , 21, theTherefore, as mentioned by  the climThe overall population genetic differentiation (PhiPT) value is (0.11) indicating the presence of moderate differentiation between the genetically clustered subpopulations . SimilarThe presence of weak geographical or agro-ecological structure for Ethiopian barley landraces , 67, 68 injera, and bread) in this area, and a special dish and beverage (tihlo and korefe), which are exclusively prepared from barley, are commonly consumed in this area [From the three genetically distinct clusters, cluster 1 is explained by the Welo predefined subpopulation.  describehis area . Thus, aCluster 3 mainly contains landraces from Gamo-Gofa, and the production of barley in Gamo-Gofa is mainly on highlands with an altitude higher than 2,500 meter above sea level . Such hiinjera) [Shewa is located in the central part of Ethiopia, with best road facilities, and high consumer demand. Farmers usually produce barley for home consumption and market; and  reportedinjera) . A signiinjera)  due to sCluster 3 is a diverse cluster based on the results of genetic indices . 86% of Genetic structure and diversity of 260 Ethiopian barley landraces, comprising 239 accessions from EBI, and 21 barley breeding lines of the national barley improvement program of the HARC, were investigated based on data obtained from the barley 50k iSelect SNP array . The preS1 FigA: Filtered 10,644 SNP markers; B: Highly informative 983 SNP markers.(TIF)Click here for additional data file.S1 TableAccessions descended from similar ‘EBI accession code’ are highlighted by yellow color.(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.