Prediction of spontaneous preterm birth (sPTB) in asymptomatic women remains a great challenge; accurate and reproducible screening tools are still not available in clinical practice. We aimed to investigate whether the maternal serum metabolome together with clinical factors could be used to identify asymptomatic women at risk of sPTB. We conducted two case-control studies using gas chromatography-mass spectrometry to analyse maternal serum samples collected at 15- and 20-weeks’ gestation from 164 nulliparous women from Cork, and 157 from Auckland. Smoking and vaginal bleeding before 15 weeks were the only significant clinical predictors of sPTB for Auckland and Cork subsets, respectively. Decane, undecane, and dodecane were significantly associated with sPTB (FDR < 0.05) in the Cork subset. An odds ratio of 1.9 was associated with a one standard deviation increase in log (undecane) in a multiple logistic regression which also included vaginal bleeding as a predictor. In summary, elevated serum levels of the alkanes decane, undecane, and dodecane were associated with sPTB in asymptomatic nulliparous women from Cork, but not in the Auckland cohort. The association is not strong enough to be a useful clinical predictor, but suggests that further investigation of the association between oxidative stress processes and sPTB risk is warranted. Although sPTB is prevalent in both high and low/middle-income countries, the major burden of sPTB is concentrated in Asian and African countries where about 85% of preterm births occur5. Short- and long-term consequences of preterm birth include bronchopulmonary dysplasia, neurodevelopment and cognitive impairment, retinopathy, as well as substantial impact on the functional, mental and social health of the infant and its family8. Despite an increase in the research conducted into sPTB and advancements in the implementation of management and prevention strategies, significant reductions of sPTB have not been achieved10.Spontaneous preterm birth (sPTB) due to spontaneous onset of labour or premature rupture of membranes (PROM) is a major cause of neonatal mortality and morbidity14, but their benefits are disputed. According to systematic reviews and meta-analyses, the use of such interventions in selected women can reduce the risk for sPTB, but they are less likely to reduce the risk for perinatal morbidity19. The identification of a more accurate screening tool to predict which women are most at risk of sPTB could improve the selection of women that would benefit from interventions, increasing the likelihood of success and reducing the costs to the healthcare system. However, current screening tools used to identify women at risk of developing sPTB fail to accurately predict preterm birth in asymptomatic women.Natural progesterone , 17α-hydroxyprogesterone (intra-muscular), pessary and/or cervical cerclage have been variously recommended to prevent sPTB in some guidelines12, approximately 2/3 of women with short cervical length will have a term birth21. Chemical biomarkers for sPTB have also been sought, with fetal fibronectin (fFN), insulin-like growth factor-binding protein 1 (IGFPB-1), and interleukin-6 (IL-6)27 among the most studied. A systematic review of sPTB biomarkers found more than 200 studies published between 1965 and 2008, reporting more than 100 potential biomarkers27. However, no single biomarker has proven to be a reliable predictor of sPTB. The authors of the systematic review concluded that there are many heterogeneities between the studies in terms of their experimental study design, timepoint of sample collection, and sample processing methods. One of the largest prospective cohort studies to date evaluated the performance of serial transvaginal cervical length measurements and quantitative vaginal fFN levels for predicting sPTB in a sample of approximately 10,000 nulliparous women with singleton pregnancies28. Despite determination of cervical length and fibronectin levels at three different gestations, the model still showed low predictive accuracy for sPTB.Cervical length is possibly the most employed indicator for sPTB risk in clinical practice. While there is evidence for an increased risk of sPTB in women with a shortened cervix29. For instance, the cervical remodelling process, which involves softening and ripening of the cervix, invariably occurs in the spontaneous onset of labour and is considered its endpoint30. However, it does not occur in the same period of pregnancy for all women, which limits its potential as a marker for the prediction of sPTB31. The underlying complexity of the drivers of sPTB demands a robust analytical technique capable of taking into consideration the multiple pathways potentially affected in the development of sPTB.sPTB is a condition with a multifactorial aetiology, a long pre-clinical phase, and adaptive mechanisms during pregnancy35. Metabolomic measurements provide a biochemical snapshot of the physiological state of the organism36; their close relationship with the biological phenotype means they have the potential to reveal underlying mechanisms of disease. Due to the biochemical complexity of the metabolome, hyphenated analytical techniques are used. This involves subjecting sample extracts to a high-resolution separation technique such as gas chromatography (GC), high-pressure liquid chromatography (HPLC), or capillary electrophoresis (CE). Identification is then carried out using mass spectrometry (MS), or nuclear magnetic resonance (NMR).Metabolomics, the study of low molecular weight compounds in a biological system, has previously been used to successfully investigate pregnancy complications such as fetal growth restriction, preeclampsia, and gestational diabetes mellitus37, in collaboration with the SCOPE (Screening for Pregnancy Endpoints) study. SCOPE was an international multicentre prospective cohort study that collected standardized data and samples from more than 5,690 low-risk pregnant women40. The main objective of SCOPE was to develop a robust database and biobank to identify predictors and develop screening tests for preeclampsia, sPTB and small-for-gestational-age newborns. The current study describes the results of Preterm SAMBA phase 1, which used the SCOPE biobank to investigate the metabolomic profile, at 15- and 20-weeks’ gestation, amongst SCOPE participants who went on to have sPTB, when compared to matched controls. Parallel analyses were performed using participants from the Irish and New Zealand’s centres of the SCOPE study.The Preterm SAMBA study (Preterm Screening and Metabolomics in Brazil and Auckland) has been established to identify and validate metabolomic biomarkers for sPTB41.Two case-control studies were conducted, using a sub-set of women from the Auckland, New Zealand and Cork, Ireland centres of the Screening for Pregnancy Endpoints (SCOPE) study40. Collection of data and biological samples complied with standardized procedures in all participating centres and was conducted in accordance with the principles of the Declaration of Helsinki. Cases of sPTB were defined as those women who delivered before 37 weeks’ gestation, and controls as those who delivered at or after 37 weeks’ gestation.Cork, Ireland.2)*.The subset of SCOPE participants included in this study from the Cork, Ireland centre consisted of 55 cases of sPTB and 102 controls, matched to cases according to maternal age (±3 years) and maternal body mass index  05/02/08, Clinical Research Ethics Committee of the Cork Teaching Hospitals; New Zealand: study number AKX/02/00/364, Northern X Reginal Ethics Committee) and all participants gave their written informed consent. Details on enrolment and inclusion criteria for the SCOPE study have been published elsewhere2)*.The subset of SCOPE participants included in this study from the Auckland, New Zealand centre consisted of 55 cases of sPTB and 109 controls, comprising 56 controls matched to cases according to maternal age (±3 years), and 53 controls matched to cases according to both maternal age (±3 years) and BMI .Demographic information including maternal age and BMI was collected at the time of recruitment (15 ± 1 weeks’ gestation). History of vaginal bleeding and any infections during the pregnancy were also recorded via interview at the initial visit. The infant characteristics were recorded at birth and included birth weight, customised birth weight centile, and whether the infant was small, normal, or large for gestational age.Spontaneous preterm birth was defined as any birth that occurred before 37 weeks due to spontaneous onset of labour or premature rupture of membranes. Gestational age (GA) was estimated by last menstrual period (LMP) and confirmed by an ultrasound dating before 16 weeks of gestation. A discordance of seven or more days between LMP and ultrasound dating or an unsure LMP led to the estimation of GA exclusively by early ultrasound parameters. Term birth for the control group was determined as delivery after 37 weeks of gestation. Early sPTB before 34 weeks was considered for subgroup analysis.42.Maternal blood samples were collected in two 6 mL vacutainers by venepuncture at 15 and 20 weeks (±1 week). Following clot formation, the vacutainers were centrifuged at 3,000 rpm for 10 min at 4 °C, followed by a second centrifugation at 4,000 rpm for 10 min at 4 °C. After centrifugation, the resulting serum (supernatant) was pipetted into a sterile tube and aliquots of 250 µL were dispensed into cryotubes. All samples were stored at −80 °C. The study followed standard best practice procedures for repositories in all steps of sample collection and storage, registering all SPREC  data accordingly in an online databaseFor this study, two 250 µL maternal serum samples were obtained from the biobank for each participant, one from each time point; 15- and 20-weeks’ gestation (±1 week).et al.43. Briefly, samples were thawed on ice at 4 °C and transferred from cryotubes to 1.5 mL microcentrifuge tubes. An internal standard  was added to all samples and the sample-IS mix was vortexed for 1 min. Samples were dried for 4 h at 0.8 HPa in a centrifugal vacuum concentrator with a −104 °C refrigerated vapour trap (Thermo Fisher Scientific Savant SC250EXP SpeedVac Concentrator with Savant SP5121P Refrigerated Vapour trap). Metabolites were extracted using 50% and 80% cold methanol-water solution . Specifically, 500 µL of 50% cold methanol-water solution was added to all samples, followed by vortexing for 1 min and centrifugation at 3,000 rpm for 5 min at −4 °C. After centrifugation, the supernatant was transferred to a fresh chilled microcentrifuge tube (kept on dry ice). Then, 500 µL of 80% methanol-water solution was added to the pellet and this was centrifuged at 3,000 rpm for 5 min at −4 °C. The supernatants obtained from both extraction steps were combined and dried in the centrifugal vacuum concentrator for 4 hours at 0.8 HPa, with a −104 °C refrigerated vapour trap. Dried extracted samples were stored at −80 °C prior to derivatisation.The serum samples underwent extraction and derivatization procedures based on the 2010 protocol of Smart Negative controls were produced by subjecting an empty microcentrifuge tube to the same processing as the samples. Pooled quality control samples (QC) were produced by pooling a small amount from every sample, mixing, and then making aliquots of the same volume as the samples.Derivatization was carried out using methyl chloroformate (MCF). Samples were derivatised in batches of 18–24, on the same day that they were analysed on the GC-MS. Samples were re-suspended with 400 µL of 1 M sodium hydroxide and were transferred to silanised glass tubes, followed by addition of 334 µL of methanol and 68 µL of pyridine. The sample was placed on a vortexer for the remainder of the derivatization process at ~1,500 rpm. The rate limiting step began from the addition of 40 µL of MCF. A second addition of 40 µL of MCF was made, 30 sec later. After another 30 sec, 400 µL of chloroform was added to extract the alkylated derivatives from the reaction mixture. After 10 sec, 400 µL of sodium bicarbonate (50 mM) was added. Centrifugation was used to separate the aqueous layer from the chloroform layer. After centrifugation, the aqueous layer was removed and the remaining chloroform extract was dehydrated by the addition of sodium sulphate (~0.3 g). The remaining liquid was then transferred to an amber glass GC-MS vial with a glass 33 µL insert.et al.43, with modifications. One microliter of sample was injected for analysis. The injector was set to 290 °C in splitless mode. The column flow was maintained at 1.0 mL min-1 in constant flow mode. The column was a fused silica ZB-1701 30 m long, 0.25 mm inside diameters, with a 0.15 µm stationary phase constituting of 86% dimethylpolysiloxane and 14% cyanopropylphenyl (Phenomenex). Instrument grade helium  was used as the carrier gas for the analysis. The detector was run in positive-ion, electron-impact ionisation mode, at 70 eV electron energy. Identification of compounds was carried out using mass spectra acquired in scan mode from 38 to 550 atomic mass units. The Cork samples were analysed on an Agilent 7890 A gas chromatograph coupled to an 5975 C inert mass spectrometer. The Auckland samples were analysed on a Thermo Scientific Trace GC Ultra gas chromatograph coupled to an ISQ mass spectrometer.The GC-MS instrument parameters were based on Smart http://www.amdis.net/)44 from an in-house mass spectral library for MCF-derivatised metabolites (~210 compounds) developed by Silas Villas-Boas. The library contained mass spectra predominantly from certified reference standards. In addition to the in-house library, the National Institute of Standards and Technology (NIST) mass spectral library  was also used to identify peaks in the raw chromatograms. Since AMDIS is not able to batch deconvolute with the entire NIST library, a NIST subset library was constructed employing a method developed by Elizabeth McKenzie using pooled quality controls.Data processing was semi-automated. The raw files obtained from the GC-MS were converted into common data form (cdf) format for analysis and were deconvoluted and identified using the Automated Mass Spectral Deconvolution and Identification System  developed by Ting-Li Han. MassOmics used XCMS and the AMDIS report to integrate the peak areas for each of the identified metabolites. The summary report obtained from running the MassOmics script was then checked, and peaks with low ID hits, or with large retention time shift, and laboratory contaminants were removed.The NIST subset was produced using the top five results for each feature from the Agilent Chemstation PBM (Probability Based Matching) deconvolution program. MassOmics in house software (version 2.3) was used to create the subset library. MassOmics is an R script based on the XCMS R packageData was checked against negative controls to identify and remove background contaminants. Peaks that were not extracted correctly with XCMS were integrated separately using the Ion Extractor feature in MassOmics. Co-eluting peaks underwent manual integration. The relative abundance obtained for each metabolite was normalised to the internal standard (Alanine-d4). After internal standard normalisation, the remaining technical variation was corrected for by analytical batch median centering using the samples. An analytical batch was defined as ~25 injections, comprising ~18 samples, 4 QC’s, one Alkane series, one negative control, and one standard mixture.https://www.R-project.org)46. Data was analysed separately for each site. Clinical predictors were analysed for univariate associations with preterm birth, and a multivariate logistic regression model predicting preterm birth with these predictors was selected using stepwise logistic regression with AIC, starting from an intercept only model, followed by backward elimination of variables with multivariate p > 0.05. Mann-Whitney tests were used to analyse the difference in metabolite levels between cases and controls. Ratios between the 20 and 15-week levels of each metabolite were also assessed in this way, to identify situations where changes from baseline rather than absolute metabolite levels were associated with case status. To adjust for multiple comparison testing, false discovery rates (FDR) were calculated for each comparison using the Benjamini-Hochberg procedure47. Metabolites with an estimated FDR < 0.05 are reported.Statistical analysis of the normalised mass spectral data was performed using R 3.4.3 . The random forest was assessed via a permutation test based on the out-of-bag error rate.Where potential metabolite predictors were identified, we then built logistic regression and random forest models using the selected clinical factors and metabolites. For logistic regression, we assessed whether one or more metabolites improved the area under the receiver operator characteristic curve compared to the model based on clinical predictors alone50. The clinical variables, log transformed 15 and 20-week metabolite intensities, and 20 to 15-week ratios were used as candidate predictors. Up to three components with five predictors each were used. The number of components was selected based on the Mahalanobis distance balanced error rate using 10-fold cross validation, averaged over 10 repeats. This error rate was also used to assess the significance of this model via a permutation test.To assess whether expanding the set of predictors might improve accuracy, the sparse partial least squares discriminant analysis (PLS-DA) method from the mixOmics package was employedThe above procedures were repeated for comparing preterm birth <34 weeks to the entire control group.Participant demographic and outcome characteristics are shown in Table In the Cork samples, 176 compounds were detected. Of these, 77 were identified using an in-house library of reference standards and the remaining compounds were identified using mass spectrum alone . Of the in-house library matches, 61 were putatively identified (80–100% match to a reference standard) and 15 were tentatively identified . Of the NIST14 library identifications, 25 were putatively identified , 58 were tentatively identified , and three were unknown .In the Auckland samples, 142 compounds were detected. Of these, 50 were identified using an in-house library of reference standards and the remaining compounds were identified using mass spectrum alone (NIST 2014). Of the in-house library matches, 41 were identified, eight were tentatively identified, and one was unknown. Of the NIST14 library identifications, 51 were putatively identified, 34 were tentatively identified, and seven were unknown.Table Similarly, the random forest model using log(undecane) and vaginal bleeding was significantly better than random (p = 0.002) although the classification success was modest (70%). However, models also including log(decane) or log(dodecane), or both, had error rates similar to those based on permuted data .Sparse PLS-DA also produced a 1-component model for the Cork preterm birth data with an error rate that was significantly better than error rates produced for random permutations (p = 0.01). This model again included undecane, dodecane, decane, and vaginal bleeding. A fifth predictor, stearic acid measured at 15 weeks’ gestation, was present; however, its loading was small, and it did not have an odds ratio significantly different from 1 (p = 0.07) when incorporated into the logistic regression with vaginal bleeding and undecane.No metabolites or 20–15 week ratios met the false discovery rate threshold for the Cork <34 weeks data, for either preterm <37 weeks or <34 weeks in the Auckland data, nor were the sparse PLS-DA models significant .Log transformed Undecane did not meet the FDR threshold for association with preterm birth <34 weeks in the Cork data (compared to birth ≥ 37 weeks), but was associated with preterm birth <37 weeks. We therefore examined the sensitivity of our model to the threshold for preterm birth. The odds ratio associated with a 1 sd change in log(Undecane), in a model also including vaginal bleeding, when the divider between preterm birth and term birth was reduced to 36, 35 and 34 weeks were 2.1 (p = 0.0009), 2.2 (p = 0.0014), and 1.7 (p = 0.0571) respectively.We have analysed potential predictors of sPTB using serum samples and clinical data from Cork and Auckland participants of the SCOPE study, an international cohort of low-risk nulliparous women. An untargeted metabolomics approach was applied to serum samples collected at 15 and 20 weeks of gestation. More than one hundred metabolites were identified in each subset (Cork and Auckland). As expected for the metabolomics method employed, the most common classes of metabolites were fatty acids, followed by amino acids. Only three metabolites from the 20-week serum of Cork participants were found to be significantly associated with sPTB. Vaginal bleeding before 15 weeks, and smoking during pregnancy were the only clinical factors associated with sPTB, in Cork and Auckland subsets, respectively. In the Cork cohort, adding undecane to a multivariate logistic regression model for predicting sPTB improved its performance over a model with vaginal bleeding alone. This improvement is robust to the definition of preterm birth, persisting when the preterm threshold was decreased to 36 or 35 weeks; at 34 weeks the improvement is no longer significant. We note that, as depicted in Fig. 53, and oxidative stress has been associated with elevated alkane levels in gastroenteric disease, lung disease, and other chronic diseases of metabolism56. Glutathione, an important intracellular antioxidant, has been found to be decreased in maternal and umbilical cord blood of very low preterm neonates and their mothers57. Preterm birth seems to be associated with depletion of glutathione, reinforcing a possible increased oxidative status and lower antioxidant capacity. However, failure to demonstrate elevated alkanes among sPTB cases in the Auckland cohort limits our confidence, and there was no evidence these alkanes were associated with early sPTB (<34 weeks) at either study site. In addition, hypotheses describing the role of reactive oxygen species generation, metabolic and inflammatory imbalance and many other downstream mechanisms  caused by oxidative stress activation51 do not clarify whether those mechanisms are trigger factors or consequences of underlying conditions/alterations resulting in preterm PROM and/or spontaneous onset of preterm labour.There is a biologically plausible explanation for observing elevated alkanes  in the serum of mothers who had sPTB. Oxidative stress may lead to degradation of cell membranes by lipid peroxidation, followed by conversion of polyunsaturated fatty acids to volatile alkanes. The association of several oxidative stress-associated processes and sPTB have been previously reported59. Further investigation of individual pollutant exposure would be necessary to confirm whether elevated alkanes are associated with environmental exposure. It is also possible that technical rather than biological variability accounts for some of the differences observed across sites. Samples from the different sites, while analysed using the same protocols, were run on different platforms by different technical personnel. Thirty-four fewer metabolite species were detected in the Auckland samples, suggesting reduced sensitivity. In particular, two of the three metabolites elevated in cases in Cork (decane and dodecane) were not identified in the Auckland samples.The differences across study sites in the associations between sPTB and clinical factors such as smoking and vaginal bleeding in this case-control analysis also suggest that there are differences in the Cork and Auckland populations. The alkanes elevated in the Cork preterm birth group are present in outdoor and indoor air contaminants, which could potentially differ across study sites, providing an explanation for this observation. Association of environmental exposures with maternal and perinatal health have been reported for many years but are not yet well establishedWe also examined prediction of very preterm birth (<34 weeks). No clinical predictors were significant in the Auckland cohort, but vaginal bleeding before 15 weeks was associated with very preterm birth, as well as preterm birth, in the Cork cohort.62. Previous studies have used between 20 and 70 samples of a variety of biofluids , equally divided between cases and controls. There is also a diversity of pregnancy stages and analytical techniques, so it is not surprising that there is little overlap in the specific compounds identified. However, Virgiliou et al. also suggested that the changes in amino acids and lipids they observed could be related to oxidative stress62.To the best of our knowledge, there are few studies in the literature applying metabolomics techniques to understand sPTB in asymptomatic pregnant womenOur study has strengths and limitations. Cases and controls were selected from a large cohort comprised of low-risk nulliparous women enrolled in early pregnancy and containing a high standard biobank. Several procedures were employed to assure data, sample, and analysis compliance and reliability according to Standard Operating Protocols. Shortcomings of our study include lack of data regarding cervical length, a previously reported risk factor, and assaying of the metabolome of the two cohorts at different times on different equipment. Genetic factors may also be contributors to PTB risk however these were not assessed in this study. In addition, we have not investigated predictive metabolites for the different preterm birth subtypes (spontaneous onset of preterm labour or preterm premature rupture of membranes), focusing instead on an early sPTB (<34 weeks) subgroup, due to the increased morbidity in this specific group. Using data and samples from women of different populations , enabled us to compare reproducibility of our technique and also to discuss possible local drivers for sPTB . Cork and Auckland samples were analysed by different laboratory experts and instruments and as such, the reproducibility and sensitivity differed across the sites.While our finding of an association between elevated alkanes and sPTB at the Cork site is preliminary, it raises several interesting questions to pursue in the future. What differences are typical or expected across geographically distant study sites? What role might exposure to exogenous pollution sources, including alkanes, play in preterm birth? And finally, how might oxidative stress trigger, or be triggered by, processes leading to spontaneous preterm birth?Supplementary Figures


The current study aimed at genomic characterization and improved understanding of genetic diversity of two Indian mithun populations  using genome wide genotype data generated with Illumina BovineHD BeadChip. Eight additional populations of taurine cattle (Holstein and NDama), indicine cattle (Gir) and other evolutionarily closely related species  were also included in this analysis (N = 137) for comparative purposes. Our results show that the genetic background of mithun populations was uniform with few possible signs of indicine admixture. In general, observed and expected heterozygosities were quite similar in these two populations. We also observed increased frequencies of small-sized runs of homozygosity (ROH) in the farm population compared to field mithuns. On the other hand, longer ROH were more frequent in field mithuns, which suggests recent founder effects and subsequent genetic drift due to close breeding in farmer herds. This represents the first study providing genetic evidence about the population structure and genomic diversity of Indian mithun. The information generated will be utilized for devising suitable breeding and conservation programme for mithun, an endangered bovine species in India. Bos frontalis), also known as gayal, a unique bovine species, has a limited geographical distribution primarily restricted to the North-Eastern Hilly (NEH) region of India, Myanmar, hilly provinces of Bangladesh, Bhutan and Yunan province of China1. Though it is difficult to ascertain the actual population of Mithun in the world, India possesses 0.30 million mithun which constitutes ~97.57% of the world population 2. Mithun has a very special socio-cultural status among the indigenous tribal population of NEH region of India. Having the very high value in the barter system, owning mithun is considered as a sign of prosperity.Mithun  and quantity (higher dressing %)6. Mithun have higher feed conversion efficiency than local cattle7. The current population size of mithun in India is not large. However, considering its importance in widening the biodiversity base and adaptation to a humid sub-tropical climate and a hilly topography, it could be exploited as an alternative means of livelihood as well as for improved food and nutrition security in its present habitat and in similar environments elsewhere.Mithun are traditionally raised as a meat animal in a free-range system in the sub-tropical rain forest with almost zero input. As a part of socio-cultural practices, mithuns are sacrificed during rituals and festivals to offer feastGenetic improvement of mithun, however, is impeded by the way mithun is presently reared. Under traditional system of rearing, mithuns are let loose in the forest with minimal intervention. Recording of traits and pedigree information is almost non-existent. The size of a typical mithun herds ranges from 50 to 100. Herds are generally served by a few dominant bulls. The mithun population may therefore suffer from inbreeding. Under these circumstances, the genetic diversity of the mithun population needs to be described. This will assist ongoing mithun conservation and genetic improvement programme and for devising a suitable breeding policy.8. The availability of high-throughput SNP genotyping platforms makes it feasible to undertake high-resolution scans by using large numbers of SNP markers distributed across the whole genome. SNPs are useful in studying livestock genetic diversity and population structure9. Although a large number of SNPs have been identified in bovine genome-sequencing projects, few of these have been validated outside Bos taurus, as for example in mithun10. The Illumina BovineHD BeadChip  with 777k SNPs was introduced and utilized for genotyping studies in various breeds of cattle, but has also been used in other members of the bovidae, for example, yak, gaur, buffalo and wild anoa16.Among a wide range of molecular markers developed, single nucleotide polymorphisms (SNPs) are the most abundant, widely dispersed throughout genomes, and have variable distribution among species17. Hence, we used the unsupervised clustering analysis (carried out with the ADMIXTURE software)18 to estimate individuals’ ancestries from SNP genotypes to assess the extent of admixture in mithun with other cattle species  and expected (He) heterozygosities estimated in the Indian mithun population (farm and field population) ranged from 0.25 to 0.17, and 0.25 to 0.18, respectively (Table IS) based on the observed versus expected number of homozygous genotypes, were found to be 0.06 ± 0.02 and 0.02 ± 0.01 for the farm and field animals  in the autosomes of 48 farm mithun and 24 field mithun animals were determined using PLINK1.9The total length of ROH per animal was averaged within population in six sized windows: 250 kb–1 Mb, 1 Mb–2 Mb, 2 Mb–4 Mb, 4 Mb–8 Mb, 8 Mb–16 Mb and >16 Mb. The average total length of ROH per animal was 823.7, 242.1, 92.30, 47.03, 66.22 and 110.9 Mb in the farm and 735.8, 245.0, 121.3, 84.4, 86.0 and 87.4 Mb in the field population for these six length categories.Summary statistics of ROH observed are outlined in Table Unsupervised clustering analysis using ADMIXTURE software program were carried out for 10 populations . The admixture analysis including other species grouped mithuns from farm and field together, and were fairly homogeneous Fig. , K = 4–7The principal component analysis was done using 50k markers for eight Bos species and two mithun populations (firm and field). The first two principal components (PC1 and PC2) grouped farm and field mithuns in a cluster with gaur, yak and bison, separated from three other major clusters of two taurine cattle (NDama and Holstein), and indicine cattle  positioned mithuns along with gaur, yak, bison and Bali cattle in distinct clusters from indicine and taurine cattle populations. Principal component 2 (PC2) separated two taurine breeds into separate clusters. Principal component 3 (PC3) further separated Gir from other populations Fig. .Figure 520 analysis was used to study the population splits and subsequent gene flows. We constructed a phylogenetic tree of all the bovine species without adding any migrations and assuming wild buffalo (OWB) as outgroup using Treemix analysis . Myanmar , Bangladesh , China  and Bhutan  also home small populations of mithun in Asia2. Information regarding genetic diversity and population structure of Indian mithun remains scanty. A conservation programme for mithun has been initiated at Khonoma and Thevopisu villages, Nagaland in their native breeding tracts in India. Genetic improvement in mithun through a systematic breeding program could not be initiated under field conditions due to the small number of animals, uncontrolled mating, and their scattered distribution across remote and inhospitable hilly terrain.India has the largest mithun population (~97.57%) in the world , observed heterozygosity (Ho) and estimates of inbreeding (FIS and FST). FST between two populations was low (0.03) indicating a close genetic connection between them21. Mithun is assumed to be a close evolutionary relative of gaur23. Hence, the BovineHD BeadChip was used to genotype mithuns and available literature on cattle was referred for comparison in the present study. Genetic diversity in terms of average heterozygosity in both mithun populations (0.17–0.25) was found to be similar to zebu and African taurine cattle14, and was in the same range as for taurine cattle from West Africa (from 0.18 for African Lagune to 0.22 for Somba cattle) or East Africa (0.24 for Sheko cattle)10. However, SNPs that are included in the analyses are polymorphic within Mithun, but originate from bovine and are thus expected to be old SNPs (polymorphic in the common ancestor of Mithun and Bos taurus) leading to biased allele frequency spectrum.Genetic diversity of two groups of Indian mithun population (farm and field groups) were assessed by expected heterozygosity  than in the farm population (0.06). We see some evidence of mating between relatives in the farm population, while there is little evidence of mating among close relative in the field population. The FIS values in our study were similar to that reported24 in mithuns from Bangladesh using the Illumina BovineSNP50 BeadChip, mean expected heterozygosity of 0.148 ± 0.14 with a heterozygote deficiency of 0.06 (FIS).We used Fo and He of mithun population in our study compared to taurine and indicine cattle breeds as reported by the Bovine HapMap Consortium13 and Korean cattle populations16 may also be attributed to ascertainment bias in SNP discovery of the BovineHD BeadChip. Previously low heterozygosity estimates of Tunisian cattle populations were likewise attributed to ascertainment bias in the design of the BovineSNP50 BeadChip as polymorphic sites of African origin present in the genome of Tunisian cattle were not included in the chip14. The Bovine HapMap Consortium reported that nucleotide diversity in (Brahman) cattle, a crossbred of indicine breeds, is more than twice that observed within Holstein and Angus breeds26 as expected.Lower heterozygosity values H29.Our study revealed a higher number of short and medium ROH  than longer categories (8–16 Mb and >16 Mb) in both the farm and field mithun population. This reflects ancestors shared between the parents long ago, can be indicative of selective sweep, ancient inbreeding or bottleneck. The farm population showed greater ROH length and numbers compared to field population, which was as per expectation due to higher level of inbreeding and small population size parental population in the farm. Similar observations in various cattle breeds were reported as short ROH are generally due to older haplotype relatedness, while longer ROH result from more recent inbreeding31. Long ROH indicates consanguinity between an individual’s parents27. Shorter ROH indicate more distant demographic and selective events after repeated fragmentation of chromosome segments by recombination27. In particular, recent inbreeding resulting from the mating of closely-related ancestors leads to a high occurrence of long ROH. On the other hand, very long ROH sometimes occur in outbred populations32.Length and frequency of ROH provide information about demographic history as well as recent inbreeding in individuals33. Level of inbreeding can be estimated accurately in cattle population without knowledge of the pedigree using Illumina BovineHD SNP genotyping assay34. FROH, the genomic inbreeding coefficient, was found to be highly correlated with pedigree inbreeding in a small group of populations34.In human, short ROH (<1.5 mb) is due to ancient linkage disequilibrium through inheriting parental common haplotypes, whereas, bigger ROH is due the history of related parents in recent timesROH for the various categories  in the farm and field mithun population were generally low, probably indicating larger effective population sizes in the past. We observed relatively higher mean FROH for the 250kb–1Mb category in the farm mithun (0.335). Such short ROH reflect events at least 25 generations ago. With generation interval of approximately 5–10 years in mithun, this translates to selection 100 or 200 years in the past. It seems that probably mithun population has experienced a population bottleneck in the past.Estimates of mean FROH) with lengths above 1 Mb was in intensely selected Brown Swiss breed (0.151–0.156)35 and in Holstein cattle (0.116)29, while lower FROH for unselected cattle breeds Pinzgauer and Tyrolean Grey domestic cattle 35, and the lowest in unselected, preserved Polish Red breed (0.057)29.Our results fit well with other genotyping studies in various breeds of cattle using Illumina BovineHD BeadChip and validated through Illumina BovineSNP50 BeadChip, viz. average autozygosity calculated from ROH  and a diverse field population (n = 24) from four locations of North Eastern Hill Region  falling in the native mithun breeding tracts of India. All the experiments were performed in accordance with relevant guidelines and regulations approved by the Institutional Animal Ethics Committee, ICAR-NRC on Mithun, Nagaland.Blood samples were collected from jugular vein in a vacutainer tube (BD) containing EDTA by a qualified veterinarian and were transported to the laboratory in cool pack as soon as possible. The DNA was isolated using a standard blood DNA isolation kit (Promega #A1620) as per the manufacturer’s instructions.Genomic DNA from each mithun was quantified to assure a concentration of at least 50 ng/µl genomic DNA required by the Illumina® Infinium® SNP genotyping platform. A total of 72 mithun  were genotyped by Sandor Lifesciences Pvt. Ltd., Banjara Hills, Hyderabad, India with the Illumina® BovineHD BeadChip assay, following the manufacturer’s protocol. Genotypes were called using the validated standard cluster file provided by the manufacturer. Only the autosomal SNPs were considered in this study.https://support.illumina.com/array/arraysoftware/genomestudio/documentation.html). Genome Studio was also used to generate PLINK1.9 data files (in.ped and.map format)19 for further analyses. The SNPs located on sex chromosomes, not polymorphic or without known position in the cattle genome were excluded from further analysis. The Bos taurus genome assembly  was used as a reference genome due to the absence of a published mithun genome.Samples and marker based quality control was performed using the Illumina’s Genome Studio software . SNP markers showing deviation from Hardy-Weinberg proportions (HWP) based on parental genotype data (p < 0.001) were removed. The attributes considered for quality control finally include filtering of SNPs with call rate ≥95%, MAF ≥5% and HWP ≥0.001 using PLINK1.9 software. After this filtration 139,350 polymorphic SNPs remained for analysis.To identify closely related individual, a pair-wise identity-by-state (IBS) distance analysis was performed using PLINK1.9The parameters estimated to study the genomic diversity in mithun populations included estimates of heterozygosity, detection of runs of homozygosity (ROH) and estimates of inbreeding coefficients.o) and expected (He) heterozygosities, as well as the estimates of inbreeding for mithun populations were estimated using PLINK1.9 software19. The FST was estimated using PLINK software, which uses method introduced by Weir and Cockerham (1984)36.The observed  were detected to determine the extent of autozygosity in the mithun populations. A ROH is defined as a contiguous length of homozygous genotypes. A ROH of sufficient length indicates the two copies of the chromosome in this region are identical-by-descent (IBD)19 was used to identify ROH by running a sliding window that scans the genomic distribution of SNP data to identify stretches of homozygous SNPs. A minimum number of 10 consecutive homozygous SNP and zero heterozygotes were allowed in each window. A maximum gap between SNP of 1,000 kb was allowed. With high-density SNP data this approach mostly detects truly autozygous segments37. Moreover, this strategy is particularly suitable for livestock populations because they have much higher levels of autozygosity than model organisms making identification of longer ROH easy37.PLINK1.9Detected ROH were classified in windows: ROH in the range of 250 kb-1 Mb, 1 Mb–2 Mb, 2 Mb–4 Mb, 4 Mb–8 Mb, 8 Mb–16 Mb and >16 Mb.ROH of each individual was estimated as the sum of length of autosomal ROH divided by the total length of the autosomes covered by markers34.In the present study, genomic autozygosity, Fhttp://widde.toulouse.inra.fr/widde/]38 and were included along with two mithun populations to explore the evolutionary relationship of mithun with other bovine species  was included assuming contribution of gaur to mithun genomes as its presumed wild relative; while yak , bison  and Bali cattle  were taken into account to find out possible genetic contribution towards mithun genome, if any. As it is assumed that there might be some introgression due to crossing between mithun and cattle under the field conditions, the indicine cattle, Gir (Bos indicus) genotype data was included. Taurine cattle  were included to identify any taurine introgression and anoa, the wild buffalo  was included as an outgroup in the present study.The main goal of the present study was genomic characterization of two mithun populations (farm and field). Another eight Illumina® BovineHD BeadChip or BovineSNP50 BeadChip genotype data sets were collected from previously published work, available at [es Table . The wil19 if they did not have call rate of at least 95%. No filtering was done at this step for MAF, except SNPs monomorphic across all breeds. Summary statistics such as heterozygosity and inbreeding coefficient were computed and results are presented  showing admixture were removed from the Treemix analysis. Nodes robustness was estimated with 500 bootstrap replicates and plotted using the Treemix bootstrap function of BITE40. Admixture detection was done using the F3 statistics computed using threepop program from the Treemix software package.Phylogenetic analysis was carried out using TreemixApproval of Institutional Animal Ethics Committee was obtained for this study including collection of mithun blood samples and DNA extraction from the Institute research farm. Blood samples of the mithun were collected from the farm and with the permission of the mithun owners from the field by qualified veterinarians.Data supporting this paper were generated by ICAR-NRC on Mithun. The phenotype and genotype data are available with the Data Cell of the Institute and should be requested directly from the corresponding authors or the ICAR-NRC on Mithun.


Perrault syndrome is a rare autosomal recessive disorder that is characterized by the association of sensorineural hearing impairment and ovarian dysgenesis in females, whereas males have only hearing impairment. In some cases, patients present with a diversity of neurological signs. To date, mutations in six genes are known to cause Perrault syndrome, but they do not explain all clinically-diagnosed cases. In addition, the number of reported cases and the spectra of mutations are still small to establish conclusive genotype–phenotype correlations.TWNK gene.Affected siblings from family SH19, who presented with features that were suggestive of Perrault syndrome, were subjected to audiological, neurological and gynecological examination. The genetic study included genotyping and haplotype analysis for microsatellite markers close to the genes involved in Perrault syndrome, whole-exome sequencing, and Sanger sequencing of the coding region of the TWNK gene in the three affected subjects: c.85C>T (p.Arg29*), previously reported in a case of hepatocerebral syndrome; and a novel missense mutation, c.1886C>T (p.Ser629Phe). Mutations segregated in the family according to an autosomal recessive inheritance pattern.Three siblings from family SH19 shared similar clinical features: childhood-onset bilateral sensorineural hearing impairment, which progressed to profound deafness in the second decade of life; neurological signs , with onset in the fourth decade of life in the two females and at age 20 years in the male; gonadal dysfunction with early cessation of menses in the two females. The genetic study revealed two compound heterozygous pathogenic mutations in the TWNK mutations will manifest neurological signs in adulthood. Molecular and clinical characterization of novel cases of recessive disorders caused by TWNK mutations is strongly needed to get further insight into the genotype–phenotype correlations of a phenotypic continuum encompassing Perrault syndrome, infantile-onset spinocerebellar ataxia, and hepatocerebral syndrome.Our results further illustrate the utility of genetic testing as a tool to confirm a tentative clinical diagnosis of Perrault syndrome. Studies on genotype–phenotype correlation from the hitherto reported cases indicate that patients with Perrault syndrome caused by  Perrault syndrome (PRLTS) is an autosomal recessive genetic disorder that is characterized by the association of sensorineural hearing impairment and ovarian dysgenesis in females , 2. AffePRLTS is genetically heterogeneous. To date, mutations in six genes are known to cause different genetic types of the syndrome Table .Table 1GHSD17B4, the first gene whose involvement in PRLTS was reported [HARS2 [LARS2 [CLPP [TWNK [ERAL1 [d [HARS2  and LARS2 [LARS2  code forPP [TWNK  encodes K [ERAL1  codes foHSD17B4 and TWNK present with neurological manifestations  [CLPP or LARS2 may result in clinical types I or II [HARS2 or ERAL1 [TWNK gene, one of which is novel whereas the other was previously reported in a case of hepatocerebral syndrome. Implications of these findings in the genotype–phenotype correlations of TWNK mutations are discussed.The number of cases with mutations in each of the six known genes is still small to establish definite genotype–phenotype correlations. So far, all patients with PRLTS due to mutations in type II) , 10–14.  I or II , 14–20. or ERAL1 , 9, 14, Affected subjects from family SH19 were clinically examined by the Departments of Pediatrics and Neurology of Hospital Universitario Central de Asturias. The genetic study was performed in parallel by the Service of Genetics of the Hospital Universitario Ramón y Cajal, and by the Neurogenetics Unit of the Research Institute Germans Trias i Pujol (IGTP) in Badalona (Barcelona).DNA was extracted from peripheral blood samples from all members of each family by standard methods. Microsatellite markers flanking the genes involved in PRLTS were chosen from the literature  and elecWhole-exome sequencing (WES) was performed on genomic DNA obtained from peripheral blood. Exome capture was performed using SureSelect All Human Enrichment Target Exon (Agilent Technologies) for 71 Mb according to the manufacturer’s protocol. Paired-end, 101-nt long reads were generated on a HiSeq 2000 platform (Illumina Inc.). Read alignment was performed using BWA (Burrows-Wheeler Aligner) . VariantTWNK gene are listed in Table Primer sequences for PCR amplification of all exons and exon–intron boundaries of the Mutation nomenclature is annotated based on cDNA sequence (GenBank accession number NM_021830.4) and according to the current Human Genome Variation Society rules as implemented by the Mutalyzer 2.0.3 program .Family SH19 included four siblings who were born from non-consanguineous parents  had been diagnosed with Parkinson’s disease, and the father (I:1) had cavus feet. Family history did not show any other relative with similar traits.The genetic study was requested in parallel to the Service of Genetics of Hospital Universitario Ramón y Cajal (HURyC), because of the hearing impairment, and to the Functional and Translational Neurogenetics Unit  (IGTP), because of the neurological signs that were observed.HSD17B4, HARS2, CLPP, LARS2 and ERAL1, but showed that the three siblings were haploidentical for markers close to TWNK  and c.1886C>T (p.Ser629Phe) , a missense mutation that is predicted as pathogenic by all bioinformatic algorithms  [In the IGTP, the genetic cause of the polyneuropathy that was observed in the affected siblings was investigated by massively-parallel DNA sequencing. WES of subject II:3, with a ≥ 20× average coverage of 88.7% of the target regions, identified 72,511 variants within 1396 candidate genes under a recessive model of inheritance. Upon excluding those DNA variants with a minor allele frequency ≥ 1% in known databases, variants were prioritized by their predicted deleterious effect, amino acid conservation, and associated disease heritability. Six of these candidate variants were located in genes related to ataxia phenotype including causing) –30, and Mutation p.Arg29* is expected to result in a very small truncated protein lacking both domains of Twinkle, primase-related and helicase, or in no protein at all because of mRNA degradation through nonsense-mediate decay. Mutation p.Ser629Phe has not been reported previously in any database, it affects a highly-conserved residue Fig. c, and itMost patients with rare diseases face diagnostic delays, because of the difficulties in noticing the association of clinical signs that define their particular disease . In PerrHSD17B4, TWNK, CLPP and LARS2 genes [CLPP and LARS2, there are also reports of patients without neurological signs [HARS2 or ERAL1 who have been reported to date [HARS2 or ERAL1, still underrepresented. Moreover, different mutations in some of the six PRLTS genes may result in other diseases. For example, mutations in LARS2 have been reported to cause hydrops, lactic acidosis, sideroblastic anemia, and multisystem failure in a female newborn [TWNK have been reported in autosomal dominant progressive external ophthalmoplegia  and in three autosomal recessive disorders.Genetic testing can contribute to clarify the diagnosis, but it is also complicated by the genetic heterogeneity of PRLTS . In spital signs , 17–19,  to date , 9, 14.  newborn . Also, dTWNK gene codes for the 684-aa monomer of the mitochondrial DNA helicase twinkle, which localizes to the mitochondrial matrix and is thought to play a key role in mtDNA replication [TWNK gene. Whereas there is no overlap between the mutations causing dominant or recessive phenotypes, this overlap does exist among the recessive diseases [TWNK.The lication , 37. Thelication . The linlication . Most mulication , 41. Thelication , 41. On lication . They relication  and hepalication , althouglication –44, wherlication –14, as adiseases . For exadiseases , but alsdiseases . Moreovediseases  and in Pdiseases . It is cTWNK mutations will manifest neurological signs in adulthood. Recessive disorders caused by TWNK mutations constitute a phenotypic continuum, so that establishing genotype–phenotype correlations is a challenging task that still needs identification and molecular and clinical characterization of novel cases.Genetic testing is a useful tool to confirm a tentative clinical diagnosis of Perrault syndrome, although the genetic cause remains unidentified in over half of the cases. Studies on genotype–phenotype correlation from the hitherto reported cases indicate that all patients with Perrault syndrome caused by Additional file 1: Table S1. Relevant mutations obtained by whole-exome sequencing of subject II: 3.


Chihuahua cheese is a traditional cheese produced in Northwest Mexico that is consumed shortly after production. Cheeses prepared during autumn, winter and summer were collected from five dairies, and analyzed to determine seasonal influence on proximate analysis, texture profile and the microbiological dynamic during a ripening period of 270 days. Coliforms, coagulase-positive staphylococci, molds, yeast, as well as presumptive mesophilic lactobacilli, thermophilic lactobacilli, lactococci, thermophilic cocci and enterococci, were enumerated by plate count on selective agar. Manufacturing dairy had an effect on Chihuahua cheese composition and texture profile. Seasonality influence on the microbial dynamic was observed, since the highest initial counts of coliforms (5.14 log CFU/g), coagulase-positive staphylococci (4.13 log CFU/g) and mesophilic lactobacilli (7.86 log CFU/g) were detected on summer samples. Also, ripening time affected the survival of coliforms and presumptive lactococci after 270 days (1.24 and 5.89 log CFU/g respectively) while from day 90th, coagulase-positive staphylococci were absent. Microbial changes and seasonal influence provide information on the microbiota that can influence the sensorial characteristics of Chihuahua cheese. Cheese is the result of the metabolic action of the microorganisms already present or added to milk , and theConsumption of cheeses prepared with raw milk represents a health risk, due to the presence of pathogenic microorganisms; but during ripening, their presence or elimination is determined by such factors as moisture content, salt concentration and pH . The comIn Mexico, Chihuahua cheese is the fifth type of cheese manufactured nationally, with an annual production in 2017 of 40,700 tons ; it is pThere are no reports on the microbiological changes that occur during aging of Chihuahua cheese, and the existing reports have been focused only on the study of physicochemical changes that occur in a maximum period of 16 weeks ,14. The Blocks of Chihuahua cheese manufactured with raw milk without the addition of starter cultures or chemical additives were obtained from five artisanal dairies (A–E). Cheese manufacturing plants were visited in one occasion during autumn, winter and summer (2013–2014) and included two Mennonite farmers (B and C) and three dairies operated by non-Mennonite farmers . Cheeses were manufactured by a process that is based from the beginning to the end, on the experience of each producer . SamplesChemical analysis of Chihuahua cheese was done using AOAC methods  for moisv/v); the external layer of the cheese was discarded and a portion of 10 g was taken. The sample obtained was placed inside of a sterile plastic bag and 90 mL of sterile sodium phosphate buffer  were added. A food homogenizer  was used during 1 min to get a uniform mix, and was used to prepare decimal dilutions in PBS which were used to inoculate specific media for each microbial group.Cheeses were analyzed 24–48 h after their elaboration (referred as time 0), once every 30 days during 6 months, and finally at 9 months of ripening (270 days). For aging, samples were vacuum packaged and maintained at 6.5 °C. Prior to microbial analysis, cheese surface was aseptically cleaned with ethanol 70% (w/v) (1.5 mL/100 mL) tartaric acid and incubated for 5 days at 25 °C. Presence of coagulase-positive staphylococci was determined spreading 100 µL of each dilution on Baird Parker Agar plates  enriched with egg yolk tellurite emulsion incubated at 37 °C for 2 days. Presumptive colonies were tested for catalase, coagulase and Gram strain, in order to identify them as coagulase positive staphylococci [Enumeration of total coliforms was done using Violet Red Bile Agar  incubated on aerobic conditions during 24 h at 37 °C; molds and yeast were determined aerobically on Potato Dextrose Agar  acidified with 10%  for 2 days at 25 for mesophilic and 42 °C for thermophilic species. Also, presumptive enterococci were determined using Kanamycin Esculin Azide Agar, or Bile Esculin Agar  incubated on aerobic conditions during 2 days at 37 °C [For the enumeration of LAB groups, plates of MRS (for determination of presumptive lactobacilli) and Elliker Agar (for determination of presumptive lactococci), were inoculated with 100 µL of the dilutions prepared from cheese sample . Plates were incubated on a reduced oxygen atmosphere . Regarding springiness, there were differences among seasons , but not between dairies (p > 0.05). The only parameter that presented highly significant difference among seasons  and dairies was chewiness (p < 0.01).Chihuahua cheese is considered a semi-matured cheese, with only a short period of ripening before being offered to the consumer (one month) . It is uCheese texture is the result of a complex interaction of factors such as cheese composition, manufacturing process and ripening conditions. Considering that cheese was manufactured with raw milk, conditions such as herd race, lactation time and quality of the animal feed, will influence cheese properties . The rheMicrobiological changes during aging promote the abundance and diversity of distinctive groups of microorganisms on the cheese matrix , whose mp < 0.05). Changes in the LAB groups during the 270 days of analysis, are included in Lactobacillus species. Based on statistical analysis, season of cheese production was the main factor that affected the dynamic of all LAB groups analyzed; a strong influence of ripening time (p < 0.05) was observed only in the cases of presumptive mesophilic lactobacilli, lactococci and thermophilic cocci.Statistical analysis of LAB microbial counts considering all samples collected  showed that counts of all LAB groups were affected by the season in which the sample was obtained  was observed in the case of thermophilic lactobacilli and presumptive enterococci, as can be observed in In addition to ripening time and season in which cheese is manufactured, variation between dairy producers (p < 0.05). The highest counts were observed on cheeses from dairies A and E (6.9 log CFU/g), while the lowest was of factory C (5.8 log CFU/g). The presence of this microorganism in cheese has been associated with the use of raw milk for cheese manufacturing [Levels of presumptive enterococci were strongly influenced by season, the manufacturing dairy and maturation time  at early stages of ripening; but the cheese produced in autumn, had the lowest counts of presumptive mesophilic and thermophilic lactobacilli as well as lactococci counts  of all dairies tested. On the contrary, there is no explanation of why cheeses from different dairies  had similar pattern of some microorganisms, as was the case of cheeses A and C from autumn that had undetectable counts of presumptive lactococci and thermophilic cocci after 6 months of maturation, and the lowest levels of presumptive mesophilic lactobacilli at the end of the study.p < 0.05), as shown in Raw milk from a healthy udder should be sterile, but microorganisms from different sources are incorporated into milk ,3,31. CeElevated counts of coliforms on freshly prepared cheeses (time 0) were observed on summer samples, whose media of counts was 6.9 log CFU/g; followed by the ones sampled during winter (6.4 log CFU/g) and autumn (5.9 log CFU/g). From all the 15 samples, cheese B obtained during summer sampling had the highest count of coliforms (7.6 log CFU/g), while the lowest was cheese C manufactured during autumn (5.2 log CFU/g). Although coliform bacteria are common members of microbial community of cheeses made from raw milk, their presence in cheese is mostly associated with deficient hygienic practices during milking and manufacturing ,30, unrep < 0.05), on microbial count of total coliforms. In 2O2), in addition to the physicochemical changes produced in the cheese matrix [There is a strong influence of ripening time and dairy plant . After 60 days of maturation, coagulase-positive staphylococci were undetectable in cheese C from winter; and after 90 days on autumn cheeses A, B, C and D, and in winter cheese B. The trend of coagulase-positive staphylococci counts over time can be observed in Highest levels of coagulase-positive staphylococci were noticed on all cheese samples at the beginning of the ripening stage; cheese samples from dairies D and E manufactured during autumn registered the highest counts of this microbial group , as well as cheese manufactured during summer on farm E (6.6 log CFU/g). Like coliform, coagulase-positive staphylococci levels were drastically affected by the maturation time and were also related to the dairy plant where the cheese was manufactured , but season had no effect (p = 0.09). On freshly prepared cheeses, the highest counts of molds were observed on samples from cheese C prepared during autumn (5.9 log CFU/g), while the lowest were obtained from winter cheese E (1.6 log CFU/g). At the end of the ripening period analyzed, molds were undetectable on cheeses A, D and E from the autumn, but in cheese B from winter, counts were high (5.0 log CFU/g). Similar profile of molds during ripening was observed in Canestrato Pugliese cheese [The population of yeast and molds increased during the first 60 days of ripening, time at which they reached their maximum level ; however, while counts of molds tended to decrease after that time, yeasts counts slightly decreased and remained constant during the rest of the maturation period. Strong influence of factory and ripening time was observed on counts of molds (p < 0.05) but not by the ripening time (p = 0.09). Initial yeast counts from cheese C manufactured during autumn had the highest counts (6.7 log CFU/g), while cheese D from winter had the lowest (3.9 log CFU/g). After 270 days, elevated counts in cheese E from winter were observed (6.4 log CFU/g), while they were absent in cheese from dairy A manufactured in summer. In other cheese varieties, such as Canestrato Pugliese, a similar behavior on the dynamics of yeast and molds was reported [The counts of yeast were affected by season . Further investigation is necessary in order to identify strains with high potential to be used in the elaboration of Chihuahua cheese from pasteurized milk.Chihuahua cheese manufactured with raw milk in different dairies located in the state of Chihuahua, Mexico, showed differences in proximate analysis depending on the manufacturing location, and their texture profile after four months of ripening was different only for chewiness. Seasonality was an important factor to determine the initial counts of indicators and LAB microbial groups.


Surgery is frequently recommended for removing large tumors, but it is associated with a high risk of cancer recurrence and metastasis. Here, we applied PPTT before surgery, which showed improved treatment for large tumors. We divided the animals (eight cats/dogs) into two groups: Group I (control), where three cases were solely treated with surgery, laser, or AuNRs alone, resulting in recurrence and metastasis; and Group II, where animals were treated with PPTT before surgery. In Group II, four out of the five cases had tumor regression without any recurrence or metastasis. Interestingly, we observed that applying PPTT before surgery displayed reduced bleeding during tumor removal, supported by histopathology that showed altered blood vessels. In conclusion, our study showed that applying AuNR-assisted PPTT (AuNRs-PPTT) before surgery could significantly affect blood vessels inside the tumor, leading to a decreased amount of bleeding during surgery, which can potentially decrease the risk of metastasis and blood loss during surgery.For localized tumors, gold nanorod (AuNR)-assisted plasmonic photothermal therapy (PPTT) is a potentially effective alternative to traditional surgery, in which AuNRs absorb near-infrared light and convert it to heat in order to kill cancer cells. However, for large tumors (volume ≥ 20 cm The N by PPTT . During  by PPTT ,13. In a by PPTT ,8. There by PPTT . 3), PPTT could be hard to achieve because of an uneven distribution of injected AuNRs that causes an inhomogeneity of the heat in the tumor. In these cases, surgery is usually recommended to remove primary solid tumors. However, the surgical resection of primary breast cancer tumors commonly has a risk for metastatic recurrence [For large tumors  × 6 (±1) nm (length × width) were used in this study, as they showed enhanced efficacy of PPTT . AuNRs w2 intensity for 2 min). The optimized conditions were conducted multiple times (2 weeks apart) until complete regression via apoptosis was shown, and this apoptosis proved to be better than necrosis [3 by combining surgery with PPTT. In our earlier studies, we optimized the PPTT conditions for treating dogs and cats, including the AuNR dosage and laser conditions  and were followed by surgery after the last PPTT session. The decrease of bleeding might be explained by the histopathology of the tumor bed vasculature B,D. WithIn addition, the regimen of PPTT before surgery has shown to be effective for achieving complete tumor regression, as shown in 2, iron, Cr3+-doped zinc gallate, and silver, could accumulate in the lung, and might cause toxicity [Three cases among the five in Group II died a few months after PPTT and surgery treatment because of viral pneumonia, but evidence of tumor recurrence or metastasis was not observed. Earlier, we have shown in our study on mice that there was no toxicity after 15 months of AuNRs injection, whose bio-distribution mainly locates at the liver and spleen . Howevertoxicity ,23,24,25In this report, we observed that PPTT affected the tumor blood vessels that, in turn, decreased the blood flow inside the tumor. The reason is still not fully understood. Previous reports have shown that AuNPs could affect blood vessels and tumor angiogenesis ,27,28. I4  was mixed with 5 mL of 0.20 M cetyltrimethylammonium bromide , followed by adding 250 μL of 4 mM AgNO3 (Sigma-Aldrich Co.) and adjusting the pH of the solution to be 1–1.15 by 37% HCl. Then, 70 μL of 78.8 mM ascorbic acid (Sigma-Aldrich Co.) was added to the solution until the solution became clear. A total of 15 μL of 0.01 M ice-cold NaBH4 (Sigma-Aldrich Co.) was injected into the growth solution immediately, and the solution was left unstirred for 6 hours. To remove the extra CTAB and prepare for surface modification, the AuNRs were centrifuged at 19,000 rcf for 1 hour, and the pellet was redispersed in deionized water and centrifuged at 14,000 rcf for 15 min. The AuNRs were rinsed with water, then conjugated with different surface ligands (polyethylene glycol (PEG) thiol and Arg−Gly−Asp (RGD) peptides). For surface modification, methoxy PEG thiol  was added to AuNRs and stirred overnight to achieve a concentration of around 1000 PEG molecules per AuNR. For preparation of the AuNRs@RGD, the PEGylated nanoparticles (1 nM) were treated with RGD (1 mM) to achieve 10,000 ligands on each AuNR. Afterward, the solution was kept overnight to be shaken at normal temperature, and the extra ligands were removed by centrifugation. A UV–VIS spectrometer was used to confirm the conjugation. AuNRs were prepared according to the seedless method . BrieflyA JEOL 100 CX transmission electron microscope (TEM)  was used to measure the size and homogeneity of the samples. A Cary 500 UV–VIS spectrometer  was used for measuring the absorbance of the AuNRs. To characterize the surface conjugation with PEG, a ZetaSizer 3000 HAS  was used for measuring the surface Zeta potentials. In addition, Ellman’s reagents (Sigma-Aldrich Co.), which react with free-Thiol groups , were used to quantify the number of PEG molecules boun to the surface of the AuNRs. All animals were handled in accordance with Association for Assessment and Accreditation of Laboratory Animal Care and Office of Laboratory Animal Welfare guidelines under the direction of the Institutional Animal Care and Use Committee at Cairo University. The pet animals were admitted to the Department of Surgery Clinic of the Faculty of Veterinary Medicine at Cairo University. This research was approved by the Institutional Animal Care and Use Committee (CU-IACUC) Cairo University (code: CU II F 9 16).All pets’ owners claimed that their animals did not receive any treatment before their arrival at the university. Written informed consent was provided by the owners of the pets for the treatments. Eight female animals were treated in this study, including two canines and six felines, with a total of 21 tumors with varied grades (I to III). The tumor dimensions were measured using calipers. Histopathology tests were used to diagnose the tumor types/grades. At the tumor site, the animal’s hair was shaved, and subsequently, radiographic recordings were taken with an X-ray machine . The radiographic setting factors were 58–70 kVp, 10 mAs, and a 90 cm focal spot film distance. The radiographic exposures were conducted dorsoventrally and right laterally. Blood loss was quantified by measuring the blood volume and weighing surgical sponges used for blood collection before and after the surgery. General anesthesia was applied for animals during the surgical mastectomy and postsurgical application of PPTT. Under general injectable anesthesia, each animal was pre-medicated with atropine sulphate  and xylazine , and then anesthesia was induced using ketamine HCl  and maintained by ketamine HCl ,33. 2 and a spot size of around 5.6 mm2. An effective dose of AuNR solution (7.5 nM AuNRs) for each 100 cm3 was used for 2 min, and the amount used was scaled up based on the volume of the tumor, and then injected directly into the tumor. Five minutes after injection, the entirety of the tumor was irradiated with the laser. The AuNR concentration was decreased by 50% for each subsequent treatment. The temperature increase of a tumor during the laser irradiation was measured by placing a 33-gauge hypodermic thermocouple  needle directly inside the tumor (42–44 °C). Each animal was subjected to three sessions of PPTT treatment in 2 week intervals using an 808 nm diode laser with a power of 0.5 W/cmThe detailed pathologic evaluation of tumors was conducted by members of the pathology department of the Faculty of Veterinary Medicine at Cairo University. Histopathological analysis was performed on 5 μm sections from tumor tissue that were fixed in 10% buffered formalin. The samples were stained with H&E to assess pathology. 3 [3), we applied PPTT before surgical resection to naturally occurring tumors in the mammary glands of dogs and cats. Five cases were treated with this regimen and showed complete remission without any recurrence after therapy. Three cases died in the few months following treatment, but in two cases from three, there was no evidence of any tumors upon examination . Histopathology results showed a decrease in cancer grades compared to before (variant grades from 1 to 4) and after 2 weeks of treatment via PPTT and surgery (grade 0). X-ray diffraction revealed an absence of metastasis 1–2 years after treatment. In conclusion, our study demonstrates the feasibility of applying PPTT before surgery to large tumors in dogs and cats. Applying AuNRs-PPTT before surgery in treating large tumors could significantly affect blood vessels inside the tumor and potentially avoid the risk of bleeding during surgery. PPTT could be incorporated before the surgery to decrease the bleeding and potentially avoid the risk of bleeding during surgery that could lead to excessive blood loss and metastasis. Surgery is often used for tumor removal; however, it might trigger metastasis . PPTT co3 . TherefoThis work has been filed in the US-Patent Publication of US20190008964A1.


Degenerative cervical myelopathy (DCM) is caused by degenerative or congenital changes to the discs and soft tissues of the cervical spine, which leads to chronic compression of the spinal cord. The current treatment for moderate to severe DCM consists of surgical decompression, which, while effective in most cases, can result in neuroinflammation and spinal cord reperfusion injury, leading to perioperative neurological complications and suboptimal neurological recovery. The primary objective of this study was to assess, in a translationally relevant animal model of DCM, the efficacy of perioperative methylprednisolone (MP) in enhancing neurological recovery and to evaluate its effect on the inflammatory response following decompression.DCM was induced in C57BL/6 mice. Briefly, an aromatic polyether material was implanted underneath the C5-C6 laminae to cause progressive compression of the cervical spinal cord due to focal ossification. Decompressive surgery was undertaken at 12 weeks post initial biomaterial implantation. Animals received one dose of MP (30 mg/kg) or vehicle 30 min before decompression and at 2 weeks after decompression. Acute analysis of secreted cytokines and spinal cord microvasculature was complemented with immunohistochemistry for glial and neuronal cell markers. Locomotor outcomes were measured using the CatWalk system. The composition of circulating white blood cells was analyzed by flow cytometry.p < 0.05) and reduced the incidence of perioperative motor complications, without affecting the composition of circulating white blood cells. Histological assessment of the spinal cord showed significant neuronal preservation and a modest reduction in parenchymal inflammation.A single dose of MP before decompression significantly sped locomotor recovery (*Our data suggest that MP reduces perioperative neurological complications following decompressive surgery for DCM by protecting neurons from inflammation, without compromising the composition of circulating immune cells. We propose that MP, which is commonly used for neurological disorders including spinal cord injury, be considered as a perioperative adjunct to decompressive surgery to attenuate neurological complications. Degenerative cervical myelopathy (DCM) is an overarching term used to describe the most common forms of non-traumatic cervical spinal cord myelopathy . DCM, which increases in prevalence with aging, is caused by degenerative or congenital changes to the discs and soft tissues of the cervical spine, which leads to chronic compression of the spinal cord . ImportaDCM is associated with significant neurological dysfunction, including gait impairment, loss of manual dexterity, and pain . The curIn the present study, we hypothesized that reducing neuroinflammation following decompression for DCM would attenuate perioperative neurological decline and promote enhanced neural recovery. To examine this hypothesis, we used a mouse model of DCM that is associated with severe neuroinflammation . This moIn the present study, we examined the effectiveness of MP treatment to reduce inflammation following decompression in a mouse model of DCM at the C5-C6 level. We observed perioperative MP treatment accelerated locomotor recovery by preserving the number of neurons, while modest effects on inflammation were observed. There was also a reduction in the incidence of perioperative motor complications  following MP treatment. Importantly, no harmful side effects (including increased incidence of wound infection and death) or changes in the peripheral white blood cell composition were observed after MP treatment.The Animal Use Committee at the University Health Network  approved the study protocol, and experiments were carried out in accordance with the committee recommendations. Adult 8-week-old female C57BL/6 mice were purchased from the Ontario Council Institute (Canada) for use in this study. Investigators remained blinded to the treatment groups for the duration of the study.DCM was induced in mice as previously described . BrieflyAnimals were decompressed at 12 weeks following compression and randomly assigned to one of two experimental groups: (1) decompression with saline treatment  and (2) decompression with MP treatment (30 mg/kg). MP treatment was given intravenously (i.v.) 30 min before surgical decompression and 2 weeks following decompression  was used to assess locomotion of DCM and decompressed animals, as described previously . Stance Repeated blood sampling of mice was performed via saphenous vein puncture without anesthesia. Blood samples were collected using a 23G needle with ethylenediaminetetraacetic acid (EDTA)-coated tubes to avoid blood coagulation  and wereCervical spinal cord homogenates from a 0.3 cm long section of tissue centered at the compression area were prepared following transcardial perfusion with PBS, as previously described . Levels + cells were automatically quantified over a 3240 μm area centered at the lesion epicenter in gray matter using the cell counter plug-in from ImageJ, whereas Iba1+ cells were manually counted in the dorsal and ventral horns. The area of the dorsal and ventral horns evaluated for GFAP immunoreactivity was traced using ImageJ software in a constant square region of 89 × 89 μm, as described before . Ho. Ho25]. Major surgical procedures can lead to alterations to the hemodynamic, endocrine, and immune functions of the body. Specifically, this leads to an initial activation of the peripheral immune system along with enhanced blood flow to muscle, liver, and ischemic organs . These c+ cells and astrogliosis within the spinal cord. Albeit modest, these changes could lead to a more permissive environment that will allow neuronal preservation and an improved rate of functional recovery. This was reflected by an increased number of neurons in the MP-treated group, as compared to saline-treated animals. Similarly, in experimental models of traumatic SCI and ischemic optic neuropathy, MP treatment has been shown to preserve the number of neurons from apoptosis . In. In+ celreceptor  or the fOur study has certain limitations that should be acknowledged. Firstly, although mouse models are commonly used in research, the composition of peripheral white blood cells between humans and mice is different. Human white blood cells are enriched in granulocytes and monocytes, whereas mouse white blood cells are rich in B and T cells . TherefoImportantly, the incidence of motor complications was reduced after decompression with MP treatment. Given that 1 out of 3 (34.9%)  patientsIn conclusion, the current study provides a deeper understanding of the peripheral immunological response following delayed decompression, and a detailed assessment of the role of MP in locomotor recovery following delayed surgical decompression. Larger studies, including prospective controlled trials in DCM patients, will be needed to better understand MP effects on the incidence of complications and enhancement of locomotor recovery, as well as the potential use of other anti-inflammatory drugs.


From the perspective of Central and South America, the peopling of the New World was a complex process lasting thousands of years and involving multiple waves of Pleistocene and early Holocene period immigrants entering into the neotropics. These Paleoindian colonists initially brought with them technologies developed for adaptation to environments and resources found in North America. As the ice age ended across the New World people adapted more generalized stone tools to exploit changing environments and resources. In the neotropics these changes would have been pronounced as patchy forests and grasslands gave way to broadleaf tropical forests. We document a late Pleistocene/early Holocene stone tool tradition from Belize, located in southern Mesoamerica. This represents the first endogenous Paleoindian stone tool technocomplex recovered from well dated stratigraphic contexts for Mesoamerica. Previously designated Lowe, these artifacts share multiple features with contemporary North and South American Paleoindian tool types. Once hafted, these bifaces appear to have served multiple functions for cutting, hooking, thrusting, or throwing. The tools were developed at a time of technological regionalization reflecting the diverse demands of a period of pronounced environmental change and population movement. Combined stratigraphic, technological, and population paleogenetic data suggests that there were strong ties between lowland neotropic regions at the onset of the Holocene. Lack of knowledge of the Paleoindian period in southern Mesoamerica, a critical early migration bottleneck, has impeded our understanding of the peopling of the Americas and how early New World migrants adapted to emergent tropical environments. Here we present new archaeological and chronological data from stratigraphic excavations in unusually well preserved rockshelter contexts in southern Belize. We securely reassign the chronology of a stone tool technocomplex to 12,000–9,300 years ago linking it to changes in stone tool technology in North America and tropical Central and South America. This is the first securely dated Paleoindian tool technocomplex for southern Mesoamerica.Our data indicate the late Paleoindian period is characterized by movement away from technological uniformity towards increasing diversification and the establishment of regional traditions and support genetic evidence of strong relationships between Central and South America during the Paleoindian period. Prior to 13,000 calendar years before the present (BP), bands of ice age humans migrating southward from the temperate forests and plains of western North America (NA) crossed the bottleneck of the Isthmus of Tehuantepec to arrive in southern Mesoamerica ,3 in mosDuring archaeological excavations we recovered what we show below to be temporally diagnostic late Paleoindian bifacial tools. They come from stratified contexts in two well-dated rockshelters in southern Belize  where weIn southern Mesoamerica even rudimentary absolute chronologies dating Paleoindian and Archaic period stone tools are lacking. This stands in contrast to the much later Classic Period Maya where chronologies have been refined over the past century based on radiocarbon dates and hieroglyphic texts . With raBased on comparisons to NA and SA, the oldest bifacial tools found in Central America (CA) are basally thinned lanceolate and fluted Clovis projectile points ,24. The In southern Mesoamerica one of the earliest attempts to develop a Paleoindian and Archaic lithic chronology was the Belize Archaic Archaeological Reconnaissance (BAAR), directed by R.S. MacNeish, an iconic figure in New World archaeology. BAAR assigned undated examples of what we call the Lowe complex to their Lowe-Ha  and Sand Hill  phases, with some examples labeled as “Pedernales-like”, “Madden Lake-like”, and “Bulverde-like” –43, in rLowe complex. They have been recovered from diverse environments ranging from brackish coastal and swampy inland forests in the north, colluvial grasslands and alluvial gallery forests of the Belize River Valley, granitic pine barrens, and upland sandstone foothills and volcanic interior valleys of the southern Maya Mountains. Prior to this study, only seven were recovered during archaeological excavations, and of these only one was found in association with datable organic material.Since the 1990s Lowe, and a suite of technologically similar bifaces, have become synonymous with the Late Archaic . These tools are found exclusively in Belize and are called Lowe, Sawmill, Allspice, and Ya’axche’ types . To dateThough originally described as Paleoindian tools dating to 11,000–8,000 BP  and thouWe present results of excavations at two rockshelters in southern Belize where we recovered Paleoindian artifacts from well dated stratigraphic contexts. These rockshelters are located 35 km apart along perennial rivers in areas with rich soils and gallery forests. We recovered in stratigraphic context two complete and one partial Lowe complex bifaces and one distal biface fragment with features diagnostic of Lowe complex. In this section we briefly describe the two sites, excavation contexts, and age models.Pachychilus sp. (jute) snail shells, which are found in abundance in the river below and were culturally modified to facilitate consumption of the snail by removing distal spires [jute rich midden, which grades into a red clay with decreasing frequencies of jute shell. The red clay covers a yellow clay containing few cultural materials directly above limestone bedrock. The upper 30-35cm of the jute midden was disturbed in antiquity though there is no indication this was a result of fluvial erosion. Instead this was likely by horizontal removal of the midden during the Classic Period . Portions of this midden were used as a plaster applied to the back wall of the rockshelter as a frieze or mask containing jute, ceramics, lithics, and faunal bone. A single date on wood charcoal embedded in the plaster dates to calBP 10,275–10,190 2σ , indicating preceramic and ceramic-bearing contexts were mixed into the plaster material.Tzibte Yux Rockshelter (TY) is a small (37m x 4.5m) rockshelter located 8m above the Rio Blanco and 1.2km from the Classic Maya center Uxbenká . Sedimenl spires . The 110jute midden below the disturbance is highly compacted with sediments in chronological order beginning around 9,000 calBP. Seventeen 14C AMS dates . This is one of a sequence of five dates directly above and below the point ranging from calBP 8,949–8,607, 2σ  to calBP 12,391–12,087 2σ . In units 5/6 we recovered a distal end fragment of a large biface . This date is also part of a series of five sequential assays above and below the biface.A Lowe point  was recoe biface  with steσ and 2σ model errors post-8,500 calBP with age reversals in the disturbed jute midden, but excellent agreement and low 1σ and 2σ model errors in the consolidated lower levels of the jute midden and red and yellow clay layers. The basal age of Unit 7 is very early, calBP 16,939–16,474, 2σ . A third concordant date on a carbonized seed from 10cm above  was not modeled because of its larger error due to small sample size but is included in To test the integrity of our chronology we developed depositional models for both units at TY . These cMayahak Cab Pek (MHCP) rockshelter is located in an interior valley of the Maya Mountains in the Bladen Nature Reserve, a protected wilderness area where there has been minimal modern human disturbance of archaeological sites. Excavations of dry midden rich stratified deposits show that cultural use began prior to 12,000 BP and continued through the Maya Classic Period, until 1,000 BP. The shelter is east-facing with a 20m high limestone face containing caramel-colored chert lenses and cobbles. The shelter is approximately 20m above the present-day stream bed of an unnamed tributary of the Bladen Branch of the Monkey River and has cultural deposits over 3m deep.jute shell concentrations comprise up to 50 percent of the matrix.MHCP Unit 1  was a 2.jute concentrations also drop well below 30% of matrix except for at the ceramic-aceramic boundary, where they are the most abundant of any period. The overall size and the number of cobbles, expedient tools, and faunal remains tend to increase with depth. In levels below 9,000 BP sediments became increasingly silty. Within the silty matrix we found a smaller frequency of large chert flakes and crude bifaces. These silty matrices terminate on boulders intermixed with reddish clays and decaying limestone devoid of any cultural materials.The aceramic levels are organic rich black silt to silty-loam fine grained matrix with cobble clasts of limestone mixed with crude porphyritic igneous flaked tools, mostly choppers and hammer stones with battered edges. Artifact and faunal concentrations are moderate and Contexts with human remains from at least fourteen individuals have been recovered from Unit 1 and range in date between 1,000 and 10,500 BP. Although the analysis of these remains is not reported here burial MHCP.14.1.6, the disarticulated skeleton of a female, directly was dated on bone collagen to 9,300 BP  who is cσ  and one 3cm below, although out of sequence and treated as a model outlier, dating to calBP 11,701–11,266, 2σ . Conservatively, we could associate the minimum age of the point with the younger of these two dates. The partial Lowe biface was found 90cm to the east of the complete point, in the same stratigraphic level but not in direct association with any datable materials. Charcoal from 2cm below and 66cm south of the biface dates to calBP 8,609–8,451  but is likely intrusive or dating a context that is younger than the biface based on the slope of the bedrock below and its location in the sequence of dates. Both points were found below the burial 2014.1.6 [One complete and one partial Lowe point  were recσ and 2σ errors low for all age-depth samples (>85%), with the exception of one outlier mentioned above.The depositional model of MHCP Unit 1 , Table 1All our field research, exportation of archaeology samples, and destructive analysis of charcoal samples was done with permits from the Belize Institute of Archaeology (IA/H/2/19(06)), and the Belize Forest Department (FD/WL/1/19(05)) issued to KMP.AMS dates on charcoal were prepared using published Acid-Base-Acid, combustion, and graphitization methods at the Pennsylvania State University (PSU), University of California at Irvine (UCI), and University of New Mexico  and meas10/ (k/k0) which allows for depositional variability over a wide range [0,p,D)) where k0 is the base k parameter, p is the interpolation rate, and D is the prior distribution for v, and the sedimentary sequence defined in terms of depth in cm. For example, P_Sequence) defines k0 = 1cm–1, interpolation rate = 2 cm–1 (output every 5 mm), and k allowed between a factor of 10−2 and 102.Age models were developed using stratigraphic data from TY and MHCP with depths adjusted from cm below datum to cm below surface to account for surface and basal slope in excavation units. Bayesian Poisson depth models were produced in OXCAL 4.3  using pude range . It is sσ, and the deposition of the technologically related unifacially beveled biface fragment from TY minimally as calBP 12,399–12,034 2σ. There is good agreement in Poisson depositional models for all excavation contexts containing Lowe complex tools with dates on both complete Lowe points at ~10,000 calBP.Chronologies from both rockshelters bracket the minimum age of the TY and MHCP Lowe points as calBP 10,223 and 9,300 2At the end of the Pleistocene, bifacial stone tool technologies were widespread across the New World ,62. By 1The most distinctive NA technological feature in the Lowe complex is unifacial beveling on alternate edges. This appears first in the Folsom ultrathin bifacial knives as a sharpening technique with steep edge angles, centered on the Great Plains but extending southward into Chihuahua, Mexico from 12,610–12,170 calBP ,69. ThisFollowing Folsom, but in the southeastern US, Dalton  may be the first projectile points with unifacial beveling on alternate edges . It is aExperimentation with Dalton replicas suggest that beveling may promote rotation and accuracy of thrown spears  when larBasal thinning or fluting is both a NA and SA Paleoindian technology, but fluting or thinning on only one side of a biface appears to be limited to CA and SA. NA Clovis and Folsom are distinctly fluted, as are many Dalton points , with siSingle side thinning or fluting is associated with Lowe complex and with Paleoindian points in lower CA and SA. Both fluting and longitudinal thinning flake removal are observed on FPP and El Inga points , frequenBarbed bifaces, some of which have the square stems and unifacial beveling on alternate edges, are characteristic of the Lowe complex. In NA barbed bifaces do not appear until just before 10,000 BP . In SA tThe suite of traits present on Lowe complex points suggests that they served several functions. Barbs are characteristic of penetrating spears but can also be used as hooks, a handy tool in a rainforest to harvest or cut palm fruits or vines. Unifacial beveling on alternate edges with minimal thinning is one way to maintain a durable cutting edge, but on thin aerodynamic projectiles may increase accuracy. Function was likely dependent on size, thickness, and degree of rejuvenation and reduction, and during the use-life of a biface it may have served multiple functions. Further, in broadleaf forests of the early Holocene some projectiles may have been less effective as weapons given the density of trees and foliage.Transmission of Paleoindian stone tool techno-knowledge from NA to CA and SA has been proposed and discussed  but can Southern Native American” or (ANC-A) and “Northern Native American” (ANC-B) [Genetic data reveal that all Native Americans derived from a single homogeneous ancestral population that diversified sometime between 16,000–13,500 BP, likely south of the NA ice sheets . The res (ANC-B) ,95,96. T (ANC-B) . The ANC (ANC-B) . These d (ANC-B)  before tThe Lowe tradition is a technological lithic complex unique to southern Mesoamerica. It shares features with contemporaneous stemmed point types found primarily in tropical areas of SA and lower CA, but is also related to ancestral Paleoindian complexes in NA. Its development corresponds with a pattern of regionalization and diversification reflected in similar age tools found throughout the Americas  at a timLowe complex tools are basally thinned, stemmed, and barbed bifaces. They are a flexible tool type well suited for the diverse set of early Holocene environments like those that emerged in Belize. In particular, tropical forests offered new resources that were of economic importance including a wide variety of palms, tubers, and vines ,100. BroWe provide a chronology for the Lowe complex that is potentially 2700 years long, far exceeding any existing New World Paleoindian technocomplex. Unifacial beveling on alternate edges, not found on other South or Central American points and not found on earlier Clovis or lanceolate types, may be the earliest characteristic of Lowe complex. Our biface tip from TY dates to calBP 12,399–12,034 and is steeply beveled. Our Lowe point type bifaces (n = 3) all date between calBP 10,223 and 9,300, indicating they are Late Paleoindian in age. This suggests that Lowe type bifaces were manufactured over a shorter time-period, while the overall complex that includes opposite edge beveling technology of large bifaces in Central America may have lasted significantly longer, though it is less clearly defined. In this paper we argue that the Late Paleoindian period is a time of diversification in tool types as an adaptation to the range of environmental conditions found in southern Mesoamerica as well as increasing reliance on plant resources, and that this specialization is a feature you see very early on in the occupation of the neotropics, and likely in adjacent regions of South America.We lack any chronological data for Sawmill, Allspice, or Ya’axche type points which we include in the Lowe Complex based on shared characteristics we describe above. We would note the similarities between both Sawmill and Ya’axche type points and some examples of stemmed el Jobo from Venezuela , and el This reappraisal of the chronology of Lowe complex fills an important gap in the prehistory of southern Mesoamerica, a particularly diverse resource rich tropical landscape. It is also the first securely dated Paleoindian tool for this region. Shifting the chronology of the Lowe complex from 4,500 BP to as early as 12,000 BP leaves a notable temporal gap with no bifacial technocomplex for CA for the Early to Late Archaic . Middle/Late Archaic bifacial technocomplexes are notably absent from the well dated sites across lower CA and SA. In southern Belize formal bifacial tools are not present in Archaic assemblages, as is the case for El Gigante . This is