Mystacodon selenensis) indicates that the diversification of Crown Cetacea began before the Late Eocene and the divergence of Crown Delphinidae as early as the Middle Miocene. The evolution of cetaceans, from their early transition to an aquatic lifestyle to their subsequent diversification, has been the subject of numerous studies. However, although the higher-level relationships among cetacean families have been largely settled, several aspects of the systematics within these groups remain unresolved. Problematic clades include the oceanic dolphins (37 spp.), which have experienced a recent rapid radiation, and the beaked whales (22 spp.), which have not been investigated in detail using nuclear loci. The combined application of high-throughput sequencing with techniques that target specific genomic sequences provide a powerful means of rapidly generating large volumes of orthologous sequence data for use in phylogenomic studies. To elucidate the phylogenetic relationships within the Cetacea, we combined sequence capture with Illumina sequencing to generate data for  Cetaceans  have undergone the most dramatic morphological transformation of all mammals, having originated from a clade of terrestrial even-toed ungulates nt}50 Ma . The orint}50 Ma . Numerount}50 Ma .After their transition to the sea, cetaceans further diversified into two groups with unique adaptations. Toothed whales (Odontoceti) acquired echolocation to hunt using ultrasonic pulses and a highly specialized inner ear, whereas baleen whales (Mysticeti) lost their teeth and evolved a novel keratinous material for filtering aggregate prey . Modern Although the evolution of cetaceans from an even-toed “ungulate” ancestor is well understood, there are aspects of their systematics that have proven more challenging. This is particularly the case for relationships within cetacean families, some of which remain problematic . For exaadiation . Particutes e.g.  and litttes e.g. .Mesoplodon , have not robustly resolved species-level relationships  and the Ziphiidae , have also been problematic to disentangle, with several conflicting internal nodes between studies . For exaps e.g., , 2014. Tthe wild .The release of several cetacean genomes and transcriptomes in recent years has made it possible to detail the molecular differences between species, as well as identify variable regions or sites for use in population-level and phylogenetic studies . In addiDelphinus) and bottlenose dolphins (Tursiops). Importantly, our large data set also allowed us to unravel the pattern of molecular rate variation in cetaceans, and thus obtain a precise species-level timetree of cetacean divergences using our complete data set.To resolve uncertain relationships among cetacean lineages, we generated new sequence data for 3191 protein-coding genes in 68 species of cetaceans, two hippopotamids and three ruminants. By supplementing these data with available sequences from 18 taxa  obtained from a combination of published genomes, transcriptomes and other data sets, our final alignments spanned 100 individuals from 77 cetacean and 12 outgroup species. We used more than 6.5 million bp of aligned sequence from 38,167 exons of 3191 genes to construct a large-scale well-supported species tree of Cetacea using both concatenated and coalescence methods. Every node was well-resolved, including those within Ziphiidae and the problematic Delphinidae (oceanic dolphins). Our results resolve a long debate over the contentious relationships among species within the subfamily Delphininae, which includes some of the most recognizable cetaceans, such as common dolphins  of cetaceans, two species of hippopotamuses, and three species of ruminants Appendix . These DTursiops truncatus genome version turTru1 (as compared with protein-coding genes from Homo sapiens and other available laurasiatherians) was compiled using Ensembl v. 75. We included genes belonging to specific gene ontology (GO) categories based on genes of interest and added these to a larger subset of randomly selected genes. Our target loci covered a range of GO categories ranging from “regulation of centrosome cycle” to “lung development”. Official HGNC gene names were used to search the coding sequence (CDS) databases of two delphinid genomes, Tursiops truncatus (version Ttru_1.4) and Orcinus orca (version Oorc_1.1) on NCBI GenBank  were filtered using a relaxed “4” setting. We then initiated a pilot study of four cetaceans , and the pygmy hippopotamus (Choeropsis liberiensis), to determine the success of target sequence capture before proceeding further.Biotinylated RNA baits (MYbaits) of 100 nucleotides in length were designed by MYcroarray  using these 10,271 individual CDS sequences. Baits were evaluated via a MYcroarray in-house algorithm, and those with potential to cause cross-hybridization to multiple targets , and raw reads were cleaned by removing adaptors and low-quality bases using Trimmomatic 0.36 . A totalsettings . To idenxillifer , as wellcaballus . All conO. orca and T. truncatus genomes; these exons were then introduced in order for the whole alignment to remain in the correct reading frame. In addition, we removed 65 genes (665 exons) from the overall data set if the gene was difficult to align or difficult to differentiate sequences from closely related paralogues. For the remaining 3191 genes, presence of premature stop codons and/or indels that were not multiples of three nucleotides were taken as potential evidence for the presence of a pseudogene and noted for further analysis.Each individual exon was aligned separately using mafft version 7  for a toPlatanista gangetica (South Asian river dolphin) and Balaenoptera omurai , two species for which we did not have capture data and for which a whole genome is not available. This consisted of a total of 72 sequences for P. gangetica  and 67 for B. omurai . See Supplementary Table S2 available on Dryad for a list with accession numbers of these sequences and their publications.We also added isolated sequences from NCBI GenBank for P. gangetica and B. omurai, whereas Dataset B excluded these sequences. For both data sets, we conducted three concatenated maximum likelihood analyses using RAxML v8.2  and 6,527,596 bp: Dataset A and Dataset B. Dataset A contained sequences from xML v8.2 : (i) unpxML v8.2 . We perfxML v8.2 . Two unpxML v8.2  to evaluxML v8.2 .P. gangetica and B. omurai, we excluded them from all gene tree analyses. We used ASTRAL-III v5.6.1 to generate a species tree using a multi-species coalescent model . In addition, we excluded species missing Hyperoodon planifrons, Phocoenoides dalli, Berardius arnuxii, P. gangetica, B. omurai). Operational taxonomic units (OTUs) were pruned from the topology generated from our concatenated analyses , and the resulting fixed tree with 85 taxa was used as an input for downstream analyses.For our divergence dating analyses, we reduced the subset of genes and taxa used. We used only genes with no evidence of pseudogenization , reducing the number of loci included to 3096. In cases where more than one representative of a particular species was present, we retained the more complete individual; however, two representatives were retained for O. orca (an odontocete) and B. acutorostrata (a mysticete) were compiled for each gene; however, in some cases, B. acutorostrata was not present and another mysticete was used. Using pairwise distances, this data set was divided into 3 and 10 partitions of 1032 and approximately 309 genes each, respectively, representing partitions ranging from slower to faster rates of divergence. The three-partition data set was further split into “first and second” and “third” codon positions (1st/2nd and 3rd CPs) for a total of six partitions. In total, we analyzed a 3-partition scheme separated by rate of divergence, a 6-partition scheme by rate of divergence and codon position, and a 10-partition scheme by rate of divergence.Due to the computational difficulties of analyzing each gene as a separate partition, we followed the procedure outlined in Divergence dating analyses were conducted using the software MCMCTree v4.9h, part of the PAML package . MCMCTre = 0.1 (fraction of species sampled) was used to construct the prior on node ages. These parameters lead to an approximately uniform density on node ages (Test runs of the program were carried out to ensure the convergence of the Markov Chain Monte Carlo (MCMC) chains and that enough likelihood samples had been collected for Bayes factors calculation. The birth–death process with ode ages . At thison model  for both of data .MCMCTree was used to estimate divergence times on the complete data set for the 3-, 6-, and 10-partition schemes using the autocorrelated-rates model as well as the independent rates model for comparison, with both models using approximate likelihood . All parr v. 1.7  to evalur v. 1.7 .ABCA4, PTPRZ1, TNC, COL12A1, HYDIN, APOB, CENPF, C2CD3, CEP152, and LRKK2). These genes account for a total of 87,864 aligned bp, with individual gene alignments included ranging between 5,202 and 15,381 bp. To directly compare the SortaDate genes with the complete data sets, the 10 genes were ordered from slowest to fastest evolving as above, and we partitioned the data sets into 3, 6, and 10 partitions. For the 3-partition data set, three to four genes each were included in three partitions from slowest to fastest. For the 6-partition data set, these partitions were split into 1st/2nd CPs and 3rd CPs. For the 10-partition data set, each gene was analyzed separately. To assess how uncertainty in time estimates differed between analysis of the whole data set and the 10 SortaDate genes, we used the infinite-sites plot  to Stenella attenuata 38219; Supplementary Table S1 available on Dryad) with an average of Tursiops and Orcinus genomes did not appear to affect the success of sequence capture, as 21.6 million reads were obtained for the ruminant Gazella arabica. For each sample, reads were assembled into Trinity contigs numbering from 11,156 (H. planifrons) to 575,798 (Mesoplodon carlhubbsi) with an average N50 of 310 bp. After reciprocal blasting of the contigs to the Orcinus genome using blastn, we recovered between 7428 (H. planifrons) and 31,888 exons (Stenella longirostris 24923), with an average of 28,324 exons per species . Delphinids had a higher average of 30,106 exons (79% recovery). The five noncetaceans ranged from 21,259 to 26,179 exons, with an average of 24,085 exons (63% recovery).The number of reads recovered per sample ranged from Platanista and B. omurai were pruned. The phylogenomic tree resulting from the RAxML analysis with 3191 separate partitions by gene is shown in The same tree topology was generated with all concatenated analyses of Dataset A using RAxML or ExaBayes, regardless of model or partitioning scheme. In addition, topologies resulting from all analyses of Dataset B agreed with those of Dataset A when Lissodelphis and Sagmatias obliquidens + Sa. obscurus in a clade to the exclusion of Cephalorhynchus + Sagmatias australis with posterior probability of 0.8, as well as displacing T. truncatus from the clade including T. aduncus, Stenella attenuata + frontalis with high support  differs from the concatenated analyses at only three nodes, all within Delphinidae . In addi support  are suppLipotes + Inioidea, Phocoenidae + Monodontidae  and also supports its exclusion from the clade Ziphiidae + Delphinida.All analyses supported the monophyly of Cetruminantia (Cetacea + Hippopotamidae + Ruminantia), Ruminantia, Whippomorpha (Cetacea + Hippopotamidae), Cetacea, Odontoceti, Mysticeti, Synrhina, Delphinida, Physeteroidea, Inioidea, Delphinoidea, ias sensu, CephaloDifferences in posterior mean times do not differ drastically between using 10 genes (SortaDate) and using the entire data set a’, the sKogia decreased by Outside of Cetacea (and exclusive of the root), the mean age of nodes using the 6-partition model decreased using the AR model with respect to IR by an average of 8.29 Ma, with the mean age of Bovidae shifting by 14.47 Ma  as the most basal lineage, followed by Suiformes (Sus), Ruminantia , Ancodonta , and Cetacea. Phylogenomic analysis unequivocally supported a monophyletic clade which included both hippopotamuses and cetaceans (Whippomorpha) to the exclusion of other cetartiodactyls, with a mean age of 53.92 Ma , a stem mysticete previously identified as the oldest crown cetacean and used as a calibration point in numerous divergence dating analyses of cetaceans.We obtained 100% resolution for the relationships of the five major lineages of Cetartiodactyla, with Tylopoda  form an unequivocally well-supported clade  partly based on the assumption that E. japonica was more closely related to E. australis  is firmly nested within Balaenopteridae, in agreement with previous studies based on fewer phylogenetic markers  diverging earliest within the Balaenopteroidea, followed by E. robustus . According to our analyses, the radiation of Balaenopteroidea began Our analyses show that the gray whale . pattern . WhateveKogia and Physeter have sometimes been included in the same family (Physeteridae), but the divergence between these genera is earlier than the diversification of Superfamily Delphinoidea, and these genera likely warrant placement in separate families. This deep divergence also coincides with evidence of the existence of fossil kogiids in the Early Miocene  and a relict species that is part of a lineage that was much more diverse in the past, with multiple fossils known from marine deposits . Some analyses of mainly mitochondrial data have united Platanista and Ziphiidae  and Hyperoodontinae , although the monophyly of Hyperoodon + Mesoplodon is well-supported. Mesoplodon  experienced a rapid radiation beginning in the Late Miocene, with at least 13 species arising in the span of less than 5 Ma . In some molecular analyses, Lipotes was placed as the most basal taxon of the Delphinida  is well-supported with Monodontidae more closely related to Phocoenidae, as noted in previous analyses . Crown d process . Both Cr process . Within ial data .Leucopleurus acutus, O. orca, and Lagenorhynchus albirostris; O. orca as sister to the remaining delphinids to the exclusion of both L. acutus and L. albirostris , and Delphininae .Our phylogenomic reconstruction provided for a clear picture of the evolutionary relationships within Delphinidae, with high statistical support and agreement between analyses for most clades . The meaanalyses . Most otanalyses . Relatioanalyses . We findirostris . Neitherias sensu; Fig. 2.Lissodelphis, Sagmatias, Cephalorhynchus) resemble those of previous studies using both mitochondrial and nuclear data ; however, C. eutropia and C. hectori are consistently allied with Cephalorhynchus commersoni, and S. cruciger is well established as the sister species to S. australis  will need to be transferred to Cephalorhynchus pending more complete sampling.Relationships within Lissodelphininae , likely due to rapid speciation and the documented presence of viable intergeneric hybrids in this group  and the bottlenose dolphins (T. truncatus and T. aduncus), with both forming a monophyletic group with respect to the “striped” dolphin lineage. These three clades were recovered by Lagenodelphis, Stenella, Tursiops, and sometimes Sousa with Delphinus  to Delphinus, and retaining Stenella for the spotted dolphins  and TurDelphinus capensis, the “long beaked common dolphin”, has been called into question  until further detailed analyses can be completed  is moreStenella clymene. Here, S. clymene is unequivocally well-supported as the sister species of S. longirostris based on substantial genomic data , or are incredibly rare and represented by few specimens . Cetaceans are well-represented in the fossil record, and further studies will combine these new data with morphological and fossil data to produce a holistic view of cetacean evolution.We targeted and assembled 3191 protein-coding genes from 68 species of cetaceans, two hippopotamids and three ruminants new to this study, and combined them with 18 existing genomes to produce the most comprehensive phylogenetic tree of cetaceans to date, in terms of the intersection of sequence data  and species . Every node was well-resolved, including those within the problematic Delphinidae (true dolphins), although three nodes within the family differed between concatenated and coalescence analyses. Our results give clarity to a long debate over the contentious relationships among species currently contained within


Aging is a phenomenon that human's physiology and psychology is progressive decline for natural environment. Health Qigong, as a convenient and effective exercise therapy,is widely used for anti-aging. However, there are no systematic reviews or meta-analysises to evaluate the efficacy and safety of Health Qigong on anti-aging.We will systematically search for 7 English databases and 4 Chinese databases from their inceptions to August 2020. Randomized controlled trials (RCTs) using Health Qigong to anti-aging will be included. After the selection and extraction of eligible studies, a meta-analysis will be undertaken to assess the efficacy and safety of Health Qigong on anti-aging. Moreover, study selection, data extraction, and the evaluation of the methodological quality of trials will each be independently completed by at least 2 researchers. The Review Manager Software V.5.3 will be employed for meta-analysis to assess the risk of bias, data synthesis, and subgroup analysis.This review will provide the latest knowledge and evidence on the efficacy and safety of Health Qigong for anti-aging through the analysis of various evaluation scales.The conclusion of this review will help clinicians provide effective exercise therapy for anti-aging.INPLASY202090017 For example, cardiovascular disease, dementia, osteoporosis, diabetes, hypertension, and stroke are age-induced diseases. The potential mechanisms remain to be researched after many decades, and the oxidative stress and free radical accumulation theories stand out the most in the multiple theories related with aging. The antioxidant system declines as a growth of age, giving rise to destruction of the balance between radical oxygen species emergence and erasure issuing in oxidative cellular damage. Compared with other organs, postmitotic tissues such as the brain, heart and skeleton muscle are more susceptible to aging. Additionally oxidative stress, aging is also intimately related with issuing in structural and functional flaws in the immune system. The 1 of the primary feature of aging is piecemeal loss of cognition, with exhibition of descending logical thinking, memory and spacial abilities. The aging of cerebral is the primary cause of cognitive obstacle.It is well known that aging is an inevitable physiological process, but we can delay senecence by various therapy. The herb for anti-aging include Cistanches, Ganoderma Lucidum and so on. Health Qigong, containing Baduanjin, Wuqinxi, Yijinjing, Liuzijue and TaiChi, are traditional Chinese exercise, that are deemed to not only enhance the antioxidant capacity of the body but also regulate immune function. Previous researches have showed that Health Qigong has fine effects for anti-aging, including regulate mental and physical health,–14 and age-associated diseases.–17 Due to these strength of Health Qigong,doctors recommend using Health Qigong to increase vigor and vitality as well as prolong lifespan.The anti-aging interventions includes anti-aging medicine, herb, and Health Qigong. The anti-aging medicine according to mechanism include Calorie restriction mimetics, Activation of AMP protein kinase, Inhibition of growth hormone/insulin like growth factor-1 axis, Inhibition of mammalian target of rapamycin, Activation of the sirtuin pathway, Hormonal replacement, Gut microbiota, Vitamin D.Although Health Qigong is widely applied to anti-aging increasing, system reviews and meta-analysis of Health Qigong are few. Therefore, we will perform a systematic review of randomized clinical trials summarizing the existing evidence and assessing the effectiveness and safety of Health Qigong for anti-aging.2The protocol of the systematic review and meta-analysis has been registered in the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY), the registration number is INPLASY202090017. It will be based on the guidelines of Preferred Reporting Items for Systematic Reviews and Meta Analysis (PRISMA-P).2.12.1.1Only the RCTs of Health Qigong for anti-aging will be included in this study, without placing the constraint on publication status and writing language. Studies without sufficient information about the randomized method or process, the animal mechanism studies, qualitative studies, uncontrolled trials and reviews and case reports will be excluded.2.1.2Subjects aged 45 to 75 years, who do not exercising daily and have no major disease will be selected. There will be no restriction on gender, race, or nation.2.1.3Our systematic review and meta-analysis will be conducted based on the RCTs that solely apply Health Qigong intervention in the experimental group and placebo or other nonpharmacological therapies in the control group. The control group has the following forms: keep previous exercise habit, restrict any exercise etc. Health Qigong include Baduanjin, Wuqinxi, Yijinjing, Liuzijue and TaiChi.2.1.42.1.4.1+,CD4+, CD8+, CD4+/CD8+).The primary outcomes include the antioxidant capacity and the immune function. The antioxidant capacity are total antioxidant capacity, superoxide dismutase and Malondialdehyde. The immune function index are tumor necrosis factor-α counts, interferon -γ counts,IL-2 counts, IL-6 counts and the level of T cell subsets  and 4 Chinese databases will be investigated from their inception to August, 2020. The search strategy for selecting the fields of topic, title, or abstract was unique referring to the characteristics of databases.The complete PubMed search strategy is in Table 2.2.2We will investgate the preplanned, ongoing, and unpublished studies by searching Google Scholar, Baidu Scholar, the International Clinical Trials Registry Platform and the Chinese Clinical Trial Registry.2.32.3.1Two researchers will be individual into 2 groups, and each group will independently scan the titles and abstracts. After deleting repetitive and irrelevant articles, the 2 groups will set up 2 lists of potential studies which will be verified against each other by superintendent(Chaohui Tang) to make a preliminary list. Next authentication of eligible articles from the preliminary list will be accomplished by 2 researchers  through using the preplanned inclusion/exclusion criteria. When divergence appear another researchers(Peng Yu) will make a judgment. The selection procedure is fully described in a PRISMA flow chart Fig. .2.3.2Two researchers  will independently work for data extraction, they will collect the undermentioned data:(1)basic characteristics, including the first author, publication year, sample size, trial location, age, geographic population, health status, duration and follow-up, frequency, intensity, Health Qigong style, control intervention.(2)(2)primary outcomes and secondary outcomes as the information showed before will also be included, including the antioxidant capacity, the immune function and cardio-cerebrovascular function.The results will be double-verified by a another researcher (Qingsong Huang). If the data is missing or unclear, the corresponding author will be contacted.2.3.3 of Systematic Reviews of Interventions, and the risk of bias (ROB) of the included studies will be assessed by 2 researchers independently using the Cochrane risk of bias assessment tool. The domains pertinent to ROB including random sequence generation, allocation concealment, blinding of participants and therapist, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. The ROB for each domain will be rated as“low risk,” “high risk,” or “unclear risk.”We will according to the Cochrane Handbook(V.5.1.0)2.3.4The meta-analysis will be performed by using the Review Manager V.5.3 (RevMan 5.3)software. We will describe the effect size with the risk Ration for dichotomous data, and the mean deference for continuous data.95% of the confidence interval will beused as an effective size for the combined analysis.2.3.52 statistic, which describes the percentage of variability in the effect estimates. I2 statistic . For more detailed explanation on potential heterogeneity among involved studies, we may also conduct subgroup analyses or meta-regression.We will calculate the heterogeneity by using the Chi-square and I2.3.6If we have a sufficient number of RCTs for inclusion in the review, we will carry out the subgroups when the heterogeneity is high. Subgroup analysis will be carried out based on the type of control interventions, type of Health Qigong, exercise frequency and duration.2.3.7We will conduct the sensitivity analysis to assess the robustness of bias on results and the sources of heterogeneity. We will delete the data of certain study 1 by 1 by comparing the recombined date with original date,and then focus on the changes merged size effects and heterogeneity.2.3.8If there are more than 10 studies are included in the review, we will build funnel plots to appraise the reporting bias. There are 2 results, it means no reporting bias existed when the funnel is symmetrical, or it means reporting bias when the funnel is dissymmetry.2.3.9We will assess the quality of evidence by using the GRADE system. The following 5 factors will be considered: risk of bias, inconsistency, directness, inaccuracy, and reporting bias. Four levels will be geaded of quality of evidence: high, moderate, low or very low.3,2 Hence, it is necessary to find effective non-drug therapies to anti-aging. Health Qigong, as a traditional Chinese excercise, has a remarkable effect on improving immune function and the antioxidant capacity of the body. However, there is still no systematic review clearly demonstrating the role of Health Qigong for anti-aging.Aging is an unavoidable natural physiological process, but aging brings several diseases together will produce destructive effects on human society and breakdowns the entire health care system and economy.The results of this study will identify whether Health Qigong can delay senescence and related laboratory indicators of anti-aging, which will present an evidence for Health Qigong in applications. The small quantity of related literature and the high heterogeneity of different Health Qigong styles used in the research may attract limitations to this study. If there is enough randomized controlled double-blind experimental research, we will accomplish a subgroup analysis to identify specific Health qigong programs and exercise time. In addition, Health Qigong has a widely application in Asia, peculiarly China, but it is not well accepted in other parts of the world, so publication bias will also be considered.In conclusion, this study will investigate the efficacy and safety of Health Qigong as a treatment of anti-aging.Conceptualization: Peng Yu, Weihong Li.Data curation: Han Li, Shuang Ouyang.Formal analysis: Haiyang Cai, Jing Wu.Methodology: Peng Yu, Chaohui Tang, Qingsong Huang.Project administration: Peng Yu, Weihong Li.Software: Peng Yu.Supervision: Chaohui TangValidation: Weihong LiWriting – original draft: Peng Yu


Children aged 12–23 months were more likely to be stunted  than children aged ≤11 months. Delayed introduction of solid foods increased the odds of stunting  and underweight . Children with normal birth weight were less likely to be thin  and underweight  than children who had low birth weight. Children whose mothers had obtained secondary school education , and Grade 12 or post-Grade 12 education  were less likely to be stunted than were children of mothers who had only primary school education. Suboptimal complementary feeding predisposed children to stunting and underweight. National nutrition programs should be context-specific to improve the introduction of complementary foods among children, especially in the remote and poor areas. Despite years of interventions intended to reduce child malnutrition in South Africa, its negative effects, stunting in particular, persist mainly among children under five years old living in under-resourced regions. A cross-sectional study was conducted to determine the prevalence of malnutrition and associated factors among 404 children under age five attending childcare services with their mothers in selected healthcare facilities of Limpopo Province, South Africa. Anthropometry, socio-demographics and obstetric history were collected. Height-for-age, weight-for-age and body mass index-for-age Z-scores were used to determine stunting, underweight and thinness among children, respectively. Logistic regression analyses were performed to generate the factors associated with malnutrition. Stunting (45.3%) was the prevalent form of malnutrition among children under age five, affecting boys (51.7%) more than girls (38.8%) and children aged 12–23 months (62.4%) more than those <11 months old (40.1%), in addition to the overall prevalence of underweight (29.0%) and thinness (12.6%). Boys had increased odds of stunting (adjusted odds ratio, AOR = 2.07, 95% CI: 1.26–3.41,  Malnutrition has persisted over the past decades and continues to affect the lives of children, especially in the developing world ,2,3. In In sub-Saharan Africa (SSA), the prevalence of stunting, underweight and wasting among children under age five has been estimated at 39%, 25% and 10%, respectively . In SoutThe first 1000 days of life are critical for nutrition, and if malnutrition is not addressed by the age of five years, the child might not reach full potential in life . To addrDuring 2017/2018, approximately 11,000 children under five years were treated for severe acute malnutrition, which was an underlying cause of mortality in 30.9% of these children . ResearcThis was a cross-sectional study conducted in the Waterberg primary healthcare (PHC) facilities from July 2019 to December 2019. We used PHC facilities based on the evidence of malnutrition still observed among children under age five attending outpatient departments in facilities, as well as hospital admission and deaths resulting from severe acute malnutrition in Limpopo Province ,25.The study was conducted in Waterberg District, located in the western part of Limpopo Province, South Africa. The district has a population of 734,780 and is made up of six sub-districts, namely Bela-Bela, Lephalale, Modimolle, Mogalakwena, Mookgophong and Thabazimbi. The head count of children below five years of age seen in all PHC facilities of Waterberg District was estimated at 67,776 in the 4th quarter of 2015/16 . PHC facThe study included children who were below the age of five years, brought by their biological mothers to attend childcare services on routine immunization and nutritional status monitoring, offered in the ten selected PHC facilities of Waterberg District. These children were not sick at the time of study, based on their mothers’ reports, and were without physical disabilities that could compromise their stature.Rao software was used to calculate a sample size, taking into consideration the population size of 67,776 children, a 5% margin of error and 95% confidence level. A minimum representative sample of 378 was calculated and buffered with 10% to cater for non-response, and a sample size of 415 was obtained. One facility was randomly selected per local area, and ultimately ten PHC facilities were used in the study. On the day of data collection, after the researcher explained the purpose of the study to mothers in each selected facility, convenience sampling was used to select children whose mothers consented to participate. The difficulty in obtaining a random sample from outpatients waiting in queues for services in the facility informed the use of convenience sampling. Although convenience sampling is a non-probability sampling method, it is the most applicable and widely used method in clinical research . In thisA structured interviewer-administered questionnaire was adapted from studies conducted in a population living in a similar socioeconomic context in Limpopo Province ,33. The Anthropometric measurements of height and weight for each child were determined using WHO standard procedures . Weights2) = weight (kg)/height (m2)). Normal BMI is within 18.5 to 24.99 kg/m2. Underweight is defined as BMI <18.5 kg/m2, overweight as BMI of 25 to 29.99 kg/m2 and obesity as BMI ≥30 kg/m2. Furthermore, the mothers’ waist (WC) and hip (HC) circumferences were measured to the nearest 0.1 cm using a non-stretchable plastic tape. Abdominal obesity was defined at WC ≥88 cm and waist–hip ratio (WHR) equal to or greater than 0.85 [Weights for mothers were measured to the nearest 0.1 kg using a calibrated smart D-quip electronic scale, and heights to the nearest 0.1 cm using a stadiometer, according to WHO . Three mhan 0.85 ..Stata Statistical Software: Release 14. College Station, TX, USA). Complete case analysis was used to identify participants with missing data during analysis. Skewness and kurtosis tests for normality were performed to check the distribution of data for children  and for mothers . All variables had Chi (2) p-values less than 0.05, which indicated skewed data, except for the height of mothers. The medians for HAZ, WAZ and BAZ of the children were compared using the Mann–Whitney test by sex. The results are presented as median (interquartile range (IQR)). The Kruskal–Wallis test was used to compare the medians for HAZ, WAZ and BAZ by age, and the results are presented as median (interquartile range (IQR)). Following the Kruskal–Wallis test, the Dunn test was further applied for multiple pairwise comparison of medians, and Bonferroni adjusted p-values between groups are reported. A Chi-square test was used to compare the prevalence of malnutrition indicators, stratified by sex and age, and the results are presented as frequency (n) and percentage (%). Fisher’s exact was applied to variables with expected values less than five (5) in a cell. Univariate and multivariate logistic regression analyses were used to determine the association of malnutrition indicators with child and maternal variables. The purposeful selection process began with a univariate analysis of each variable. Any variable having a significant univariate test at some arbitrary level was selected as a candidate for the multivariate analysis. We based this on the Wald test from logistic regression and a p-value cut-off point of <0.20. A stepwise backward elimination procedure was used to eliminate confounders. Adjusted odds ratios (AOR) with a 95% confidence interval (CI) were generated and used to determine the independent strength of the relationship. Significance was considered at p < 0.05.Statistical analyses were performed using STATA 14  (SMUREC/H/45/2019: PG). Furthermore, this study received permission from the Department of Health in Limpopo Province, South Africa, and written consent was obtained from the mothers.In this study, 404 children aged under five years participated with their mothers, and the response rate was 97%. The median age (IQR) of the children was eight months , ranging from 2 to 59 months. Although most of the children were still breastfed at the time of the study, mixed feeding was also practiced and some children were not yet on solid foods .The median age (IQR) of mothers was 28 years  when presenting at the clinic. Mothers were characterized by singlehood (54.2%), unemployed (78.7%), lived on child grant (78%), but at least had secondary education (59.4%). Households with five family members and above were observed, and most had lower monthly income . Some mop = 0.002) and underweight (p = 0.004) compared to girls, while the prevalence of thinness was not significantly different (p = 0.776). It is noteworthy that some children had a growth problem and some were at a risk of overweight, while overweight and obesity were less prevalent . The sample size for age groups of the children was not evenly distributed, especially in the group of children >24 months. This could be because of the schedule of the expanded immunization program occurring frequently from birth, in weekly intervals, until the children are 18 months. As a result, children aged ≥24 months visit the facilities less frequently until they reached five to six years of age [In s of age .p < 0.20). After controlling for potential confounding, the final multivariate logistic regression analyses showed that stunting was associated with child’s age and sex, mother’s education level and introduction of solid foods. Underweight was associated with child’s sex, birth weight and introduction of solid foods, while thinness was associated with birth weight  and the risk of malnutrition are worsened by continued breastfeeding not accompanied by adequate complementary feeding at the appropriate age, due to nutritional demand ,47,48. BSouth Africa is not on track to meet the global target for exclusive breastfeeding; however, the SADHS (2016) has reported some notable improvements in exclusive breastfeeding among infants less than six months of age . Then agLiterature documents that mothers may prolong the duration of breastfeeding, especially if they do not know when to commence giving complementary food or due to poverty-related barriers to adequate complementary feeding . Some ofThe current population was characterized by poor socioeconomic status based on the prevalence of unemployment, dependence on child social grants and singlehood with no spousal support, which may lead to food unavailability in households. In the context of food insecurity when adequacy and accessibility of food are impaired, the mother’s decision-making ability for effective infant feeding is also disrupted . ComplemSeveral low- and-middle-income countries, including Ethiopia, Nepal and Senegal, have dramatically reduced child stunting . Strong The current study further showed that stunting was associated with a child’s sex and age and maternal education, while underweight was associated with a child’s sex and birth weight, and thinness was associated with birth weight. The biological differences of stunting among boys and girls have been attributed to morbidity during early life . The difPersistent early life malnutrition has been implicated to fuel the burden of non-communicable disease, an emerging public health concern in South Africa . Similarn = 11) [We acknowledge the limitation of using a cross-sectional study design, which could only report on inferences and not on causality. The other limitation is that we used convenience sampling. While our sample may not be representative of children who attended the PHC in Waterberg District of Limpopo Province, the large sample size used in our study provides valuable and useful information for comparisons with other published studies from developing countries. Missing data were excluded using complete case analysis, a commonly used method, although its limitations of affecting sample size and leading to reduced statistical efficiency of estimates while increasing the potential for bias have been reported . Certainn = 11) . We acknn = 11) , which wn = 11) ,33 from n = 11) ,33, and Persistent malnutrition, in particular stunting and underweight, among children under five years of age in Waterberg district of Limpopo Province, are a public health concern. In addition to other factors associated with malnutrition indicators, delayed introduction of solid foods during the complementary feeding period predisposed children to stunting and underweight more so than children who had optimal complementary feeding. These findings clearly indicate a need to improve the introduction of complementary foods among children, through national nutrition programs that are context-specific, especially in the remote and poor areas. Moreover, modifiable maternal vulnerabilities such as a poor socioeconomic status should be considered in the intervention programs.


Bacillus thuringiensis (Bt) cells by using surface-enhanced Raman spectroscopy (SERS) are presented. Bt has been chosen as a harmless surrogate of the pathogen Bacillus anthracis (Ba) responsible for the deadly Anthrax disease, because of their genetic similarities. Drops of 200 μL of Bt suspensions, with concentrations 102 CFU/mL, 104 CFU/mL, 106 CFU/mL, were deposited on a SERS chip and sampled after water evaporation. To minimize the contribution to the SERS data given by naturally occurring interferents present in a real scenario, the SERS chip was functionalized with specific phage receptors BtCS33, that bind Bt (or Ba) cells to the SERS surface and allow to rinse the chip removing unwanted contaminants. Different chemometric approaches were applied to the SERS data to classify spectra from Bt-contaminated and uncontaminated areas of the chip: Principal Component Regression (PCR), Partial Least Squares Regression (PLSR) and Data Driven Soft Independent Modeling of Class Analogy (DD-SIMCA). The first two was tested and trained by using data from both contaminated and un-contaminated chips, the last was trained by using data from un-contaminated chips only and tested with all the available data. All of them were able to correctly classify the SERS spectra with great accuracy, the last being suitable for an automated recognition procedure.In this work, the results on the detection and identification of  Nowadays the rapid detection and identification of microorganisms in the environment, including aerosol, water and food, is a major issue to prevent large numbers of infections  for publFor this purpose, fast analytical techniques, not requiring time-demanding samples preparation and suitable to be applied in a continuously running device, are strongly desired.In this context Surface-Enhanced Raman Spectroscopy (SERS)  has beenSERS) and the chemical one (ChemSERS), whose features are briefly summarized in the following.The SERS enhancement comprises two multiplicative contributions: the electromagnetic enhancement  [SERS plays an important role because it determines the spectral pattern of the SERS spectrum  which could significantly differ from the standard Raman pattern of the same substance [Chemnsitions . ChemSERgstroms)  between ubstance ,12.SERS is particularly suitable for a real time detection of bio-hazards because the typical time required to acquire a spectrum is of the order of seconds or minutes ,3,4. ForBa, with the following objectives:(1)good selectivity ;(2)detect-to-warn response time within 60 min;(3)minimal use of chemicals and reagents (for long-term unattended operation);(4)state of the art sensitivity (towards one unit limit);(5)man-portability with a ruggedized design for use in defense and security scenarios(6)suitability for a broader biological set of samples, that is, extendable to other microorganisms and toxins.RAMBO aimed at developing advanced methods, instrumentation and sensing strategies/protocols for continuous monitoring of air particles against biological threats, in particular Ba, which is classified as a biosafety level 3 bioagent and responsible for Anthrax, a severe, fortunately rare, acute infection involving the skin, the gastrointestinal tract and the lungs [The underlying idea is that a rapid detection of the threat allows to activate precautionary measures, including prompt prophylaxis against he lungs .Bacillus cereus group, of which Ba is member. Indeed, in addition to Ba, the Bacillus cereus group includes six very closely related species: B. cereus (in strict sense), B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis [B. thuringiensis, (Bt) exhibits such a high genetic affinity to Ba [To reach these objectives, two sensing techniques are employed in sequence in RAMBO: SERS and Polymerase-Chain-Reaction analysis (PCRa) ,16,17. Whanensis . In partty to Ba ,21,22,23ty to Ba .Bt cells detection at very low concentration, down to the detection of a single cell by using SERS as a pre-alarm technique. According to the RAMBO specifications, the detection was carried out in few minutes, over a relatively large area of the sensor, to obtain more reproducible SERS spectra and to reduce the signal variability.This work presents the results of Bt (ATCC® 10792TM) cells were provided by the Military Institute of Hygiene and Epidemiology of Warsaw (Poland), as 1 mL suspensions of vegetative cells in physiological sterile water (0.9% NaCl) with concentration 109 CFU/mL. The samples were then diluted with pure sterile water  at concentrations down to 102, 104, 106 CFU/mL. A drop of each suspension, 200 μL vol., was then deposited on a SERS active substrate Klarite [−4, 6.4·10−2, 6.4 cells/μm2. After deposition and drying of the drop, the cells were deeply rinsed with pure sterile water, to remove residual NaCl and cells not anchored on the SERS surface. Klarite  and drieKlarite substrates consist of regular arrays of inverted pyramidal pits, realized depositing a sputtered gold layer on a silicon substrate with an ordered nanostructure produced by electron beam lithography. The full chip active area is 4 mm × 4 mm. Each pit has an aperture of 1.5 μm × 1.5 μm and a total pitch size (aperture plus distance to the next microcavity) of 2 μm × 2 μm with a fixed apex pit angle of 70.5°. The cavity depth is 1.06 μm. The regular arrangement of the nanostructures favors a uniform enhancement of the weak Raman signal over the whole excited area, providing a uniform distribution of “hotspots” [Bt cells deposited on the substrate.BtCS33 phages [SERS surfaces were first coated using carboxy-aniline electrodeposition through cyclic voltammetry .The grafted carboxylic acid function was activated using carbodiimide (EDC 0.4M + NHS 0.05M for 8 min at room temperature).The phages were then brought in contact with the activated surface .The activated surface was then neutralized using 1 M ethanolamine, pH 8.5.The substrates were functionalized with 3 phages ,27 at thBetween each step, the SERS surface was washed with MilliQ sterile water (18 MΩ), air dried and stored under vacuum at 4 °C.i-Raman” by Bw&Tek, equipped with a GaAlAs 785 nm diode laser (linewidth < 0.3 nm). The spectral range spans between 100–3500 cm−1 and the spectral resolution is 3.5 cm−1. The laser power was set to 60 mW, focused through a 40× objective, 0.5 N.A. in a spot of 40 μm diameter  and the laser spot diameter (40 μm) the average number of sampled cells for each dilution was between 0.8 and 8000. The SERS signal from the substrate was dispersed through a 600 lines/mm grating and recorded by a cooled (10 °C) Charge Couple Device (CCD) array detector . Each recorded spectrum was the average of 3 subsequent acquisitions of 10 s integration time, for a total integration time of 30 s.SERS measurements were performed with a table-top micro-Raman system “diameter a. The en−1; (2) fluorescence background subtraction; (3) spikes removal and (4) normalization (between 0 and 1) of the residual signal. All these steps were performed processing the raw spectra by using home-developed routines, in a Matlab™ environment. In particular, the fluorescence background subtraction was performed with an iterative procedure, similar to the one reported in [The collected SERS spectra were processed by applying the following steps (1) cropping of the spectral region between 350–1800 cmorted in , replaciAn example of this procedure, applied to a raw (as-recorded) spectrum, is shown in Bt-contaminated substrate with 102 CFU/mL; 100 sampled at 104 CFU/mL concentration and, finally, the remaining 100 at 106 CFU/mL. Spectra were collected performing 5 repetitions on 20 different points and processed by applying the described background subtraction procedure.A total of 400 spectra were acquired to apply the discrimination/classification procedures: 100 were relative to the uncontaminated substrate; 100 sampled on Bt . The high reproducibility of the SERS signal is demonstrated by the small values of the SD in each pixel, which had a mean value of 2–4% considering all the pixels and did not exceed 12.2% in any case.−1 are to be ascribed to the overall functionalization process described in Bt cells is detected and the last is inferred in the following as intensity fluctuations of the above-mentioned spectral structures.The evident spectral features at about 412, 521, 632, 834, 900, 997, 1074, 1133, 1176, 1222, 1277, 1307, 1402, 1447, 1603 cmBt cells is explained by the very superficial nature of the SERS enhancement, which is typically relevant only in a layer of few nanometers close to the surface. Then, considering that the functionalization process could result in an interfering layer increasing the distance between the cells and the chip surface, a reduced enhancement of the cells signal is expected [−1, are marked in black if positive and in red if negative. In the first case, they are supposed to represent a residual Bt signal, in the second case the presence of Bt is supposed to reduce the background signal. Error bars (in grey) are the sum of the SDs of the mean spectra. The experimental results here described highlight the sensitivity of the technique, capable to detect very few cells. However, the problem of label-free detection of large cell structures by SERS methods is well known. It is accepted that, from these measures, the selectivity, i.e., the possibility of discriminating between different types of bacilli, would be guaranteed only by the specific functionalization by BtCS33 phages, designed to bind exclusively Bt. For this reason, further tests with different types of bacilli of the Cereus group are needed and planned. However, SERS is considered in RAMBO as an early warning technique, with the task of providing high sensitivity and adequate selectivity, while the genetic identification of the bound organic material is entrusted to the PCRa technique.The weakness of the signals ascribable to expected ,11. To hBt cells appeared weak, to verify the possibility to correctly discriminate between spectra from Bt-contaminated or un-contaminated SERS substrates, two different chemometric approaches were applied: Principal Component Regression (PCR) [Bt-contaminated areas (positive entries).Although the SERS signal of the on (PCR) ,29 and Pon (PCR) . PCR invon (PCR) ,32,33 toon (PCR) ,32. PLSR6 CFU/mL group). Once built, the PCR and PLS models were projected over the whole dataset, assigning a value to the prediction variable for each spectrum. Then, the spectra were classified as negative entries if their prediction variable were closer to zero than one, positive entries otherwise. The classification performances of PCR and PLSR were tested in term of sensitivity (Se) and specificity (Sp) [Both PCR and PLSR were trained with training sets of 10, 20, 50, 80, 100 randomly chosen spectra, of which one half were composed of negative entries, the remaining half of positive entries (from the 10ity (Sp) ,35,36,37It can be observed that for PLSR both Se and Sp tend to increase with the number of spectra, while for PCR, an increasing trend is observed only for Se, whereas an opposite behavior is observed for Sp, suggesting that with larger training sets, PCR tends to build predictive models that erroneously classify negative entries. This can be confirmed also looking at the trends of TN and FP in In conclusion, both PCR and PLSR demonstrated the possibility to classify these groups of SERS spectra with high sensitivity and selectivity.A further step of automatic recognition and classification could be the implementation of a recognition procedure by using a one-class classifier method (OCC), where only a single group of data, for example the spectra from the uncontaminated samples, is needed. The advantage of an OCC technique is that the training procedure could be preliminarily performed on each new, uncontaminated SERS chip, making it ready to automatically recognize outliers, once the sensor is contaminated and used in a real scenario. For this purpose the Data Driven Soft Independent Modeling of Class Analogy (DD-SIMCA) ,38 was aTo perform DD-SIMCA on the acquired SERS data a Matlab™ tool developed by Zontov et al.  has beenBt cells was presented and discussed based on the discrimination capabilities of the chemometric techniques PCR, PLSR and DD-SIMCA. SERS spectra acquired from SERS functionalized substrates contaminated with Bt cells (simulating the deadly Bacillus anthracis) were collected and compared with those acquired from an uncontaminated one, being correctly classified as potentially dangerous with high accuracy. At the lower Bt concentration of 102 CFU/mL, the collected spectra accounted, on average, for only one Bt cell on the scanned area. PCR and PLSR required no more than 100 sample spectra to be preliminarily trained, reaching selectivity and specificity percentages close to 100% on test sets. DD-SIMCA was tested as an OCC method suitable to automatically recognize possible threats in a realistic scenario. The obtained results show that SERS spectroscopy has the potential to detect biological pathogens in real time, with minimal sample preparation and with high sensitivity down to single cell and is eligible as a pre-alarm, fast, preliminary diagnostic technique, that could be followed by subsequent unambiguous, genetic-based classification/identification techniques like PCRa in the case of the RAMBO project.In this paper, the ability of SERS spectroscopy to detect low numbers of


Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300 bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform.  A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies.  A total of 122.6 Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6 Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform. NCT02341352. The oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. This trial is registered with  The human oral microbiome comprises over 700 prevalent taxa at the species level, including a large number of opportunistic pathogens involved in periodontal, respiratory, cardiovascular, and systemic diseases –5. IdentThe emergence of the next-generation sequencers (NGS) and their sequencing by synthesis have drastically transformed the way scientists delve into the relationship between microbiome and related diseases . Since tVibrio parahaemolyticus [Third-generation sequencing (TGS), PacBio single molecule, real-time (SMRT) sequencing technology circumvented this problem by greatly increasing read lengths that have the ability to sequence the full length of the 16S rRNA gene . It invoolyticus . HoweverFive preschool children aged 63–74 months, lacking evidence of oral and systematic diseases were recruited based on a list of exclusion criteria on Nov 26, 2014. The subjects with a history of chronic antibiotic used within 8 weeks before enrollment were excluded from the study. All subjects' legally authorized representatives provided written informed consent upon enrollment. The study was approved by the Institutional Review Board of the Affiliated Stomatology Hospital of Zhejiang University in accordance with the Declaration of Helsinki principles.The subjects were instructed neither to eat and drink nor to perform any oral hygiene procedure two hours before sampling. Saliva samples were collected from all subjects in the morning between 9 : 00 am and 11 : 00 am.Unstimulated saliva samples were collected according to a protocol, modified from a previous study. The children were initially asked to rinse their mouth thoroughly with deionized water prior to sampling, followed by collection of at least 5 mL unstimulated saliva in a plastic cup. Finally, the samples were transferred into sterile cryogenic vials. Then, the samples were placed into liquid nitrogen and stored at −80°C until use.Bacterial DNAs were extracted using the E.Z.N.A.™ Soil DNA Kit , according to the instructions of the manufacturer. The enriched microbial DNAs were purified by ethanol precipitation. DNA concentration was measured using NanoDrop, and its molecular size was estimated by agarose gel electrophoresis. DNAs were stored at −20°C until use.PCR amplification of the 16S rRNA gene hypervariable V3-V4 regions was performed with universal bacterial primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The V1-V9 hypervariable region was performed with primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The products were extracted with the AxyPrep DNA Gel Extraction kit  and were then examined by agarose gel electrophoresis. According to the electrophoretic results, the PCR products were quantified by Quantifluo™-ST . Then, the products from different samples were then mixed at equal ratios for pyrosequencing on the two different platforms.Construction of DNA library was carried out by following the manufacturer's instructions (Illumina and PacBio). A 468 bp insert size library was constructed on the MiSeq platform and then applied to 300 bp paired-end sequencing. Libraries with longer insert size (1540 bp) were performed on the PacBio RS II platform, including full length of 16S rDNA gene. Barcoded 16S rRNA amplicons (V3-V4 and V1-V9 hypervariable regions) of the five Chinese children were sequenced on MiSeq and PacBio RS II platforms, respectively. Raw data were generated, and low-quality reads were then removed by quality control .We used QIIME software to cluster filtered reads into operational taxonomic units (OTUs) from PacBio and MiSeq platforms  by applyBy high-throughput pyrosequencing of 5 samples synchronously on two different platforms, a total of 122.6 Mb raw data including 244,967 high-quality sequences were generated by the MiSeq platform, while 134.6 Mb raw data including 70,030 high-quality reads were generated by the PacBio RS II platform. For the MiSeq platform, 99.99% of the clean reads distribution ranged from 401 to 500 bp, and for the PacBio RS II platform, 94.24% of the clean reads were distributed from 1401 to 1600 bp.The average lengths of quality reads were 446 bp and 1471 bp on MiSeq and PacBio RS II platforms, respectively. With accurate read lengths of 1471 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in oral cavity .A slightly higher coverage was observed in the PacBio RS II platform, and the level of coverage indicated that the 16S rRNA gene sequences identified by the two sequencing platforms represented the majority of bacterial sequences present in the oral saliva samples. The rarefaction curves and richness indices (Chao and ACE) that estimated the richness of the total oral microbiota also show that enough sequencing data were generated by the two platforms Figures  and 3.α-diversity when compared with the MiSeq platform  of unknown classes was generated (Betaproteobacteria accounted for the largest proportion of the total community in both of the two groups, while the abundance of the abundance of Bacilli and Bacteroidia were different between the two platforms.At the class level, the majority of the sequences of MiSeq belonged to enerated . The oveNeisseriales, Bacteroidales, Selenomonadales, Lactobacillales, Fusobacteriales, Pasteurellales, and Clostridiales dominated the community in both groups (At the order level, h groups . The oveh groups .Neisseriaceae, Prevotellaceaes, Veillonellaceae, Streptococcaceae, Pasteurellaceae, and Fusobacteriaceae shared 82.4% and 88.0% of oral microbiome by the MiSeq and PacBio platforms, respectively (At the family level, ectively . 0.39% aectively .Neisseria, Prevotella, Veillonella, Streptococcus, Haemophilus, and Fusobacterium, which contributed 79.4% and 86.8% of the MiSeq and PacBio community. The unknown and unclassified genera of the MiSeq platform accounted for 0.68% (At the genus level, the majority of the sequences of the two platforms belonged to or 0.68% . The oveAt the species level, 68 species were shared by the two platforms and 368 species were detected only by the PacBio RS II platform. Forty-two genera cannot be classified into special strains on the MiSeq platform, which accounted for nearly half of the whole community ; howeverActinomyces, 16.2% of the bacteria was unclassified by the MiSeq platform. Actinomyces odontolyticus and Actinomyces uncultured bacterium were shared by the two platforms, and 7 unique species were generated only by the PacBio RS II platform (For species of platform .Campylobacter, 2.2% of the bacteria was unclassified by the MiSeq platform. Campylobacter concisus and Campylobacter showae were shared by the two platforms, and 8 species were unique to the PacBio RS II platform (As to the species of platform .Rothia, 10.1% of the bacteria was unclassified by the MiSeq platform. Rothia uncultured bacterium was the only species shared by the two platforms, and 8 unique species were generated only by the PacBio RS II platform (For species of platform .Haemophilus, 5.7% of the bacteria was unclassified by the MiSeq platform. Haemophilus parahaemolyticus, Haemophilus parainfluenzae T3T1, and Haemophilus uncultured bacterium were shared by the two platforms, and 17 unique species were generated only by the PacBio RS II platform (When it comes to platform .Fusobacterium, which are among the most abundant bacteria in healthy oral cavity, 10.8% of the bacteria was unclassified by the MiSeq platform. Fusobacterium periodonticum and Fusobacterium uncultured bacterium were shared by the two platforms, and 12 unique species were generated only by the PacBio RS II platform (For species of platform .Gemella sequenced by different platforms. The comparison of sequencing results between MiSeq and PacBio RS II indicates that Gemella haemolysans was the only species shared by both the platforms and up to 98.8% species were unclassified by the MiSeq platform. Nine unique species were generated only by the PacBio RS II platform.Selenomonas, 8.6% of the bacteria was unclassified by the MiSeq platform. Five species including Selenomonas uncultured organism, Selenomonas flueggei, Selenomonas artemidis, Selenomonas noxia, and Selenomonas sputigena ATCC 35185 were shared by the two platforms, and 7 unique species were generated only by the PacBio RS II platform (For species of platform .Veillonella, 78.1% of the bacteria was unclassified by the MiSeq platform. Veillonella atypica and Veillonella sp. oral taxon 780 were shared by the two platforms, and 21 unique species were generated only by the PacBio RS II platform (As to species of platform .Prevotella, 10.5% of the bacteria was unclassified by the MiSeq platform. 11 species including Prevotella loescheii, Prevotella salivae, Prevotella sp. oral clone FW035, Prevotella sp. oral taxon 306 str. F0472, Prevotella melaninogenica ATCC 25845, Prevotella histicola, Prevotella shahii, Prevotella nanceiensis, Prevotella aurantiaca, Prevotella pallens, and Prevotella uncultured prevotella sp. were shared by the two platforms. The number of unique species generated by the MiSeq and PacBio RS II platform were 15 and 11, respectively (For species of ectively .Neisseria, which are the most abundant species of the community in this study, 71.7% of the bacteria was unclassified by the MiSeq platform. Five species including Neisseria sp. oral strain B33KA, Neisseria oralis, Neisseria subflava, Neisseria elongata, and Neisseria flavescens were shared by the two platforms. The number of unique species generated by PacBio RS II platform was up to 35 (For species of up to 35 .Streptococcus is a gram-positive bacterium belonging to the phylum Firmicutes, which is found to be associated with many kinds of oral diseases, such as caries [Streptococcus was unclassified by the MiSeq platform. Only the two species Streptococcus intermedius and Streptococcus sanguinis were shared by the two platforms. The number of unique species generated by the PacBio RS II platform was up to 77 (s caries , 27, pnes caries , 29. In up to 77 .A number of research studies have presented evidence for using childhood oral microbiome to predict future oral and systemic diseases . TherefoCompared with our previous study on the structure of oral microbiome in healthy children, the top 10 phyla, genera, and species are consistent . HoweverCompared with the traditional 16S rDNA sequencing of the MiSeq platform, the PacBio RS II technology improved its read length and annotated the nucleotide sequence of oral bacteria to the species level. PacBio RS II may be optimal for oral microbiome sequencing due to its long reads and high performance, while platforms such as Illumina MiSeq will provide cost-efficient methods for sequencing projects.Previous research studies had compared the TGS PacBio platform with the NGS Roche 454 pyrosequencing platform. Amplicons of the 16S rRNA gene from the environmental samples from streambed habitats, rocks, sediments, and a riparian zone soil were analyzed . In thisIn our study, oral microbiome of healthy Chinese children was explored. For oral microbiome studies, if the goal is identifying all species in a sample, PacBio appears to have superior performance to MiSeq. However, if the goal is to simply quantify relative differences in diversity, either platform would be appropriate. In this article, we have compared the difference between the two platforms, however, with the limited sample size, the study does not provide a statistic conclusion, and more in-depth studies with larger group sizes are needed to validate these results.


Marine organisms and their associated microbes are rich in diverse chemical leads. With the development of marine biotechnology, a considerable number of research activities are focused on marine bacteria and fungi-derived bioactive compounds. Marine bacteria and fungi are ranked on the top of the hierarchy of all organisms, as they are responsible for producing a wide range of bioactive secondary metabolites with possible pharmaceutical applications. Thus, they have the potential to provide future drugs against challenging diseases, such as cancer, a range of viral diseases, malaria, and inflammation. This review aims at describing the literature on secondary metabolites that have been obtained from Scleractinian-associated organisms including bacteria, fungi, and zooxanthellae, with full coverage of the period from 1982 to 2020, as well as illustrating their biological activities and structure activity relationship (SAR). Moreover, all these compounds were filtered based on ADME analysis to determine their physicochemical properties, and 15 compounds were selected. The selected compounds were virtually investigated for potential inhibition for SARS-CoV-2 targets using molecular docking studies. Promising potential results against SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and methyltransferase (nsp16) are presented. Marine ecosystems harbor numerous, still unexplored and taxonomically diverse micro- and macro-organisms. These marine organisms have the ability to produce novel compounds, as secondary metabolites, with a wide structural diversity and various important pharmacological potentials ,3,4,5,6.Scleractinia-associated organisms have been attracting the attention as a potential source of bioactive compounds during the past decade, reflecting the great increase in the number of the isolated compounds over the past ten years .The associated micro-organisms showed diverse geographical distribution. Associated bacteria and fungi are mainly collected from regions of East Africa up to the Southern East of Asia, as well as the East of Australia with bacterial strains being more abundant in the North Atlantic Ocean at the North of Southern America .The most studied Scleractinian family was Pocilloporidae with 51 isolated compounds, 80% of which were isolated from different fungal strains, while the least studied one was Oculinidae, which was confined only to Zooxanthellates and gave rise to six compounds .Many classes of Scleractinian-associated microorganisms were cultivated and identified but with no further chemical investigation. The bacteria attained the largest portion (86%), with the class γ-Proteobacteria being the most easily culturable one (21%), α-Proteobacteria (16%), Actinobacteria (14%), Cyanophyceae (8%), and the others being equally distributed between the respective genera. The Scleractinian-associated fungi were less easily cultivated (14%), with the two most reported classes, Dothideomycetes (9%) and Eurotiomycetes (7%) .Scopulariopsis, the other 15 compounds were obtained from the genus Gliomastix. The remaining 23 compounds were found in Aspergillus (18 compounds) and Talaromyces (five compounds), both belonging to the Eurotiomycetes.Although the largest portion of Scleractinian-associated microorganisms were only identified, a vast number was also subjected to chemical investigation and led to the isolation and structural elucidation of 134 compounds up to date. From different fungal strains, 84 compounds  were obtained, 61 of them were received from those belonging to Sordariomycetes, with most of them (46 compounds) isolated from the genus Streptomyces, six from Pelmonas and only one from Nesterenkonia. This was followed by the class α-Proteobacteria, from which twelve compounds were obtained from Erythrobacter, and the class Cyanophyceae, from which eight metabolites were obtained . The least studied bacterial class was the γ-Proteobacteria with only three isolated compounds from the two studied species Pseudoalteromonas (two constituents) and Microalbu (one compound). The zooxanthellae were the least studied Scleractinian-associated microorganisms with only six isolated compounds (5%) . From Actinobacteria, 21 compounds were isolated, with 14 metabolites from nds (5%) .Diverse classes of compounds were isolated from Scleractinia-associated microorganisms, with xanthones and anthraquinones being the most abundant metabolites (15%), all of which were isolated from fungal strains .The metabolites isolated from Scleractinia-associated organisms exhibited diverse biological activities, with more than 50% of the isolated compounds having cytotoxic properties (33%), followed by antibacterial (22%) potencies .Scopulariopsis sp., which had been isolated from the inner tissue of the host coral Stylophora collected from the Red Sea in Egypt, 29 different compounds were detected. They comprised of eleven xanthones (compounds 1–11), five sesquiterpene derivatives 12–16, four phenyl ethers 17–20, five alkaloids 21–25, and four miscellaneous compounds 26–29  and the lack of activity for the remaining xanthone derivatives 1–2 and 4–11, indicated that both the dihydroxanthenone nucleus and a C-12 free hydroxy group are operational landscapes that are imperative for the cytotoxic activity. Comparison between scopularide A (24) and scopularide B (25) suggested that the length of the aliphatic side chain and hence possibly the lipophilicity are also important for the activity. Concerning the cytotoxicity of the biphenyl ether derivatives violaceol I (17) and violaceol II (18) and the absence of such activity for diorcinol (19), led to the hypothesis that increasing the number of hydroxy groups in these molecules boosted the cytotoxic activity ..74 and 780) and verruculosin B (81), along with three known analogues, bacillisporin F (82), duclauxin (83), and xenoclauxin (84), all of which possess a unique octacyclic skeleton, were obtained from the marine-derived fungus Talaromyces verruculosus, which was isolated from Goniopora sp. (family: Poritidae)  inhibitory activities, with IC50 values of 0.38, 0.4, 0.75 and 0.26 µM, respectively; as a reference compound, Na3VO4 was used with an IC50 value of 0.52 µM. The outcomes designated oligophenalenone dimers as a tool for screening of the new natural CDC25B inhibitor candidates.Two oligophenalenone dimers, verruculosin A (ritidae)  37]. In. In80) a84, which exhibited the strongest activity, with an IC50 value of 0.24 µg/mL, compared to afatinib as a reference drug of 0.0005 µg/mL ..105), th108 showed strong antibacterial inhibition against Ralstonia solanacearun with a MIC value of 2 µg/mL . Al. AlPseudectively . Both ofne 7 mm) .Z,4E)-3-Methyl-2,4-decadienoic acid (126) is a new fatty acid, detected as a major metabolite in a butanol culture broth extract of a marine bacterium Microbulbifer sp. C46, isolated from the stony coral Porites sp. (family: Poritidae), obtained from an aquarium vendor in Nagasaki . Remarkably, it was known as a synthetic compound . In. InOculintration .Streptomyceces cyaneofuscatus and was sequenced being designated as Streptomyceces cyaneofuscatus M-157. The phylogenetic tree revealed the evolutionary relationship of strain M-157 with a group of known Streptomyces species [Phylogenetic analysis of the strain M-157 was performed by PCR via amplification of the 16S rDNA of strain M-157. After Basic Logic Alignment Search Tool (BLAST) sequence comparison, strain M-157 showed 100% identity to  species .Scopulariopsis belonging to class Sordariomycetes is considered as the most heavily studied fungal strain which has been isolated from Stylophora sp. From this species, 46 compounds have been isolated, of which only 20 are biologically active. The compounds included eleven xanthones, five triterpenes, seven sesquiterpenes, four phenylether derivatives, three alkaloids, two cyclodepsipeptides, two α-pyrones, one chromone, one sterol, three polyketides and seven nitrogenous compounds, most of which exerted antibacterial, cytotoxic and immunosuppressive potencies. The fungus Aspergillus belonging to class Eurotiomycetes is isolated from Galaxea sp., from which 18 compounds were isolated including: two prenylcandidusins, three candidusins, seven terphenyllin derivatives, and six anthraquinones, from which only 13 compounds were biologically active, mostly with antibacterial and cytotoxic activities. The fungus Gliomastix belonging to class Sordariomycetes was isolated from Stylophora sp., from which 15 hydroquinone derivatives were isolated from its fungus, only six of which were bioactive exhibiting cytotoxic and antitubercular activities.Investigating bacterial strains isolated from different Scleractinian species, Erythrobacter sp., belonging to class Alphaproteobacteria, which was isolated from Acropora sp., is chemically investigated to give twelve carotenoids, of which only three were active with cytotoxic and radioprotectant potencies. The class Actinomycetes was intensely investigated, where Streptomyces isolated from Porites sp. led to isolation of eight compounds (five anthracyclines and three tirandamycine), all of which showed antibacterial and cytotoxic activities. The same bacterial strain, isolated from Lophelia sp. and other different corals led to isolation of the cytotoxic compounds, lobophorin and 5-hydroxyquinaldic acid derivatives, two of which were cytotoxic. Moreover, the chemical investigation of Nesterenkonia isolated from Platygyra sp. led to the isolation of a polyhemiacetal with RXRα activity. The class Betaproteobacteria has rarely been investigated. As an example, Pelmonas sp. was isolated from Acropora sp. and subjected to chromatographic analysis to give four imidazole alkaloids and two cyclopeptides, most of them were inactive. The class Gammaproteobacteria has been studied relatively less intensively, here both Pseudoalteromonas sp. and Microbulbifer sp. were isolated from Montipora sp. and Porites sp., giving rise to the isolation of two cytotoxic compounds and one antibacterial fatty acid, respectively. The class Cyanophyceae was also investigated, and both Synechocystis sp. and Phormidium corallyticum were isolated from Acropora sp. and Porites sp., to give two active cytotoxic cyclic C11 compounds and two antiproliferative fatty acid amides, respectively. From the same class, Roseofilum reptotaenium was isolated from Montastraea and investigated to result in the identification of two inactive macrolides.Moreover, investigating some bacterial strains isolated from different Scleractinian species, as Oculina sp., were also investigated, leading to the isolation of six sterols, which were found to be cytotoxically inactive.Furthermore, the zooxanthellae, isolated from 1–134) are presented, together with their chemical categories and their biological sources (marine organisms), as well as the locations of collection of the studied marine organisms.In 3. In all cases the co-crystallized ligand was redocked to validate the docking method and its score was used as a reference to compare the tested compounds. Root mean square deviation (RMSD) between docked and crystallized ligand in the validation step was accepted when it was less than 2 Å using the DockRMSD server [Physicochemical properties of the investigated compounds were studies using SwissADME  and a boD server .From the 134 compounds investigated, ADME highlighted the compounds that can be absorbed orally but without crossing the blood brain barrier (BBB) to avoid possible side effects. These target compounds are found in the egg white in the following boiled-egg diagram .Based on the above criteria, 15 compounds were selected for investigating their potential effect against the above mentioned SARS-CoV-2 targets. The results of the docking are shown in pro)  . Main prmologues . The cry with N3 . These rO position. This modification helps to hide the viral RNA from the host immunity, with the methyl donor being S-adenosyl-L-methionine (SAM) [18, 103, 98, 104, and 124 attained docking energies better than that of SAM.Another important SARS-CoV-2 target is the non-structural protein 16 (nsp16). Nsp16 forms a complex with another protein, nsp10, which is resposible for the methylation of the viral RNA ribose at the 2′-ne (SAM) . A cryst103, 98, and 104 are closely similar in structure and their binding poses were also found to be very similar, which suggested the selective recognition of nsp16/10 towards this class of compounds. The docking pose of this compound in the active site of nsp16/10 is shown in The top 3 compounds The third investigated target is the SARS-CoV-2 RNA dependent RNA polymerase . This enzyme, in a complex form with nsp7 and nsp8, plays a key role in the replication of the positive strand viral RNA. Remdesivir in its triphosphate form is a known inhibitor to this class of enzymes . Nsp12, 124, 103, and 104 were docked in the same pocket which is formed at the interface between nsp12 and the RNA sequence as seen in 124 exhibited noticeably good bonding energy  and several hydrogen bonds with the active site residues, which include T687, S759, D760, R555, and with Mg2+.The co-crystallized ligand as well as the highest-docked compounds 103 and 104 showed similar binding behavior, but flipped binding poses  has shown promising results against RdRp, which further implicate the importance of natural products repository as a source of new leads for the design of inhibitors against SARS-CoV-2. Scientific efforts should be continued for the purpose of exploring the stony coral environment in the search for new bioactive compounds for the development of new drugs getting benefit of the rising development of oceanographic science and metabolome screening techniques.Scleractiniae and their associated microorganisms have so far been largely studied as a promising source of metabolites with varied new chemical structures displaying a variety of interesting biological activities. These compounds present an enormous potential for the discovery of new therapeutic leads for drug development to fight the current global health problems with increasing numbers over the past years . Interes


Peri-implant diseases are one of the main complications of dental implants. There are no well-established guidelines regarding laser parameters for implant decontamination. The aim was to compare two different settings of irradiation of the Er,Cr:YSGG laser on dental implants regarding surface alterations and determine the best settings for less damage on the surface. An in vitro study was performed and 30 areas of dental implants were irradiated with two different regimes of energy per pulse 50 and 84 mJ (1.5 W/30 Hz and 2.5 W/30 Hz). A total of 30 sites of implants were irradiated with three different tips (10 surfaces per tip): conical (RTF3-17 mm), side firing (SFT8-18 mm) and cylindrical (MGG6-6 mm). The following descriptive classification on surface damage was employed: no damage (class A), minimal effects (class B), metal fall with melting (class C), and destruction with carbonization (class D). The assessment was made through a descriptive scanning electron microscope (SEM) analysis. Side firing and conical tips at 50 mJ were classified as class A. Side firing at 84 mJ and cylindrical tips 50 mJ and 84 mJ were classified as class B. Finally, class C defects were found in the areas where the conical tip was used at 84 mJ. Side firing and conical tips at 50 mJ do not seem to damage the implant surface. Oral rehabilitation using implants is a highly documented treatment in scientific literature and has a high success rate with more than 10 years of follow-up . Peri-imAccording to two papers by Cortés-Acha et al. ,7, implaA recent ex vivo study concluded that laser irradiation of titanium implant surfaces using a 9.3 mm carbon dioxide laser with an average power of 0.7 W showed no increase in temperature of the implant body and surrounding tissues, as well as no evidence of implant surface damage . AlthougTherefore, the aim of this study was to compare two different settings of irradiation of the Er,Cr:YSGG laser on implants regarding surface alterations and to determine which application tip of the above-mentioned laser provides less damage to the dental implant surface.®, Irvine, CA, USA), was used in 15 Avinent® Coral HE implants . The implants had a diameter of 3.3 mm, were 10 mm long and had a surface sandblasted by alumina and oxidized with calcium and phosphorus.A Waterlase iPlus laser, a class IV Er,Cr:YSGG laser with 2780 nm wavelength  on one side, as recommended by the manufacturer to treat the peri-implantitis, and 2.5 W (84 mJ per pulse) on the other side . Three different tips were used. Thus, there were 3 subgroups of 5 implants treated with the 3 different tips , the side firing tip of 800 μm (SFT8-18 mm), and the cylindrical tip of 600 μm (MGG6-6 mm)), each implant being lased on opposite sides with 1.5 or 2.5 W/30 Hz.The website The irradiation of the implants was carried out to replicate a clinical scenario of peri-implant diseases treatment. The side firing and the conical tips were kept parallel and in contact with the surface of the implant, and continuous vertical movements were made from top to bottom , with a 2 mm per second speed. The irradiated area was of approximately 2 squared cm. In this way, the application resembles what occurs in patients with peri-implant diseases without opening a surgical flap.On the other hand, the cylindrical tip was applied perpendicular and oblique to the implant surface, thus mimicking the situation when a surgical flap is raised , The irrA gold bath on the implant surfaces using diode sputtering  was deposed. After sputtering, the implants were placed, one by one, in a Quanta 200 SEM  in an insulating cavity at a 55 degrees angle. The xT Microscope Control Software  was employed to process the digital images. A chemical analysis of the implants was performed using the EDAX Genesis program .The alterations of the implant surface were assessed and classified according to the following criteria:Class A: No visible damage.Class B: Minimal effects .Class C: Metal fall with melting of the surface, considerable fusion and crystallization, visible cracks and small crater-like defects.Class D: Complete alteration of surface including carbonization with large deep craters.The classification was carried out observing images at different magnifications  in each sample and comparing it with a control implant. To reduce the risk of bias when assessing this variable, two blinded researchers, unaware of the employed parameters and tips, analyzed different photos of samples with different magnifications (including the control). In case of discrepancy, a consensus was reach between the researchers.Chemical analysis of the implants disclosed several elements: titanium, calcium, aluminum, gold (coating) and phosphorus. .There is currently a great discussion about the best way to decontaminate the surface of implants in the treatment of peri-implantitis. The use of local or systemic medications, laser application, mechanical and/or chemical decontamination, and implantoplasty have been suggested in previous reports ,12,13. AAlthough a randomized clinical trial concluded that Er:YAG treatments might reduce bleeding on probing around implants in comparison with subgingival debridement alone , the avaPark et al.  studied A study  evaluate®, Zurich, Switzerland) or an Er:YAG laser on three different implant surfaces, machined, sandblasted and acid-etched (SLA) and hydroxyapatite (HA). Er:YAG laser irradiation was reported as the best option for decontamination of the HA-coated implants. The Er,Cr:YSGG laser is more effective in calculus removal and caused less surface roughness compared with citric acid application [Regarding of the decontamination methods, a recent study  evaluatelication . TherefoA study showed that the Er,Cr:YSGG laser with 1 Watt of power, for 30 s, is capable of the complete elimination of A.baumannii and P.aeruginosa biofilm on the implant surface without damaging the surface topography . RegardiThe present study examined different tips mimicking a clinical situation of implant debridement. It seems that the side firing and conical tips using 1.5 W with 40% water and 50% air at 30 Hz during 60 s are less likely to produce alterations to the implant surface. The calculated parameters estimate that the side firing tip (SFT8-18 mm) had less power density in comparison with the other tips, which, in our opinion, is an advantage. Another positive aspect of this tip is that, due to the lateral irradiation, it could be used to introduce the peri-implant sulcus without raising a flap.More studies are needed to confirm that the Er,Cr:YSGG laser, applied with the above-mentioned parameters and tips, effectively eliminates bacteria. Moreover, it would be interesting to assess whether the surface roughness modification reduces the bacterial growth after laser irradiation. These issues are paramount to determine the value of laser irradiation in the treatment of peri-implant diseases.Based on the present findings, an Er,Cr:YSGG laser with side firing and conical tips at 50 mJ (1.5 W/30 Hz), 50% air, 40% water, for 60 s, do not seem to produce implant surface alterations. A higher degree of damage should be expected when the energy per pulse increases to 84 mJ (2.5 W/30 Hz), especially when the conical tips are used.


Samples from longissimus thoracis, trapezius, triceps brachii and biceps femoris muscles, with different prevalences of red and white fibers, were selected. Among the fifty-three metabolites quantified in each of them, carnitine, carnosine, creatine and taurine are known for their bioactive properties. Twelve molecules were found to be differently concentrated in relation to muscle type. Longissimus thoracis, compared to biceps femoris, had higher concentrations of carnosine and formate and lower concentrations of mannose, inosine, threonine, IMP, alanine, valine, isoleucine, tyrosine, phenylalanine and leucine. A metabolic pathway analysis suggested that the main pathways differing among the muscles were connected to the turnover of amino acids. These results contribute to a deeper understanding of yak raw meat metabolism and muscle type differences, which can be used as an initial reference for the meat industry to set up muscle-specific investigations. The possibility of simultaneously quantifying several bioactive compounds suggests that these investigations could revolve around meat’s nutritional value.Yak represents the main meat source for Tibetan people. This work aimed to investigate the metabolome of raw meat from Jiulong yaks, focusing on specimens farmed and harvested locally through traditional procedures. Untargeted nuclear magnetic resonance spectroscopy ( Bos grunniens) bred in the world, 92% are located in the Himalayan highlands region [Of the 14 million specimens of yak  in a public abattoir (without modern slaughtering equipment) located in the pastoral area of Litang County , Sichuan, China, at the end of August. Yaks are generally slaughtered at this time of the year at three years of age, because they reach the highest weight and have optimal health conditions.post-mortem, one side of each carcass had been hung by the Achilles tendon and the other side had been hung using the pelvic suspension.The yaks had been transported to the abattoir and held in lairage for 24 h with water supplementation until 3 h before slaughtering. The yaks had been sacrificed following the local traditional manual procedures . As desctriceps brachii (TB), 5 samples of biceps femoris (BF), 5 samples of longissimus thoracis (LT) and 3 samples of trapezius (TP). The samples, weighing approximately 10 g, were immediately placed in bags at 4 °C. The discrepancies in the number of samples are linked to practical reasons during the slaughtering procedures. In detail, the TP muscle was generally removed together with the head, so that only three samples were collected. In the case of the other muscles, some samples could not be collected due to the impossibility to slow down the slaughtering procedures. This resulted in a total of 20 samples for metabolomics investigation. We transported all samples in dry ice and stored them prior to analysis at −80 °C.Two hours later, from 10 consecutively slaughtered animals we collected 7 samples of 1H-NMR analysis, we added two grams of meat to 12 mL of distilled water, and then we homogenized the mixture for 5 min by means of a high-speed disperser . Then, we centrifuged 1 mL of the mixed sample at 18,630 g and 4 °C for 15 min. To get rid of the fat fraction, we transferred 0.7 mL of the supernatant fraction to a new Eppendorf tube containing 0.8 mL chloroform, we vortex mixed for 3 min and we centrifuged again at the above conditions. We added the supernatants (0.5 mL) to 0.2 mL of a D2O solution, buffered at pH 7.00 ± 0.02 with a 1 M phosphate buffer, containing 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM, used as NMR chemical-shift reference, and NaN3 2 mM, used to avoid microbial proliferation. We finally centrifuged each sample at the above conditions.To create yak meat samples suitable for 1H-NMR spectra at 298 K by means of an AVANCE III spectrometer  operating at a frequency of 600.13 MHz. According to Zhu et al. [1H-NMR spectra processing is shown in We acquired u et al. , we suppu et al. , implemeu et al. . We consu et al. , appliedp < 0.05). Due to the low number of samples in TP group, we investigated the differences between LT and TP groups focusing on fold change, as suggested by Wang et al. [For statistical analysis we employed R computational language (ver. 3.6.1.) . We trang et al. .In order to obtain an overall of the trends underlying the metabolome of the samples, we calculated robust principal component analysis (rPCA) models  for the We highlighted the most relevant metabolic pathways differing among the muscle types by pathway analysis, performed by means of MetaboAnalyst 4.0 . For thi1H-NMR spectra of each yak muscle type are reported in 1H-NMR, carnitine, carnosine, taurine and creatine, are known as bioactive compounds. A bioactive compound is an extra nutritional constituent of food that can have a positive impact on body functions and may ultimately promote health [The e health .Twelve of the quantified molecules were significantly different among TB, BF and LT muscles, namely carnosine, formate, mannose, inosine, threonine, IMP, alanine, valine, isoleucine, tyrosine, phenylalanine and leucine .An rPCA model was built of these concentrations to obtain an overview of their trends, as shown in The PC 1 of its scoreplot A, represThe 12 molecules were used as a basis for a pathway enrichment analysis, to identify the most relevant pathways differentiating the muscles. Three pathways were highlighted: namely the valine, leucine and isoleucine biosynthesis pathway; the phenylalanine, tyrosine and tryptophan biosynthesis pathway and phenylalanine metabolism pathway.The last column of longissimus thoracis and trapezius characterized by negative PC 2 scores. PC 1 scores appeared mainly as associated to the ratio between red and white fibers, with muscles with a prevalence of white fibers characterized by positive scores.To relate the metabolome of TP muscle with those of the other muscles, we treated the TP samples as an independent test set by projecting them over the rPCA described in 1H-NMR, raw yak meat metabolome. A total of 53 metabolites were unambiguously identified, a twofold number compared to previous studies on cattle beef investigated relying on the same analytical platform [1H-NMR data reproduction is signals’ misalignment, caused by pH or ionic strength differences from sample to sample [1H-NMR spectra conveniently aligned, not requiring the application of algorithms for signal-by-signals shifting, differently from previous works [This is the first study that characterizes, through an untargeted approach by platform ,16. As ao sample . Figure us works . In addius works .LT muscle is characterized by a peculiarly high number of white, fast-twitching glycolytic fibers. However, TP muscle harbors a higher number of slow-twitching oxidative fibers, while TB and BF can be considered as intermediate between the two ,37,38. CUnivariate analysis showed that the muscle type had a profound effect on the amino acids overall profile, with significant effects on the concentration of 7 amino acids of the 14 measured. Moreover, enrichment analysis showed that all the most altered pathways were related to amino acids metabolism. A similarly profound effect of muscle type on amino acids profile has also been observed for cattle , with coThe fact that each of the seven amino acids showed a negative correlation with PC 1 scores suggests that the main reason for such trend is linked to the use of the muscles, more intense for legs and neck. The concentration of each of them was statistically different between BF and LT. Generally, TB showed intermediate values. An exception was represented by alanine, differently concentrated between TB and LT. Alanine is a non-essential amino acid, highly concentrated in muscles, where it serves as one of the major energy sources . GoldsteIMP pertains to the molecules derived from purines, often produced by ATP hydrolysis during the aging process of beef samples . DannertIn spite of potential applications of the information obtained in the present work as a base for the valorization of yak’s meat, it is important to notice that among all the molecules characterized, four, namely carnitine, carnosine, taurine and creatine, are appreciated for their bioactive properties.Carnitine is not an essential nutrient for humans, but 75% of it needs to be taken from food, mainly from meat. The major function of carnitine is to transport long chain fatty acids across inner mitochondrial membranes where the fatty acids are converted into biological energy by alpha-oxidation . Similar1H-NMR. As a primer in this context, we desired to analyze samples from the region where these animals are mainly bred and harvested through traditional procedures. This choice limited the number of samples analyzed, but a number of metabolites greater than previously reported for beef were still characterized. The metabolome profile which emerged appeared as strongly sensitive to the differences among muscles. This was particularly true for amino acids and degradation related molecules, suggesting therefore that the protocol described here could serve as a base for muscle specific studies of the transformations occurring in meat upon aging.This is the first work devoted to the characterization of the metabolome of raw yak meat by means of untargeted 1H-NMR, despite their chemical heterogeneity. The reference values listed in the present work set the basis for observations about the effects of processing practices on the nutritional value of meat, or for works aiming to valorize meat from this remarkable animal.The present experiment showed that several bioactive molecules could be simultaneously and readily quantified by