BackgroundIt is estimated that approximately 10 million individuals in Pakistan are infected with hepatitis C virus (HCV). Historically, it was very difficult not just to cure but even treat HCV as available options did not have desirable outcomes. However, the approval of directly acting antiviral (DAA) drugs has revolutionized treatment and management. These are specific proteases and polymerase inhibitors with profound capability for accomplishing elimination and overtime eradication of the virus.ObjectiveThe aim of this study was to evaluate the efficacy and safety of sofosbuvir (SOF) in combination with ribavirin (RIB) for the treatment of chronic hepatitis C virus with genotype 3.Materials and methodsThis prospective observational study was conducted at the gastroenterology section of Medical Unit IV, Jinnah Post-graduate Medical Center, Karachi and Medical Unit II, Dow University of Health Sciences, Ojha Campus, Karachi from January 2016 to December 2016. Patients aged 18 years or older of either gender having chronic active HCV infection as demonstrated by a positive Anti-HCV (ELISA) test and a qualitative polymerase chain reaction (PCR) analysis along with genotype analysis showing only type 3 were inducted into the study.Treatment was initiated with either 12-week or 24-week regimen of SOF 400 mg once daily along with weight-adjusted RIB orally. Successful treatment was indicated by the elimination of the virus, i.e., undetectable viral load/levels by PCR qualitative analysis. Rapid virological response (RVR), end of treatment response (ETR), and sustained virological response (SVR) were defined as the undetectable viral load at four, 12, and 24 weeks, respectively.ResultsA total of 300 patients were inducted into the study, predominantly female (57%). The mean age of presentation was 41.14 ± 11.48, and most (70.33%) were treatment naïve. The mean alanine transaminase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) levels at presentation were 41.89 ± 46.23 IU/l, 68.57 ± 83.62 IU/l, and 54.52 ± 77.57 IU/l, respectively.ALT, AST, and GGT levels at 24 weeks were 33.84 ± 13.60 IU/l, 32.44 ± 16.16 IU/l, and 37.59 ± 22.41 IU/l, respectively, showing significant improvement. ETR was achieved in 99.1% (209) treatment-naïve patients and 98.9% (88) treatment-experienced patients. SVR rates were almost similar with 98% (208) achieving it in the treatment-naïve group and 96.6% (86) achieving it in the treatment-experienced group.ConclusionSOF in combination with RIB is safe and remarkably efficacious in the treatment of chronic HCV, genotype 3. Not only is this regimen associated with the elimination of viral replication but it also improved transaminase levels. Outcomes are rarely, if ever, affected by previous use of antiviral medications. Hepatitis C Virus (HCV) affects about 170-200 million individuals worldwide and is the leading cause of chronic liver disease as well as decompensated liver disease in the developed world .HCV genotype-3 (GT-3) is the most common subtype in Pakistan. Previously, the only effective option for treating HCV infection was PEG-interferon. However, the outcomes with it were less than satisfactory, resulting in high morbidity and mortality especially with GT-3 . RecentlSofosbuvir (SOF) was the first DAA to be approved in December 2013 . Since tThis prospective observational study was conducted from January 2016 till December 2016 at Gastroenterology section of Medical Unit IV, Jinnah Postgraduate Medical Center, Karachi and Medical Unit II, Dow University of Health Sciences, Ojha Campus, Karachi. Written consent was taken from all patients. Patient confidentiality was made certain. All international standards were met, and the study was approved by the ethical board committee.Inclusion criteriaA total of 300 patients were enrolled. Patients aged 18 years or older of either gender having chronic active HCV infection as demonstrated by a positive Anti-HCV (ELISA) test and a qualitative polymerase chain reaction (PCR) analysis along with genotype analysis showing type three only were inducted into the study.Exclusion criteriaPatients with any of the following were ineligible for induction:1. Cirrhotics with compensated or decompensated liver disease Cirrhosis was ruled out on the basis of history with clinical  and biochemical markers 2. Patients who have already used SOF or any other DAAs for HCV3. Patients treated for previous malignancy or going under chemotherapeutic regimens.4. Active chronic hepatitis B Virus (HBV) infection, any other acute viral hepatitis (HBV carriers were eligible for induction)5. Immunocompromised patients such as those with acquired immunodeficiency syndrome (AIDS).6. Alcoholics and intravenous drug abusers7. Patients with other liver disorders such as autoimmune hepatitis, Wilson’s disease, etc.Primary outcomeThe primary outcome was to evaluate the efficacy of the regimen used. Efficacy was shown by complete elimination of the virus from the serum as indicated by a negative/undetectable viral load via PCR qualitative analysis at four weeks since starting medication, i.e., rapid virological response (RVR), 24 weeks since starting medication i.e. end of treatment response (ETR) and six months post-treatment, i.e., sustained virological response (SVR).Furthermore, RVR, ETR, and SVR were analyzed based on two categories. Treatment naïve group consisted of patients that had never taken any medication for HCV before and treatment experience group consisted of patients that had completed a course of any of previously available options to treat HCV. The treatment-experienced group had previously received PEG-interferon almost exclusively.Secondary outcomeThe secondary outcome was to record any adverse events (AEs), i.e., the safety of the drugs used, contributable to SOF and/or RIB use only during the treatment period. AEs could either be clinical, i.e., weakness, fever, etc., or laboratorial, e.g., reduction in hemoglobin (Hb), thrombocytopenia, hyperbilirubinemia, etc. AEs were monitored throughout the duration of treatment. Due to the vast array of AEs associated with RIB, only AEs that occurred during the study were analyzed and studied. Meticulous workup including nutritional, psychiatric, dermatologic, and orthopedic consults were done to ensure the AEs were solely due to the drugs used.Drug RegimenSOF inhibits the hepatitis C NS5B protein, therefore inhibiting viral replication. It is used in combination with multiple other drugs in treating HCV. RIB is a guanosine (ribonucleic) analog used to stop viral RNA synthesis and viral mRNA capping and therefore is a nucleoside inhibitor, while indicated for other purposes as well here it was used in the context of HCV only.Tablet SOF was given at a dose of 400mg orally once (OD) daily in combination with tablet RIB 400mg orally twice (BID) daily. The treatment was continued for 24 weeks in all patients. Laboratorial AnalysisAll patients were followed up with a complete blood count (CBC), liver function tests (LFTs), prothrombin time (PT), international normalized ratio (INR), urea with creatinine levels, and serum electrolyte levels at induction, four, 12 and 24 weeks after starting medications. Ultrasound abdomen was also performed at initial evaluation. Coinfection with HBV and human immune virus (HIV) were ruled out via their respective serologies. On rare occasions, where other liver pathologies were strongly suspected, work up with an autoimmune profile, 24-hour urinary copper, serum ceruloplasmin, and iron profile was carried out.Data Analysist-test; chi-square test was used to evaluate the association of RVR, ETR, and SVR with the type of treatment, whereas repeated measures with ANOVA were applied to compare means of hematologic parameters measured at different time intervals. A p-value of <0.05 was considered statistically significant.In this study, non-probability convenient sampling technique was used. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 21.0 software . Data were presented as mean ± standard deviation (SD). The comparisons were performed with a paired Demographic characteristicsThe mean age of presentation was 41.14 ± 11.48 years. Patients were predominantly female (57%) and married (96%). Most patients presented were residents of Karachi (78%) and a high number (75%) did not have any comorbids. Demographics are summarized in Table Primary outcomeA high number of patients showed elimination of virus as evident by the excellent rates of RVR, ETR and SVR when treated with SOF+RIB. No correlation or significance was found between the treatment naïve or experienced groups. Overall only 6 2%) patients out of 300 did not achieve SVR. The high rates of SVR demonstrated great efficacy on part of the drug regimen. Primary outcomes with comparative analysis of the two treatment groups are summarized in Table % patientSecondary outcomeAdverse EventsAdverse events were far and few. The most common complains for the patients were fatigue (70.66%) and body ache 54.33%). Disease progression to decompensated cirrhosis was not seen at all. There was no incidence of melena, hemetemesis, hepatic encephalopathy, or development of ascites. Clinically, there were no signs of pallor (anemia) or shortness of breath in any of the patients (see below). Adverse events are summarized in Table 3%. DiseaSubgroup Analysis Majority of the patients were females aged between 24-45 years for both treatment-experienced and naïve groups; 263 (87.33%) had no comorbidity. All factors favor improved outcomes. Subgroup analysis are summarized in Table Laboratorial/Biochemical ParametersThe only concerning aspect was the mild decrease in hemoglobin level; this was found to be statistically significant. However, as discussed previously this did not have any clinical significance. All LFTs showed improvement over time. Analysis of labs is summarized in Table HCV GT-3 was always cumbersome to treat, having substantially lower viral elimination rates, higher relapses, and overall poorer prognosis when treated with Peg-Interferon and RIB . SVR ratThe arrival of DAAs changed all of this. SOF led the way, with cure rates of ≥90 %. An exceptionally high SVR rate of 98% has been observed in our study, as reported by Zeuzem S. et al. . There cOut rate of SVR is impressive, but few things must be taken into consideration. First, irrespective of the treatment group (naïve vs. experienced), our patients did not have signs and symptoms of cirrhosis or decompensation. Second, most patients were young or middle-aged. Thirdly, there were few comorbidities in our patients. All three factors are good prognostic markers for viral elimination.Alcohol use disorder (AUD) is rare in Pakistan and was absent among our patients to the best of our knowledge, this was again an important factor contributing to the impressive SVR. Low rates of non-alcoholic fatty liver disease (NAFLD) were seen in our study corresponding to low burden of diabetic patients, no doubt contributing to a high SVR.A vast array of AEs was seen but none were troublesome enough for patients to drop out, affect their compliance or affect their quality of life. Furthermore not all AEs such as dyspepsia, body ache and bleeding per rectum could be attributed solely to the drug regimen despite comprehensive workup (see methods). Fatigue, the most common AE that could clearly be attributed to SOF did not affect the quality of life significantly and was easily overcome.Fever was documented, mostly low to moderate grade and resolved over time, correlating with viral elimination. Abdominal pain was vaguely localized but consistently involved the right upper quadrant and showed resolution with the initiation of treatment. Significant weight loss was not seen at all.The one major concern was a mild reduction in the Hb levels. This was most probably due to RIB. The MCV demonstrated normocytic normochromic levels throughout the study period and as it did not have any serious impact nor did any patients complain of shortness of breath, this was ruled out to clinically non-significant. Hb levels were too high to be associated with fatigue.HCV is associated with deranged LFT’s . At presSOF in combination with RIB has great efficacy and safety for treatment of chronic HCV. The regimen is associated with high viral eradication rates and improvement in the liver biochemical profile with very few adverse events. It is superior to all previous medications used for HCV. SOF + RIB should now be recommended for the treatment of HCV until superseded by a newer and better regimen.


Harpago chiragra and Lambis lambis (Strombidae) were determined with the size of 15,460 bp and 15,481 bp, respectively, and both sequences contained 13 protein-coding genes, 22 tRNAs, and two rRNAs. H. chiragra and L. lambis have similar mitochondrial features, corresponding to typical gastropod mitochondrial genomes, such as the conserved gene order, a high A + T content (66.22% for H. chiragra and 66.10% for L. lambis), and preference for A + T-rich codons. The start or termination codon of same protein-coding gene in H. chiragra was consistent with that in L. lambis, except for the termination codon of cox1 gene (TAG for H. chiragra and TAA for L. lambis) and the start codon of nad4 (GTG for H. chiragra and ATG for L. lambis). Pairwise sequence alignments detected different degrees of variations in H. chiragra and L. lambis mitochondrial genomes; and the two species had lower levels of genetic distance (0.202 for nucleotide sequence) and closest relationships as compared to Strombus gigas and Oncomelania hupensis. The 13 partitioned nucleotide sequences of protein coding genes of H. chiragra and L. lambis were aligned with representatives of the main lineages of gastropods and their phylogenetic relationships were inferred. H. chiragra and L. lambis share the same gene order as Littorinimorpha species, except Vermetoidea, which demonstrate a gene rearrangement in species. The reconstructed phylogeny supports three major clades within Littorinimorpha: 1) Stromboidea, Tonnoidea, Littorinoidea, and Naticoidea, 2) Rissooidea and Truncatelloidea, and 3) Vermetoidea. In addition, a relaxed molecular clock calibrated with fossils dated the diversification of Strombidae near 112 (44–206) Mya and a possible radiation is detected to occur between 45–75 Mya, providing implications to understand the Cenozoic replacement event (65–135 Mya) of Aporrhaidae by Strombidae.The complete mitochondrial genomes of  The mitochondrial DNA has a high rate of base substitution and lacks of recombination during inheritance; besides it possesses an unique transmission mode named doubly uniparental inheritance (DUI) in molluscs5. Hence mitochondrial genomic analyses was proved as a valid molecular tool in constructing phylogenies, and has been used for phylogenetic analyses in various taxa7.Molecular phylogenetic analyses provided a different approach compared with traditional morphological methods to estimate the relationships among species based on the topological hypotheses3. Neogastropoda was widely accepted as a monophyletic group based on morphological characters; however, Tonnoidea was placed into Neogastropoda based on the molecular analyses11, which contradicted the monophyletic status of Neogastropoda. Among Littorinimorpha, Vermetidae is a peculiar snail family that shows a high rate of gene rearrangement12. Based on the molecular phylogenetic analyses, Vermetidae were regarded as the sister group of the other species in Caenogastropoda,; however, this is opposite to the morphological evidence13. Hence, the relative position of Vermetidae in the mitochondrial phylogenetic analyses has been considered spurious, although the relationship was highly supported in previous molecular phylogenetic analyses15.The derived phylogenetic relationships based on molecular data may disagree with the evolutionary hypothesis proposed using morphological data19. Species in Stromboidae varied greatly in shell shapes, which results in high morphological diversity20. Strombus  and Lambis  are the two most abundant genera in Strombidae and were once regarded as the only two genera in strombids21. However, based on the fossil record and molecular phylogenetic analyses, the genus Strombus is justified to be subdivided into several separate genera21.Strombidae species are important molluscs in shallow water of tropical and subtropical areas from past time until now19. During the course of evolution, whereas Aporrhaidae species underwent K/T mass extinction in late Cretaceous, a major genera and species radiation in Strombidae occurred during the early Cenozoic and continued to the Pliocene23.Based on the paleontological studies, Strombidae probably originated from Aporrhaidae during Cenomanian-Turonian, and evolved at low diversities during the rest of the CretaceousH. chiragra and L. lambis, and analyzed the genomic features of the two species, including their structural characters and nucleotide composition. In early taxonomical studies, H. chiragra shared close relationships with L. lambis in Strombidae, yet this was mainly based on their similar tissue anatomies 18, regardless of the great morphological difference in adults. To valid the taxonomy relation between H. chiragra and L. lambis, we attempt to determine their phylogenetic relationships based on the mitochondrial genomes. Thus, a robust phylogeny based on the concatenated 13 protein coding genes of 15 Littorinimorpha species was constructed. These data provides a framework for further evolutionary studies among Littorinimorpha.In the present study, we determined the complete mitochondrial genomes of H. chiragra and L. lambis were collected from the coastal waters of Quanfu Island, South China Sea. Total genomic DNA was extracted from the foot muscle using a modified standard phenol-chloroform procedure24 and then stored at −20 °C.Individuals of cox1 gene were PCR amplified using the universal primers LCO-1490/HCO-219825. Based on the reference genome of S. gigas26, primers were designed using Primer Premier 527 to amplify short fragments of atp6, cox3, and cytb (Supplementary Table Short fragments of 2+), 1 μL of template DNA (100 ng/μL), 1 μL of each forward and reverse primer, and 0.5 μL of TaKaRa LA-Taq DNA polymerase. The thermal cycling conditions are: 94 °C for 3 min followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 68 °C for 5 min, with a final extension step of 72 °C for 10 min. PCR products were purified using an EZ-10 Spin Column DNA Gel Extraction Kit (Sangon Biotech), and then directly sequenced using the primer walking method. DNA sequencing was performed on an ABI PRISM 3730 (Applied Biosystems) automatic sequencer.PCR was performed in a 30 μL reaction mixture containing 3 μL of dNTPs (2.5 mM each), 3 μL of 10 × LA buffer (Mghttps://www.ncbi.nlm.nih.gov/orffinder/) with invertebrate mitochondrial genetic code. The transfer RNA (tRNA) genes were identified using ARWEN (http://130.235.46.10/ARWEN/) and MITOS web servers (http://mitos.bioinf.uni-leipzig.de/index.py) using the mitochloroplast or invertebrate gene code and default search code.Sequence assembly was performed using the Seqman program, DNASTAR (http//www.DNASTAR.com). The annotations of protein-coding genes were conducted using ORF Finder (rrnL and rrnS were conducted using BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by identifying their similarity to gene sequences of S. gigas and Conomurex luhuanus. The A + T content values were computed using MEGA 6.0628 and GC and AT skews were calculated according to the formulae described before29, AT skew = (A − T)/(A + T); GC skew = (G − C)/(G + C), where A, T, G, and C are the occurrences of the four nucleotides. The relative synonymous codon usage (RSCU) values of each protein coding gene were calculated using MEGA 6.0628. The number of base substitutions per site between H. chiragra L. lambis, S. gigas, and C. luhuanus were calculated in MEGA 6.0628 using the Kimura 2-parameter model30.The gene annotation of H. chiragra, L. lambis and 13 other available mitochondrial genomes of Littorinimorpha  and Bayesian inference (BI). ML analysis was conducted using RAxML37 web server on the CIPRES Science Gateway V.3.3 (http://www.phylo.org/index.php/) based on the partitioned nucleotide alignments, with GTR + G substitution model and 1,000 bootstraps for node reliability estimation. Bayesian analyses were conducted in MrBayes38 for 200 million generations (sampling every 1000 generations) based on the partitioned nucleotide and amino acid alignments. All parameters were checked using Tracer v1.539. The first 50,000 trees were discarded as burnin, and the remaining sampled trees were used to estimate the Bayesian posterior probabilities.The phylogenetic analyses were based on the concatenated nucleotide and amino acid alignments of thirteen protein-coding genes in seventeen complete mitochondrial genomes, including 39 based on the partitioned amino acid sequences of 13 protein coding genes. A lognormal relaxed-clock model was selected as the molecular clock model. A Yule process of speciation was chosen for the tree prior. The final Markov chain was set to 100 million generations, sampling every 10,000 generations. The effective sample size of all parameters was above 200. The convergence of the chains was checked with Tracer v.1.539, and the first 1,000 generations sampled were discarded as part of the burn-in process.The estimation of divergence time of the major Littorinimorpha lineages were conducted using BEAST v.1.7.540, and the prior divergence time of Truncatellidae was set as 66.04 Mya according to the oldest fossil record of Paleocene in Belgium43. Besides, the divergence time of Tegula was 85 Mya based on the Cretaceous fossil record in United States41, and this point was used to cross-validate the accuracy of the dated tree.The posterior distribution of the estimated divergence times was specified based on the prior fossil knowledge. Two calibration points were selected, using a normal distribution of prior probability: 342.8 Mya was used as prior divergence time for Vermetoidea based on the Paleocene fossil collection in Belgium and the United KingdomH. chiragra and L. lambis are 15,460 bp and 15,481 bp, respectively  and trnS. The (−) strand encodes for the cluster MYCWQGE  and trnT  strand were both biased toward A and T. For H. chiragra and L. lambis were calculated. The A + T content of protein coding genes (PCGs), tRNA, rRNA, and non-coding regions (NCRs) is similar between H. chiragra and L. lambis (Table cox3 (60%), nad2 (68%) and nad4 (66%).The nucleotide composition of the single gene region of is Table . For sinH. chiragra and L. lambis show positive AT skews, whereas all types of protein coding genes show negative AT skews. Both the tRNA and rRNA genes of H. chiragra and L. lambis show positive GC skews. Some protein coding genes  show negative GC skews, while the other types of protein coding genes and RNA genes  show positive GC skews.Among the different types of genes, the tRNA genes and rRNA genes of H. chiragra and L. lambis encode 3,744 and 3,745 amino acids, respectively. Comparison between species of start and termination codons of protein coding genes showed that only 2 PCGs initiate or stop with different codons, which were detected in nad4 gene (initiated with GTG in H. chiragra and ATG in L. lambis) and cox1 gene (stopped with TAG in H. chiragra and TAA in L. lambis). In addition, 12 PCGs contain the same start  and termination  codons between species . The relative synonymous codon usage (RSCU) is different between H. chiragra and L. lambis, implying their larger genetic difference than previously recognized . This location was proposed as a candidate to contain the control region in other gastropod mitochondrial genomes49. Among the non-coding regions, there were 20 regions with different lengths between H. chiragra and L. lambis and 14 intervals with same length.There were 34 non-coding regions distributed in the bis Fig. . The nonS. gigas + (C. luhuanus + (L. lambis and H. chiragra))). L. lambis is the closest extant relative of H. chiragra, and this clade clustered with S. gigas and C. luhuanus. Research derived from combined phylogenetic analyses of molecular and morphological data has revealed that Lambis was monophyletic and Strombus was paraphyletic20. However, when the cladistics analyses of species in Lambis were based solely on morphological characters, the results clustered one Lambis species (L. crocata) into the outgroups of species50, suggesting that Strombus is polyphyletic and the Lambis is paraphyletic. Lambis crocata was not included in the present study since there is no complete mitochondrial genome available for this species. Although lacking a sufficient number of species for a robust phylogenetic analysis, our phylogeny is statistically supported and aims to provide a reasonable framework for further phylogenetic research within Stromboidea.The selected partition schemes for phylogenetic analyses were listed in Supplementary Tables Within Littorinimorpha, Stromboidea and Tonnoidea clustered together in the same clade, which was then clustered with (Littorinoidea + Naticoidea), as derived by the BI method. Stromboidea, Tonnoidea, Littorinoidea, and Naticoidea form a well-supported clade based on both ML and BI, confirming their close relationship within Littorinimorpha. Rissooidea was sister to Truncatelloidea, which together formed the second major clade. Vermetoidea formed the third independent well-supported clade within Littorinimorpha.Tegula. The oldest fossil record of Tegula was stated as 85 Mya41 and the documented time was coincident with the divergence time of T. lividomaculata and T. brunnea (13–309 Mya) in present study. Research documented that the number of species increased from five species within the genus Strombus during the Eocene (53 Mya) to 40 species till the Late Oligocene and the Miocene (23–36 Mya)16, indicating that the species diversification within Strombidae accelerated in the last 36–53 Mya. According to the present dated tree (Fig. 23. Furthermore, to better resolve the phylogenetic relationships and understand the replacement event, more Strombidae and Aporrhaidae mitochondrial genomes should be inserted into the phylogenetic analyses.To test the accuracy of the dated tree derived from BEAST, we made a cross-validation using the calibration point of genus Dataset 1


In conclusion, this study reports that DSL, DSL•m−1, and FI can be valid metrics to monitor fatigue related to movement strategy during a standardized submaximal intermittent exercise protocol.The aim of this study was to assess the sensitivity of accelerometer-derived metrics for monitoring fatigue during an intermittent exercise protocol. Fifteen university students were enrolled in the study (age 20 ± 1 years). A submaximal intermitted recovery test (Sub-IRT) with a duration of 6 min and 30 s (drill 1) was performed. In order to increase the participants’ fatigue, after that, a repeated sprint protocol  was performed. Following that, participants repeated the Sub-IRT (drill 2) to evaluate the external and internal training load (TL) variations related to fatigue. Apex 10 Hz global navigation satellite system (GNSS) units were used to collect the variables total distance (TD), high metabolic distance (HMD), relative velocity (RV), average metabolic power (MP), heart rate maximal (HRmax) and mean (HRmean), muscular (RPEmus) and respiratory rating of perceived exertion (RPEres), dynamic stress load (DSL), and fatigue index (FI). A Bayesian statistical approach was used. A likelihood difference (between drill 1 and drill 2) was found for the following parameters: TD (BF The quantification of training load (TL) is important for improving fitness adaptations and reducing the risk of injury in team sports . A commoTypically, GNSS-based athlete monitoring devices are also equipped with accelerometers capable of measuring linear accelerations in three axes . The comThe composite derivatives of acceleration have been previously utilized to assess a player’s fatigue in team sports . FatigueTo date, information related to other accelerometer-based training load metrics used to monitor fatigue during intermittent exercises is limited; therefore, new evidence related to transient/residual fatigue monitoring using alternative algorithms may be very important for the management of players’ training loads, recovery strategies, workload implementations, and subsequent training periodization . TherefoFifteen physically active university male students were enrolled  in this study. Inclusive criteria for participation were the absence of any injury or illness by completing the Physical activity readiness questionnaire (PARQ), and regular participation in physical activity (minimum two sessions per week). Participants were asked to maintain their habitual consumption and avoid alcohol 24 h prior to exercise. Consumption of ergogenic aids was not allowed during the protocol. The experimental protocol was conducted in accordance with the Declaration of Helsinki. The Institutional Ethics Board of the University of Suffolk  approved the experimental protocol. Written informed consent was obtained from all participants prior to participation in this study.Apex 10 Hz GNSS  units were used to collect data on a synthetic outdoor pitch, in the absence of high and large buildings in the surrounding area to enhance satellite reception . Apex unThe Apex units were turned on 15 min before the beginning of the test, while the subjects were familiarized with the equipment as well as the protocol procedures. Apex units present the following characteristics: dimension 30 mm (wide) x 80 mm (high), weight 48 g, and 100 Hz triaxial accelerometer. Prior to the experiments, Apex unit models were placed on the back of the participant, midway between the scapula. The Apex 10 Hz is a multi-GNSS augmented unit; the validity and reliability of this unit has previously been reported . GNSS da−1 (−2) (.kg−1 (HMD) were estimated using the rationale reported in the following paper  , and relng paper .k is a weighting factor and SF a scaling factor. An IMPACT is defined as the maximum composite acceleration value of every 10 accelerometer points measured, if the maximum value is greater than 2G. Fatigue index (FI) is the ratio between a player’s instantaneous speed, measured by the GNSS sensor, and the linear acceleration values. Raw sensors’ readings are converted to a common scale by using a convex weighting function, similar to DSL. Therefore, FI is as follows:K is a weighting factor. Reliability of accelerometer data has previously been reported . Acceleration values were then aggregated over the defined period to obtain a measure of dynamic stress load using the following equation:reported .Participants’ internal TL was quantified using maximum HR (HRmax), which was recorded during each drill, while mean HR (HRmean) was the average HR recorded during each drill. HR was recorded during drills using Polar T31 belts  . At the a err prob. = 0.05, power = 0.8), participants = 15, estimated power = 0.82  on TL variables. Estimates of median standardized effect size (ES) and 95% credible interval (CI) were calculated  was set as BF10 > 3 and evidence for null hypothesis (H0) was set as BF10 < 1/3. BF10 was reported to indicate the strength of the evidence for each analysis. The BF10 was interpreted using the following evidence categories: 1 < BF10 < 3 = anecdotal evidence for H1; BF10 ≥ 3 = moderate; BF10 ≥ 10 = strong; BF10 ≥ 30 = very strong; BF10 ≥ 100 = extreme  software version 0.9.1. Data are presented as mean ± SD. The reliability between drill 1 and drill 2 was assessed using the typical error of measurement and expressed as percentage of the coefficient of variation (CV) . A CV(%)r = 0.82 . Externar = 0.82 . A pairelculated . ES was ge > 2.0 . Evidenc extreme .Apex 10 Hz GNSS data recorded during drill 1 and drill 2 and reliability are reported in 10 = 0.33, 0moderate per H), HMD , RV , MP , accelerations , HRmax , HRmean , RPEmus , RPEres , DSL , DSL•m−1 , and FI .Bayesian analysis related to differences between drill 1 vs. drill 2 reported the following strength of the evidence: TD  and TL monitoring in sports.The aim of this study was to assess the sensitivity of novel accelerometer-based metrics  at detecting player’s fatigue recorded using 10 Hz GNSS units, with an embedded accelerometer, during an intermittent submaximal standardized exercise protocol. This study is the first to report that DSL, DSL•m−1, and FI) increased in drill 2 vs. drill 1, demonstrating sensitivity when monitoring physical variations related to fatigue. These results may have great applicability in professional team sports, where such player’s fatigue variations may have an impact on fitness adaptations and injury risk reduction  did not change between drill 1 vs. drill 2 , while ieduction . The valeduction . Moreoveeduction . These reduction . DSL mayeduction .A limitation related to this study is the sample utilized that is composed of physically active university male students. Therefore, the results reported in the current study should be considered carefully when elite and professional football players are monitored since fatigue may be associated with physical activity level and sport specificity. This is supported by previous evidence that showed a different energy cost between sporting populations during intermittent running exercises .In conclusion, accelerometer-based metrics like DSL and FI are sensitive for monitoring player’s fatigue during a submaximal intermittent exercise protocol. Future studies could replicate the current study involving elite and professional players to clarify if such metrics may be valid when monitoring fatigue in a sport-specific population..m−1, and FI by GNSS-embedded accelerometers during a Sub-IRT in order to monitor in a valid and quick way (throughout the season) athletes’ fatigue. This approach may offer further support to practitioners’ decision-making process.DSL can be used as an external TL metric with confidence when monitoring team sport athletes . Practitioners may evaluate DSL, DSLMB and MC have designed the study. MB, KD, and BC have recorded the data. MB and MC have performed the data analysis. MB, KD, BC, and MC have written the paper.MC was employed at the time of the study by STATSports.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microlaena stipoides) in Si-supplemented and untreated soils.Grasses are hyper-accumulators of silicon (Si) and often up-regulate Si following herbivory. Positive correlations exist between Si and plant water content, yet the extent to which Si uptake responses can be mediated by changes in soil water availability has rarely been studied and never, to our knowledge, under field conditions. We used field-based rain-exclusion shelters to investigate how simulated grazing (shoot clipping) and altered rainfall patterns  affected initial patterns of root- and shoot-Si uptake in a native Australian grass  of Results highlight the importance of soil water availability for Si uptake and suggest a role for both active and passive Si transport mechanisms. Such manipulative field studies may provide a more realistic insight into how grasses initially respond to herbivory in terms of Si-based defence under different environmental conditions.The online version of this article (10.1186/s12898-018-0208-6) contains supplementary material, which is available to authorized users. Investment in defence requires resources that could otherwise be invested in growth or reproduction, and are thus costly to the plant. In order to mitigate these costs, many plant defences are induced in response to damage instead of being constitutively expressed , 2. Many foliage . While g foliage ).Many grasses are hyper-accumulators of silicon (Si), sometimes accumulating up to 10% of their dry mass, more than any other inorganic constituent . Their cWhile changes in the Si-accumulating responses of grasses have been linked to grazing , 12, and4) from soil water by the roots [Plants can only acquire Si via uptake of silicic acid (Si(OH)he roots . Thus, dhe roots , 20, havhe roots –23, yet he roots , 24, whehe roots , 26.Microlaena stipoides Labill.) respond to water stress (drought and elevated precipitation), simulated grazing (i.e. clipping) and the application of Si to the soil, with a particular focus on changes in silicification of above- and below-ground tissues. Microlaena stipoides is common within grazed native pastures in the high rainfall zone  of south-eastern Australia [M. stipoides roots and shoots, which would be enhanced by increased levels of precipitation. Moreover, Si might substitute for C as a cheaper plant structural resource [Using a field-based rain-exclusion experimental facility, this study aimed to determine how the growth and chemical characteristics of weeping grass  located at the Hawkesbury campus of Western Sydney University near Richmond, NSW , as described by Johnson et al. , were us88 cm locMicrolaena stipoides (var. Burra) seeds were sown and grown in 288 individual seed cells (38 × 57 mm) under field conditions. After 4 weeks (on 26 September 2016), four cells were transplanted into each mesocosm pot. Shelters were assigned at random to one of three rainfall treatments giving six shelters per rainfall regime. The ambient treatment was set at 65 mm per month, which was the average precipitation in Richmond between September and November over the previous 30 years . The drought and elevated precipitation treatments comprised 50% and 150% of the ambient rainfall amounts, respectively. This translated to an application of 586 mL (ambient precipitation), 293 mL (drought) and 878 mL (elevated precipitation) of rainfall water collected at the site three times per week. Soil moisture measurements were taken weekly using a 12 cm Hydrosense II probe .Six 2SiO3.9H2O) into the water applied under each watering regime. Four weeks after transplantation, grasses in two of the four mesocosms (one with Si applied and one without) were clipped, using hand shears, to leave approximately 40% of their aboveground biomass. The factorial treatment design under each shelter therefore comprised one mesocosm with Si applied alone (Si), one mesocosm with clipped grasses alone (Clipped), one mesocosm with both treatments applied (Si + Clipped), and one mesocosm with neither . Carbon (C) and nitrogen (N) concentrations were determined with a Carlo Erba CE1110 elemental analyser and total Si concentrations (% of dry soil mass) were determined with an X-ray fluorescence spectrometer , as described in I Hiltpold, L Demarta, SN Johnson, BD Moore, SA Power and C Mitchell . The “upnlme statistical package [glht function in the multcomp package [cor.test in the package stats.The R statistical interface v3.3.3  was used package , with me package . Where nSoil moisture was significantly affected by the interaction between rainfall and Si-application Table , whereby−1). The application of Si had no significant effect on the concentration of Si in the roots or shoots of M. stipoides, however. When plants were clipped, the concentrations of Si in the roots and shoots increased by 12 and 41%, respectively, compared with those that were not clipped , which increased by 84% under elevated precipitation (11.87 ± 0.75) and decreased by 40% under drought (3.89 ± 0.35) relative to ambient rainfall conditions (6.46 ± 0.76). In general, average soil moisture content was positively correlated with root  and shoot Si concentrations  and negatively correlated with root  and shoot C concentrations . Root N concentrations increased and decreased under elevated precipitation (1.68 ± 0.03%) and drought (1.32 ± 0.03%), respectively, relative to ambient rainfall (1.53 ± 0.04%), whereas shoot N concentrations did not vary between treatments. Rainfall, Si-application and clipping had no significant effects on soil C or N concentrations , though treatment concentrations were very low (1 mM) and field soil has the capacity to dissipate impacts. In any case, plants would most likely be unaffected as uptake of Na tends to decrease as plants take up Si [The application of Si to the soil increased the retention of water in the soil, especially as rainfall increased, which was likely responsible for the increase in plant height in Si-supplemented plots observed before the clipping treatment was applied. The observed increase in soil water could be associated with either a physical effect of Si-application on the soil profile  or an ike up Si .4SiO4 by M. stipoides may already be sufficient in the natural soil environment for optimal growth [Silicification of leaves has been shown to reduce water loss by as much as 30% in rice  and incrl growth . Under sl growth .M. stipoides shoots relative to that of their roots was maintained under drought but was significantly higher under elevated precipitation, which was also evident in plants that had been clipped. The maintenance of shoot Si deposition under drought may be caused by or responsible for the drought tolerance that M. stipoides is generally known for [Root and shoot Si concentrations and Si uptake efficiency were positively correlated with soil moisture, demonstrating the importance of plant water status in driving Si uptake and storage . The accnown for , likely nown for . Other gnown for  or reducnown for , 46. Spenown for . Alternanown for .M. stipoides responded to clipping by storing Si in its roots and shoots, indicative of an induced defensive response, which was consistent across all watering regimes. Kindomihou et al. [Silicification patterns in roots are understudied, despite evidence that Si concentrations can occur at highest levels in these tissues . This stu et al.  also ideu et al.  identifiu et al. . The effMicrolaena stipoides may therefore show an even greater defence response in the form of higher grazing-induced uptake of Si when subjected to herbivore attack. High levels of rainfall may further enhance Si uptake by increasing transpiration overall [Simulated herbivory may not mimic natural damage exactly but it enables researchers to control the type, timing and intensity of damage with fewer confounding effects , which i overall , with im overall . Above-gThis study provides clear evidence that water availability influences Si-based responses to damage in plants under field conditions. In particular, Si uptake responses to simulated grazing were enhanced when soil water availability was not limited, which are likely linked to a combination of active and passive uptake mechanisms. Field-based approaches such as this are fundamental to our understanding of how biotic and abiotic factors contribute to silicification in plants . StudiesAdditional file 1: Table S1. Chemical composition of excavated soil (N = 12) used in the experimental study.


Ostrya rehderiana and its widespread congener O. chinensis. Both species have similar demographic histories prior to the Last Glacial Maximum (LGM); however, the effective population size of O. rehderiana continued to decrease through the last 10,000 years, whereas O. chinensis recovered to Pre-LGM numbers. O. rehderiana accumulated more deleterious mutations, but purged more severely deleterious recessive variations than in O. chinensis. This purging and the gradually reduced inbreeding depression together may have mitigated extinction and contributed to the possible future survival of the outcrossing O. rehderiana. Our findings provide critical insights into the evolutionary history of population collapse and the potential for future recovery of the endangered trees.Increased human activity and climate change are driving numerous tree species to endangered status, and in the worst cases extinction. Here we examine the genomic signatures of the critically endangered ironwood tree  Here, Liu et al. compare the genomic signatures of two tree species, one of which had undergone population collapse. The declining species had low genetic diversity, but had more strongly purged severely deleterious recessive variations, likely due to inbreeding and perhaps mitigating extinction risk. For example, during the last glacial maximum (LGM), the ranges of most temperate species moved south and contracted as the temperature decreased6. Although many species recovered at the end of LGM and rapidly expanded north with Holocene climate warming, numerous species became extinct7 or failed to recover to pre-LGM population sizes9. At the same time, human populations expanded rapidly in the Holocene10 and negatively affected plant and animal recoveries through both hunting and land clearing12. Increasing human disturbance and climate warming in the modern era are raising further concerns of accelerated worldwide extinction rates2.The combined effects of changing climate and human activities are assumed to have reduced species throughout the world to critically small population sizes13 and provide a detailed empirical description of the impact of population collapse on genomic diversity and genetic load14. In endangered Felide, for instance, population declines predate the beginning of the Holocene16, and these and subsequent bottlenecks have resulted in the accumulation of genetic load and excessive runs of homozygosity that span the genome18. Similar patterns of long-term decline are apparent in the genome of Mountain Gorillas, which caused extensive homozygosity and increased genetic load19. Paradoxically, this study also revealed a reduction in severely deleterious loss-of-function variants, perhaps resulting from the increased efficacy of purging of deleterious homozygous recessive alleles in small populations20, which may have helped Mountain Gorillas survive at a low population size for thousands of generations. To date, genomic studies of critically endangered species have been conducted on animals, but plants have unique aspects to their life histories that may result in different outcomes as population size declines3. For instance, most plants are capable of self-pollination21, which even at low levels provides a mechanism of systematic inbreeding not present in animals that will strongly contribute to purging of deleterious recessive alleles22.Studies of the demographic history and extent of the genomic erosion leading to endangerment may both inform management and policy decisions23. Their extinctions will inevitably lead to additional extinctions of species that rely on trees as a component of their fundamental niche. A total of 1208 trees are currently listed as critically endangered24; however, it remains unknown how these foundation species became endangered2 and why they have survived longer than has been predicted3. The most effective way to increase the population size and conserve endangered trees is to directly plant wild-collected seeds or clonally propagate genotypes with stem cuttings25, but these strategies require oversight because they may exacerbate inbreeding if parental genomes are not well represented, especially if the source population is extremely small. Here we aim to address these fundamental questions through comparing genomic patterns of diversity between the critically endangered Ostrya rehderiana (IUCN Red List)24 and the widespread O. chinensis. O. rehderiana is native to southeastern China where rice was first domesticated27. This region has been heavily populated by humans for thousands of years. Although seeds from the remaining few large trees have been successfully germinated and grown to maturity29, wild populations may soon be extinct. The close relative O. chinensis (=O. multinervis), however, has relatively large wild populations, which are distributed from southeastern China to the high mountains of southwestern China30, an area that was only recently colonized by humans and remains sparsely populated32. Wood produced by both species is extremely hard and is highly prized for construction of boats and religious temples33. Both of these species are deciduous, monecious, primarily outcrossing, and rarely reproduce clonally34, so factors impacting historical fluctuations in population size likely are associated more with extrinsic factors associated with the locations of their ranges than differences in life history.Trees comprise the dominant elements of the terrestrial landscape and are foundations for ecological stability and longevity in many biomesO. chinensis and O. rehderiana and re-sequenced 13 additional individuals of each species for population genomic analyses. Today only five O. rehderiana very old individuals (>100 years old) currently reside in a single wild population, and about 30 years ago, ~300 O. rehderiana trees were planted from successfully germinated seeds intentionally collected from the old stand, which at that time was comprised of six or seven mature trees35. Comparisons between the old and young O. rehderiana trees allowed us to contrast the very recent impacts of inbreeding on genomic diversity to longer-scale demographic impacts. Based on these genomic data, we addressed the following questions: (1) do these two species show similar demographic histories in response to the Quaternary climate change? If not, when did their demographics begin to diverge? (2) Have deleterious variations accumulated in the endangered species at a greater rate than in the widespread tree, and have they impacted the potential to recover? and (3) have more highly recessive deleterious variations been purged by drift in the endangered species? The answers to these questions will aid in identifying plant genomes that are on the cusp of demographic collapse.We sequenced and assembled de novo genomes of both O. rehderiana individual and one O. chinensis individual (>20 years old) from wild populations were sequenced to 128× and 340× depth of coverage (based on an estimated genome size of ~386 Mb), respectively  in O. chinensis with a high contiguity, coverage, and accuracy  million years ago (Mya) based on the fossil-calibrated phylogeny , and 13 additional large O. chinensis trees , the largest in the Qinghai-Tibet Plateau39 , coincident with increased temperature and precipitation in China41. In contrast to O. rehderiana, O. chinensis maintained a stable population size throughout the remaining Holocene and currently is not endangered.Based on re-sequencing data from the assembled genome, four additional large (>100 years old), and nine additional younger O. rehderiana displayed extremely low observed sequence diversity  compared to O. chinensis , Betula pendula (π = 8.84E−3)36, and most other trees  across intergenic regions, introns, and coding regions were significantly lower for O. rehderiana than O. chinensis , higher frequencies of derived alleles, and an elevated Tajima’s D when compared to O. chinensis , was higher in O. rehderiana (FROH range 0.31–0.45) than in O. chinensis  decay. Half the maximum r2 was not attained until ~444 kb, whereas half the maximum r2 for O. chinensis was attained at ~29 kb  and the number of deleterious mutations, was detected in both O. rehderiana and O. chinesis  and tolerated (TOL) sites compared to synonymous (SYN) sites in O. chinensis, but not in O. rehderiana . In O. rehderiana, an average of 5350 DEL variants were homozygous across 3793 genes in each individual, whereas in O. chinensis 4359 DEL variants were homozygous across 3793 genes in each individual , suggesting that reproductive fitness was reduced in the younger trees as expected from inbreeding depression  showed a lower number of heterozygous-derived sites, a slightly higher number of homozygous sites, and a similar number of total derived alleles compared to the five old trees, but not all comparisons were statistically significant . Although our sample size for young trees was small (n = 9), all of them were half-sibs, indicating that without intervention, future kin-mating among these planted trees could further enhance the inbreeding bottleneck of this endangered tree. Future management efforts should focus on reducing inbreeding, which may have already led to a decrease in female cymule development , 14 O. chinensis (synonym to O. multinervis), two C. cordata, one C. fangiana, and one Ostryopsis nobilis individual using the CTAB method59. One large O. rehderiana individual and one O. chinensis individual located in Tianmu Mountain, Zhejiang and Zehei Township, Yunnan were selected for assembling de novo genomes, respectively. High coverage shotgun-sequencing was conducted for the Illumina HiSeq2500 platform with the short, medium and long insert libraries. Statistics for the obtained reads are given in Supplementary Table High molecular weight genomic DNA was extracted from mature leaves of 14 O. rehderiana and extracted using a CTAB procedure59 for transcriptome sequencing. cDNA libraries with insert sizes of 200 bp were constructed, and sequencing was conducted using the Illumina Genome Analyzer platform. After trimming adaptor sequences and filtering out low-quality reads, the RNA data was assembled using Trinity v2.060 for all reads combined, and for each of the four tissues separately  were collected from the wild tely See .https://github.com/vsbuffalo/scythe) and trimming low-quality sequences using SICKLE (https://github.com/najoshi/sickle). We employed SOAPec61 to minimize the influence of sequencing errors with low-frequency cutoff of 4. A total of 49.56G (or 128.39×) and 133.36G (or 345.48×) of data were retained for assembly of the O. rehderiana and O. chinensis genomes, respectively. The two Ostrya genomes were assembled de novo using Platanus v1.2.462, which is optimized for high-throughput Illumina sequence data and heterozygous diploid genomes. Briefly, Platanus assembles reads through three modules: de Bruijn graph-based contig assembly, scaffolding, and gap closing. The final assemblies were polished by GapCloser61 to further close the gaps in the Platanus assemblies.Before assembly, we filtered the low-quality sequencing reads, including removing adaptor sequences by SCYTHE 63 and functional assignments for all genes were generated by aligning their CDS to sequences available in the public protein databases including KEGG, SwissProt, TrEMBL, and InterProScan  from NCBI were used to generate clusters of gene families  alignments. 1896 single-copy genes were identified within the 10 species, and subsequently used to build a phylogenetic tree. Coding DNA sequence (CDS) alignments of each single-copy family were created based on the protein alignment, using MUSCLE65 software. The phylogenetic tree was reconstructed with PhyML66 software under the GTR + gamma model using only four-fold degenerate sites, which are less likely to be influenced by selection and more likely to evolve in a manner consistent with a molecular clock. To estimate divergence times, the approximate likelihood calculations were conducted using the PAML67 mcmctree program assuming a correlated molecular clock model and a REV substitution model. After a burn-in of 5,000,000 iterations, the MCMC process was performed 20,000 times with sample frequency of 5000. Convergence was checked by Tracer v1.4 (http://beast.community/tracer) and confirmed by two independent runs. The following constraints were used for time calibrations:68,140–150 Mya for the monocot–dicot splitVitis–Eurosid split69,94 Mya as the lower boundary for the A. thaliana and C. papaya split (http://www.timetree.org),54–90 Mya for P. persica and J. regia split (http://www.timetree.org).90–106 Mya for All protein sequences from eight species  under a random birth and death model for each branch of the phylogenetic tree using a maximum likelihood method. In addition, GO and KEGG enrichment for genes in gene families that expanded and contracted in O. rehderiana, O. chinensis, and their ancestor lineage were also calculated using GOEAST71.To study gene gain and loss, Computational Analysis of gene Family Evolution (CAFÉ)O. rehderiana and 13 O. chinensis individuals were selected for whole genome resequencing. Adapter sequences were trimmed from the raw reads and low-quality sequences  were filtered using SCYTHE  and SICKLE (https://github.com/najoshi/sickle). Filtered reads were mapped to either the O. rehderiana or O. chinensis genome (scaffolds length > 2 kb) by BWA-MEM software with default parameters. Sequence alignment/map (SAM) format files were imported to SAMtools v0.1.1972 for sorting and merging, and Picard v1.92 (http://broadinstitute.github.io/picard) was used to assign read group information containing library, lane, and sample identity. The Genome Analysis Toolkit 73 was used to perform local realignment of reads to enhance the alignments near indel polymorphisms in two steps. The first step used the RealignerTargetCreator to identify regions where realignment was needed, and the second step used IndelRealigner to realign the regions found in the first step, which generated a realigned binary sequence alignment/map (BAM) file for each individual.Thirteen 73 to prevent biases in SNP calling accuracy between groups with different numbers of samples. For single-sample SNP and genotype calling, a number of filtering steps were performed to reduce false positives, including removal of (1) indels with a quality scores <30, (2) SNPs with more than two alleles, (3) SNPs at or within 5 bp from any indels, (4) SNPs with a genotyping quality scores (GQ) <10, and (5) SNPs with extremely low (<one-third average depth) or extremely high (>threefold average depth) coverage. Multi-sample SNPs were identified after merging the results of each individual by GenotypeGVCFs73 to generate three datasets: dataset 1, SNPs from all O. rehderiana samples called using the O. rehderiana genome as reference; dataset 2, SNPs from all O. chinensis samples called using the O. chinensis genome as reference; and dataset 3, SNPs from all O. rehderiana, O. chinensis, C. cordata, C. fangiana, O. nobilis samples and using the O. rehderiana genome as reference. Each multi-sample SNP was first filtered by the GATK variant filter module with the following strict filter settings. For indels, “QD < 2.0 || FS > 200.0 || ReadPosRankSum < −20.0”. For SNPs “QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0”, with additional filtering steps including removing: (1) SNPs with more than two alleles; (2) SNPs at or within 5 bp from any indels; (3) genotypes with quality scores (GQ) < 10, or extremely low (<one-third average depth) or extremely high (>threefold average depth) coverage; (4) SNPs with more than two missing genotypes in either O. rehderiana or O. chinensis; and (5) SNPs showing significant deviation from Hardy–Weinberg equilibrium (P < 0.001) in either of the two species.Single-sample SNP and genotype calling were implemented in GATK with HaplotypeCallerO. rehderiana and O. chinensis genomes, separately. Genetic diversity (π) was calculated using 50 kb sliding windows in 10 kb steps for datasets 1 and 2 by VCFtools v0.1.12b74. The other detailed information for calculating FROH, LD, the population recombination rate (ρ), and population demography are shown in the Genome-wide heterozygosity, within-individual SNV incidence, and SNV density within 50-kb windows were calculated in the intergenic  and coding regions for all 32 individuals. Of 50-kb windows, 6768 and 6456 were used with a total length of 338.4 Mb (92.4%) and 332.8 Mb (86.9%) in the 47 was calculated within coding regions based on O. rehderiana annotation (dataset 3). The zero and four-fold degenerate sites were identified by iterating across all four possible bases at each site along a transcript and recording the changes in the resulting amino acid. Sites were classified as zero-fold degenerate when the four different bases resulted in four different amino acids, and four-fold degenerate when no changes in amino acids were observed. Then the ratio of zero-fold to four-fold degenerate site were calculated by each individual.The genome-wide ratio of heterozygosity  of zero to four-fold degenerate sites75, and SnpEff76 was used to classify the SNPs based on the O. rehderiana annotation. To avoid reference bias when identifying derived alleles and deleterious variants, we only called the polarity of variants when all three outgroups  had identical homozygous states. Nonsynonymous SNPs were assessed using PolyPhen2 48, PROVEAN 49, and SIFT 50 with their default settings. The intersection of those three approaches may provide more accurate predictions than any single prediction approach alone, because each approach varies slightly in its prediction procedure and assumptions51.Prior to detection of deleterious variants, all segregating sites in dataset 3 were phased and imputed using BEAGLEhttps://github.com/yongzhiyang2012/Two_iron_wood_genome_analysis).The custom scripts have deposited in GitHub (Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


P<0.001). However, this superiority was not significant from 1980–1994 (P = 0.579) or for TNM stage I BC (P = 0.927) and grade I BC (P = 0.431) from 2000–2014. In conclusion, the incidence of BC2TC has increased dramatically during the past 15 years. In addition, patients with BC2TC showed better DSS than patients with BC alone, especially in cases from 2000–2014.Breast cancer (BC) and thyroid cancer (TC) are common malignancies among females. However, the connection between TC and BC is not well understood. To explore the relationship between these two cancers and to determine the effect of second metachronous TC on BC survival, we compared BC patients with or without second primary TC using data from the Surveillance, Epidemiology, and End Results (SEER) database. We extracted data from patients with only BC or TC and from BC patients with a second metachronous cancer from 2000–2014. Differences in the clinicopathological and treatment characteristics between BC patients with or without second metachronous TC were analyzed by chi-square tests. Multivariate analyses of BC survival were performed by using Cox regression models. Comparison of disease-specific survival (DSS) curves between these cohorts was performed with the log-rank (Mantel-Cox) test. Survival analyses were also performed using data from 1980–1994. Within this dataset, we found 1,262 BC cases in which a second metachronous TC (BC2TC) developed, accounting for 3.1% of all metachronous cancers following BC from 2000–2014. No significant differences were found in molecular markers. In addition, the mean age at BC diagnosis was younger in the BC2TC group than in the BC group (55.418 y vs 60.273 y). Half of the BC2TC patients developed TC in the first three years following BC diagnosis. Patients with BC2TC showed better DSS than those with BC alone from 2000–2014 ( Breast cancer (BC) and thyroid cancer (TC) are common malignancies. Although the mortality rate of BC fell between 1989 and 2014, BC remains the second most common cause of cancer-related death among females [Many studies have attempted to demonstrate an association between thyroid disease and BC as both diseases principally appear in women. Smyth reviewed the association between thyroid disease and BC and confirmed that hypothyroidism or autoimmune thyroid disease potentially contributes to an increased risk or alters the outcome of BC . In addiThe criteria for diagnosing a second primary tumor or multiple primary tumors have been discussed for many years . MetachrIn 1973, Moossa considered that patients with BC and a history of thyroid disease had a lower average survival rate at both 5 and 10 years than those without any evidence of thyroid disease . In addihttps://seer.cancer.gov/about/factsheets/SEER_Overview.pdf). We used the following database: Incidence—SEER 18 Regs Custom Data , submitted November 2016 (1973–2014 varying)—Linked to County Attributes—Total U.S., 1969–2015 Counties, National Cancer Institute, DCCPS, Surveillance Research Program, released April 2017. The majority of patients in this study were diagnosed between 1 January 2000 and 31 December 2014. We extracted three cohorts of patients with definite survival months from this database. For cohort one, the total number of in situ/malignant tumors for each patient was 2, with BC as the first malignancy and a second metachronous cancer. In the other cohorts, each patient had only BC or TC. There were 40,520 patients in cohort one, 629,976 patients with only BC and 120,339 patients with only TC. We also extracted cases from 1 January 1980 to 31 December 1994 following the former formula with the second primary TC diagnosed before 1995. All data from SEER database were fully anonymized. The patient records used in our study are provided as supporting databases. The S1 database provides records of the BC cohort from 2000–2014. The S2 database provides records of other cohorts from 2000–2014. The S3 database includes data from all cohorts from 1980–1994.We used data from the SEER Cancer Registry database, which collects data on every case of cancer reported from 19 U.S. geographical areas. These areas cover approximately 34% of the U.S. population and are representative of the demographics of the entire U.S. population  expression, progesterone receptor (PR) expression, Her-2 expression, histopathological subtype, radiation therapy and chemotherapy history. There were 36,825 cases without definite TNM data for the BC only (BC) group and 60 cases without definite TNM data for the BC followed by TC (BC2TC) group. There were 56,532 cases without definite grade data for the BC group and 90 cases without definite grade data for the BC2TC group. There were some cases lacking ER expression data  or PR expression data . There were 214,258 cases with Her-2 expression data for the BC group and 237 cases for the BC2TC group. Radiation therapy included beam radiation, combinations of beam radiation with implants or isotopes, unspecified radiation methods or sources, radioactive implants, and radioisotopes.www.seer.cancer.gov/seerstat) version 8.3.5 to obtain the incidence rates of patients with TC only or generate lists of cases with the desired data. Data analysis was performed using GraphPad Prism 7 . The differences in clinical characteristics were analyzed by chi-square test. Multivariate analyses of BC survival were performed by using Cox regression models. Comparison of DSS curves was performed using the log-rank (Mantel-Cox) test. P<0.05 was considered significant.We used the Surveillance Research Program, National Cancer Institute SEER*Stat software   .P = 0.579)  are shown in = 0.579) . When we= 0.579)  and tumo= 0.579) , we obseIs it true that adding TC to primary BC will lead to a worse prognosis than that of BC alone? Our results indicated a better DSS in the BC2TC group than in the BC group, especially for cases between 1 January 2000, and 31 December 2014. In addition, the incidence of TC as a second malignancy following BC has dramatically increased in the last 15 years.To the best of our knowledge, this is the first paper to address the effect of second TC on BC survival.Many studies have focused on the possible relationship between BC and thyroid disease, but the results have been conflicting. Hyperthyroidism was found to be associated with an increased risk of BC compared with the general population, and hypothyroidism was associated with a slightly decreased risk of BC compared with the general population. Thus, it appears that thyroid function levels may be associated with BC risk , 14. HowIn 1994, Mazzaferri found that thyroid hormone therapy confers a distinct outcome advantage in TC; this was a very important long-term study with a major influence on TC treatment . Then, fHowever, whether TSH suppression using LT4 is the reason for the better survival for BC in the BC2TC group remains to be determined. As early as 1896, Beatson used thyroid extract to treat BC and explore the relationship between BC and thyroid dysfunction, and determined that they were correlated . Loeser BC is the most common second primary cancer following BC , in agreBecause it was based on data from the SEER database, our study was retrospective. We found that patients in the BC2TC group had better DSS, but we can only speculate that TSH suppression therapy is the reason for the better survival for BC in these patients. In addition, stage I and grade I BC did not show any survival advantages for subsequent TC; thus, studies to identify the BC patients who might in fact achieve improved survival for second metachronous TC should be performed. The results of this study should be verified in a prospective population-based study, and the underlying mechanisms should be investigated.In conclusion, despite these limitations, our results showed that the incidence of TC as a second malignancy following BC has dramatically increased during the past 15 years. Notably, our study found better DSS in the BC2TC group than the BC group, especially in cases diagnosed between 1 January 2000, and 31 December 2014. We speculate that TSH suppression therapy is the underlying reason for better DSS in the BC2TC group. TSH suppression therapy may be another potential regimen for BC.S1 Table(XLSX)Click here for additional data file.S1 FigBC vs BC2TC with radiation therapy (A); BC vs BC2TC without radiation therapy (B).(TIF)Click here for additional data file.S2 FigBC vs BC2TC with chemotherapy (A); BC vs BC2TC without chemotherapy (B).(TIF)Click here for additional data file.S1 Dataset(RAR)Click here for additional data file.S2 Dataset(RAR)Click here for additional data file.S3 Dataset(RAR)Click here for additional data file.