Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings.Chloroquine remains the mainstay of treatment for P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310.A systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified Of 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8–35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall  and in children younger than 5 years . Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1–7·7) by day 42, which is lower than with chloroquine alone .P vivax.Chloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant Wellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation. Plasmodium vivax for over 60 years.P vivax were published in 1989,P vivax was present in most vivax-endemic countries.8Chloroquine has been the mainstay of treatment for P vivax include optimising chloroquine regimens or changing policy to a more effective blood schizontocidal agent. In countries where high-grade chloroquine-resistant P vivax is prevalent, national treatment guidelines have been revised to a universal policy of artemisinin combination therapy (ACT) for all species of malaria.P vivax, the treatment regimen can potentially be optimised, either by increasing the dose or duration of chloroquine, or by combining chloroquine with an additional drug with blood schizontocidal activity or the ability to reverse chloroquine resistance.P vivax at doses below 25 mg/kg, higher doses are well tolerated and might provide increased efficacy.13Treatment options for chloroquine-resistant Evidence before this studyPlasmodium vivax malaria. A systematic review and meta-analysis showed that there was evidence of reduced chloroquine efficacy for P vivax present in most P vivax endemic countries. No reviews or pooled analyses had assessed the effect of chloroquine dose on the risk of recurrence.Using the search terms “vivax” and “chloroquine”, MEDLINE, Web of Science, Embase, and the Cochrane Database of Systematic Reviews were searched for articles published before Nov 29, 2017, that assessed the efficacy of chloroquine, with or without primaquine, for uncomplicated Added value of this studyP vivax clinical trials so far. Our findings highlight the substantial benefit of increasing the dose of chloroquine in children younger than 5 years and the additional benefit of adding primaquine to chloroquine.Our pooled analysis of individual patient data from 37 studies across 17 countries is, to our knowledge, the largest individual pooled analysis of Implications of all the available evidenceP vivax recurrence within 42 days in children younger than 5 years who are not given primaquine. The risk of P vivax recurrence was reduced by an even greater degree by the addition of primaquine to chloroquine in all age groups, through prevention of relapse and probably improvement in blood schizontocidal efficacy. These measures warrant consideration by regional and global policy makers to reduce the risk of early P vivax recurrence.Chloroquine is currently under-dosed in children younger than 5 years. Increasing the target dose of chloroquine from 25 mg/kg to 30 mg/kg could significantly reduce the risk of P vivax clinical trials to investigate the effect of chloroquine dose and primaquine co-administration on the risks of P vivax recurrence between day 7 and day 42.To explore alternative strategies for improving chloroquine efficacy, we did a pooled analysis of individual patient data from prospective P vivax malaria published in any language between Jan 1, 1960, and March 22, 2017, were identified using the following search terms:  AND . Further details are provided in the We searched MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement . ProspecThe review process was done by two independent investigators (RJC and RNP), who also extracted the data. Disagreement was resolved through discussion. To ensure results were relevant to the current clinical landscape, only studies published after 2000 were included. Principal investigators were contacted and invited to share individual patient data and any additional unpublished data.P vivax monoinfection treated with chloroquine alone or chloroquine plus primaquine during the first 28 days after treatment were included. Individual patient data were uploaded into the WorldWide Antimalarial Resistance Network (WWARN) secure repository, anonymised, and processed according to a data management plan.Studies assessing patients with All data included in this analysis were obtained in accordance with ethical approvals from the country of origin. The data are fully anonymised and cannot be traced back to identifiable individuals; these do not require review from an ethics committee according to the guidelines of the Oxford Central University Research Ethics Committee.Chloroquine and primaquine doses were calculated from the number of tablets given to each patient. If these data were unavailable, doses were calculated based on the study protocol and assuming complete adherence. Individual patient records were excluded if the course of chloroquine was incomplete, the course of chloroquine or primaquine was intermittent, or if information on dose, parasitaemia, age, or weight were unavailable. Early primaquine was defined as the first dose of primaquine administered in the first 3 days of treatment .P vivax parasite rate <1·5%), moderate (≥1·5% and <4·0%), or high (≥4·0%) based on transmission estimates obtained from the Malaria Atlas Project and observed study site reinfection rates  when a linear association with outcome was not present. Figures of risk of recurrence were estimated according to chloroquine dose and primaquine co-administration, adjusted for other confounders and assuming no study site effect.The associations between chloroquine dose and microscopy-detectable parasite positivity in patients treated with chloroquine alone were analysed by logistic regression, with study sites included as a random effect. The association between the first day of parasite clearance and parasitaemia recurrence between day 7 and day 28 was assessed by Cox's proportional hazards regression.Heterogeneity of studies was assessed by removal of one study site at a time and calculation of the coefficient of variation around parameter estimates. Additionally, baseline characteristics of included studies were compared with targeted studies that were not included.Statistical analyses were done in Stata (version 15.0) and R (version 3.4.0), according to an a-priori statistical analysis plan.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. RNP had full access to all the data in the study and had final responsibility for the decision to submit for publication.P vivax clinical trials were identified, 134 of which included patients treated with chloroquine and were published between Jan 1, 2000, and March 22, 2017 , with 450 (8·6%) aged younger than 5 years . 3773 7. The corIn the 2990 patients from 26 studies treated with chloroquine alone, the median total dose of chloroquine was 25·4 mg/kg  with 1041 patients (34·8%) receiving less than the WHO recommended target dose of chloroquine of 25 mg/kg . This prInformation on acute vomiting was available in eight studies (n=557), with 20 (4%) of 557 patients vomiting at least one dose of chloroquine within 60 min of administration. After adjusting for age, sex, baseline parasitaemia, presence of baseline fever, and relapse periodicity, chloroquine dose was not a significant risk factor for vomiting  of 298 recurrences within this period occurred by day 28. The cumulative risk of recurrence was 10·4% (95% CI 9·3–11·6) at day 28 and 32·4% (29·8–35·1) at day 42. The risks of recurrence for individual studies are presented in the After controlling for age, parasitaemia, regional relapse periodicity, and sex, a 5 mg/kg increase in chloroquine dose reduced the rate of recurrence between day 7 and day 42  of 2070 patients had cleared their detectable parasitaemia, increasing to 2095 (80·9%) of 2590 on day 2 and 2369 (94·8%) of 2499 on day 3. Low chloroquine dose (<25 mg/kg) was a risk factor for parasitaemia on day 1 in the univariable analysis , as were male sex, older age, and higher baseline parasitaemia . After cP vivax between day 7 and day 28, compared with 229 (9%) of 2657 who had already cleared their parasitaemia (p<0·0001). After controlling for confounding factors, parasite clearance on or after day 3 was associated with an increased rate of recurrence between day 7 and day 28 compared with parasite clearance on day 1  of 139 who were parasitaemic on day 3 had recurrent In 17 studies, 1790 patients were treated with chloroquine and early primaquine. 917 (51·2%) patients from 11 of these studies had a target dose of primaquine between 3·5 mg/kg and less than 5·0 mg/kg, and 873 (48·8%) from six studies had a target dose of at least 5·0 mg/kg. Overall, patients were administered a median dose of primaquine of 4·7 mg/kg  at day 28 and 4·9% (3·1–7·7) at day 42. When patients treated with chloroquine plus early primaquine were added to the previous Cox regression model, the addition of early primaquine was associated with a reduction in the rate of recurrent parasitaemia ; increasing the total mg/kg chloroquine dose reduced early recurrences if primaquine was not given, especially in children younger than 5 years; and, the risk of early recurrent parasitaemia was markedly reduced by co-administration of primaquine.P vivax.P vivax.P vivax relapses, with the added benefit of providing additional blood schizontocidal activity.P vivax recurrence substantially in children younger than 5 years.Increasing reports of declining chloroquine efficacy have highlighted the need for alternative treatment strategies for Previous pharmacokinetic studies have shown that chloroquine is under-dosed in children and have suggested that an increase in the chloroquine dose or dosing based on body surface area would be more appropriate and effective.Plasmodium falciparum were well tolerated in children younger than 15 years.Giardia lamblia (10 mg/kg twice daily for 5 days).Our data are in keeping with these findings and suggest that increasing the total chloroquine dose from 25 mg/kg to 30 mg/kg in children younger than 5 years would decrease the risk of early recurrence by more than 40% if chloroquine was used alone. Although increasing the target dose might reduce tolerability, substantial data support the safety of 30 mg/kg in children. In Guinea-Bissau, chloroquine doses of 50 mg/kg against drug-resistant P vivax parasitaemia: recrudescence, relapse, and new infections.Current molecular analyses cannot differentiate reliably between the three causes of recurrent The addition of primaquine to chloroquine reduced early recurrences before day 42 by 90% compared with chloroquine alone; probably in large part as a result of prevention of early relapse related to primaquine. However, addition of primaquine probably also reduces recrudescence through its blood schizontocidal activity, potentially in patients with low-grade chloroquine resistance. In the current pooled analysis, the reduction with chloroquine and primaquine did not vary before and after day 21, consistent with a reduction in both recrudescences and relapses.P falciparum malaria.P vivax.Delayed parasite clearance predicts treatment failure in Our study has several limitations. First, the analysis only included about 20% of patients from the clinical trials targeted. However, a sensitivity analysis in which one study site was removed at a time revealed no apparent bias relating to individual study sites that were included, and baseline characteristics of patients included had similar characteristics to those from all targeted studies . Second,P vivax after chloroquine monotherapy is high, it can be reduced by a modest increase in the dose of chloroquine, particularly in children younger than 5 years, and by the additional administration of primaquine. As reports of chloroquine treatment failure for P vivax increase, we recommend that the dose of chloroquine be increased to 30 mg/kg in children younger than 5 years, and health-care providers should be encouraged to provide adjunctive primaquine radical therapy to reduce the risk of both recrudescent and relapsing infections. Alternatively, a universal policy of ACT for uncomplicated malaria, with additional primaquine for vivax malaria, should be considered in regions where there is a high risk of recurrent P vivax after chloroquine treatment.In summary, although the risk of early recurrence of


Extra virgin olive oil (EVOO) has elevated commercial value due to its health appeal, desirable characteristics and quantitatively limited production, and thus it has become an object of intentional adulteration. As EVOOs on the market might consist of a blend of olive varieties or sometimes even of a mixture of oils from different botanical species, an array of DNA-fingerprinting methods have been developed to check the varietal composition of the blend. Starting from a comparison between publicly available DNA extraction protocols, we set up a timely, low-cost, reproducible and effective DNA isolation protocol, which allows an adequate amount of DNA to be recovered even from commercial filtered EVOOs. Then, in order to verify the effectiveness of the DNA extraction protocol herein proposed, we applied PCR-based fingerprinting methods starting from the DNA extracted from three EVOO samples of unknown composition. In particular, genomic regions harboring nine simple sequence repeats (SSRs) and eight genotyping-by-sequencing-derived single nucleotide polymorphism (SNP) markers were amplified for authentication and traceability of the three EVOO samples. The whole investigation strategy herein described might favor producers in terms of higher revenues and consumers in terms of price transparency and food safety. Olea europaea L. subsp. europaea var. europaea) is a species that originates from regions surrounding the Mediterranean Sea [Olive  has elevated commercial value due to its health appeal, desirable characteristics and quantitatively limited production. As a high-value product, a certification label added to a bottle of EVOO allows food fraud to be prevented and consumers to trace oil “from tree to table”. The European Commission Regulation 1151/2012 disciplines quality schemes for agricultural products and defines guidelines on the labeling of foodstuffs.Italy is the first ranked country in terms of the number of protected designation of origin (PDO), protected geographical indication (PGI) and traditional specialty guaranteed (TSG) certifications. As for EVOOs, these include 42 PDO and 4 PGI . Given tSeveral approaches based on chemical analysis have been proposed for EVOO traceability ,10,11,12To have a reliable DNA extraction protocol from EVOOs is the key for a successful molecular fingerprinting. A critical aspect to take into consideration concerns the recovery of an adequate amount of DNA from filtrated oils. DNA is usually degraded and present in a very low amount when a food matrix is used for its extraction ,23. So fWithin this motivating context, our work presents a case study aiming at set up a timely, low-cost, reproducible and effective DNA extraction protocol from three filtered commercial EVOOs, which allows an adequate amount of DNA to be recovered. Different authors showed the effectiveness of extraction buffer containing CTAB and/or hexane in DNA isolation from different vegetable oils ,26,27,28Analyses were performed on three EVOO samples , provided by an Italian mill, which underwent two filtration steps in order to extend their shelf-life. The first step was carried out with two filters made of cellulose and diatomaceous flours; the second filtration step was performed using a polishing paper. The only available information on those samples was that they were bottled in Italy. Three bottles of one liter each were collected and stored at 4 °C before the analysis.p1 Spadoni et al., 2019 ;p2 Muzzalupo et al., 2002 ;p3 Busconi et al., 2003 ;p4 Consolandi et al., 2008 .We tested four protocols  for DNA isolation:One mL per sample was vigorously mixed for 5 min and transferred into a 2 mL microcentrifuge tube, then it was shaken with 500 µL of hexane for 5 min and centrifuged .v/v Tween 20).The oily supernatant was transferred to a fresh 2 mL tube and the pellet was mixed with 400 µL of lysis buffer .The two aqueous phases from the oil/buffer mix and the pellet were transferred into separate 1.5 mL microcentrifuge tubes, being careful to avoid contact with the interface between oil and buffer.The pellet derived from treatment of the aqueous phase at point 4 was discarded.The three phases were transferred into separate 1.5 mL microcentrifuge tubes and the samples were gently mixed with 500 µL of cold isopropanol, held 30 min at −80 °C, and centrifuged again .The pellet was washed by adding 500 µL of absolute ethanol, held 30 min at −80 °C and centrifuged .The pellet was washed once more by adding 500 µL of 70% ethanol and centrifuged .After removing the supernatant, the pellet was dried in vacuum pump for 30 min and finally resuspended in 50 µL of 1× TE buffer pH 8 .After evaluating those protocols, we modified p4 as follows:A set of nine SSR primer pairs ,35,36 waAmplification products were separated by capillary electrophoresis on the ABI PRISM 3100 Avant Genetic Analyzer sequencer  using a mix containing 2 µL PCR reaction, 13,5 µL Hi-Di™ Formamide and 0.5 µL GeneScan™ 600 LIZ™ Size Standard . Electropherograms were analyzed by the GeneMapper Software v3.7 .A large panel of 470 Mediterranean olive accessions  including Italian (396), Tunisian (25), Syrian (23), and Algerian (26) accessions, was used to assess genetic similarity among olive accessions and the three commercial EVOOs.http://biology-assets.anu.edu.au/GenAlEx/Download.html). Based on PcoA, accessions with SSR-based genetic profiles more similar to the three samples under study were selected for the construction of a tree through the un-weighted neighbor-joining method, running 1000 bootstrap replicates, using DARWIN v 6.0.010 (http://darwin.cirad.fr) [http://tree.bio.ed.ac.uk/software/figtree/).Genetic similarity between the three EVOO samples and the 470 olive accessions was calculated through principal coordinates analysis (PCoA) using the GenAlEx software version 6.5 (irad.fr) . The reshttp://www.agraria.org/prodottitipici/oliodioliva.htm), were selected for SNP analysis. We took in consideration the SNP profile from a pre-existing catalogue generated by genotyping-by-sequencing [A total of six Italian olive cultivars , representative of the main PDO and PGI certifications . The cycling program was: 2 min of initial denaturation at 98 °C, 35 cycles of denaturation at 98 °C for 10 sec, annealing for 20 sec, extension at 72 °C for 30 sec and final extension at 72 °C for 5 min. Annealing temperatures ranged from 64 to 68 °C. PCR products were analyzed by 2% agarose gel electrophoresis to verify the specificity of the amplification reaction. Each amplicon was then purified through ethanol precipitation and quantified using the NanoDropTM ND2000C .PCR products obtained from DNA extracted from oils required an additional step of purification, which was performed using the Agencourt AMPure XP - PCR Purification Kit . Sequencing reaction was prepared using the BigDye™ Terminator v3.1 Cycle Sequencing Kit following the manufacturer’s instructions and the sequencing was performed by the ABI PRISM 3100 Avant Genetic Analyzer .Finally, raw data were analyzed by the Sequencing Analysis Software v6.0 and the Sequence Scanner Software v2.0. As a first step, the four protocols  were compared each other to assess their effectiveness in extracting DNA from the three filtered EVOO samples. This was done to identify the most suitable one to be modified if necessary . AlthougAll protocols share an initial step of sample incubation at 60–65 °C with a standard extraction buffer consisting of Tris-HCl and EDTA. All protocols, except p4, are based on the use of CTAB, while p1 includes the addition of the anionic detergent sodium dodecyl sulfate . AfterwaEach protocol was tested following authors’ indications. After extraction, DNA was checked for quantity and quality. In all cases, DNA was highly degraded and its amount ranged from 0.5 ng/μL (p1) to 100 ng/µL (p4).P1 was discarded because of the low amount of recovered DNA (<2 ng/μL). P2 and p3 were both based on the use of CTAB. Although both protocols start from a volume of oil higher than 10 mL, the amount of recovered DNA was lower than 10 ng/μL. Moreover, the use of CTAB, which is an inhibitor of enzymatic reactions, requires several washing steps to remove its traces. By contrast, the extraction solvent used in p4 is the hexane and this protocol resulted in a fair amount of DNA, ranging from 20 to 100 ng/µL, though starting from 2 mL of EVOO. The only drawback is that p4 was the most time-consuming. Therefore, we decided to improve p4 in order to (i) reduce the time required for the DNA extraction process; (ii) decrease the starting amount of oil; and (iii) limit the number of reagents and equipment needed. All these improvements are crucial for industrial and legal application of the method.The protocol herein proposed  was performed .The three samples resulted to be different but very close each other, suggesting the use of common and related varieties in EVOO production. Moreover, all the samples were placed in a group that includes the main Italian olive varieties. A subset of 147 accessions that lie in the fourth quadrant of the PCoA plot together with the three EVOO samples was filtered out and used to construct a tree through the un-weighted neighboring joining method . The denEight SNPs, which discriminate against six varieties, namely Frantoio, Taggiasca, Pendolino, Crastu, Leccino, and Ogliarola barese, were selected and the DNA regions harboring those SNPs subjected to PCR amplification and Sanger sequencing. PCR experiments were successful in all the samples . The presence of non-nucleic acid contaminants and inhibitors in the amplified DNA involved an additional purification step, following which sequencing reactions definitely improved as shown by the severe reduction of the background noise .All samples were characterized by the same alleles at seven SNP sites (from SNP #1 to #7) with the exception of OL3, which showed a different allele for SNP #7 . As for When olive drupes are ground, the cellular compartmentalization is destroyed and everything is mixed in the aqueous solution  where most of the DNA and proteins are. Olive oil is the water-insoluble fraction mainly lacking in DNA and proteins, but enriched in organic compounds that interfere with DNA polymerase reactions.Genetic traceability of EVOOs requires DNA to be extracted from commercial olive oils, which are all subjected to a filtration process in order to satisfy consumers’ demand .To identify an effective and robust DNA extraction protocol from filtered commercial oils, four protocols were tested and compared each other . HoweverThe effectiveness of p5 was assessed by applying PCR-based fingerprinting methods for EVOO authentication and traceability. DNA amplifications of nine SSR loci were successful for all the three EVOO samples.The presence of microsatellite multi-allelic combinations  coupled If SNP markers are used for EVOO authentication and traceability, we suggest using an additional purification step of PCR products. Indeed, phenolic compounds and residual polysaccharides could inhibit the enzymatic reactions providing irregular PCR amplifications . This inAdvances in technologies for DNA profiling have had a huge impact in combating fraud and food adulteration as well as in improving authentication and traceability of food products. The screening of multiple markers with next generation sequencing (NGS) technology has been widely applied to the olive sector, and in particular the high-resolution DNA melting technology (HRM) has been successfully used for olive oil authentication ,50. HRM Despite no information about varietal composition of the three EVOO samples under investigation was available, we were able to assert that most likely the three samples are blends of Italian varieties and generate a list of possible varieties present in each blend. The EU commission regulation 182/2009 governs the whole production process of EVOOs. The origin of olives and/or the addition of oils coming from different countries must be indicated on the label. Molecular traceability is an important tool to investigate the varietal composition of olive oils, in order to prevent fraud due to the use of oils coming from extra-EU countries not explicitly indicated on the label.Contrary to what happens for the wine market, in which there are few international varieties  that are cultivated widespread in the world, for the oil market there are cultivars strongly adapted and cultivated in each territory . In this context, the identification of varietal composition could be useful to better define the origin of the oil.Nevertheless, the comprehensive identification of all the varieties present in a blend is extremely tricky. The identification of singleton SNPs and/or haplotype blocks via NGS will allow the rapid detection of cultivars present in a blend in the near future.We developed a speedy and highly reproducible protocol able to recover whole genomic DNA from filtrated EVOOs. Then, we applied PCR-based methods for genetic tagging of filtered EVOOs of unknown origin in order to verify the effectiveness of the proposed DNA extraction protocol. The whole investigation strategy may be suitable for industrial/legal applications, as olive oil frauds are more and more rampant. Deliberate or unintentional adulteration of olive oil could seriously damage producers/providers in term of earnings and reputation and could definitely break the producer–consumer relationship. On the other hand, consumers are increasingly demanding as their preferences are moving towards the highest quality products. There is a vast range of olive oil on the market and consumers would like to have awareness of the entire olive oil supply chain, “from tree to table”. DNA-based methods for olive oil authentication and traceability are beginning to be routinely applied, and although they are still challenging due to the complexity of fraudulent practices, they can reassure consumers in terms of price transparency and food safety.


This paper studied the alterations in arterial stiffness and hemodynamic responses during resting state and immediately following acute cycling intervention at different times across 12-week supervised exercise training. Twenty-six sedentary young males participated in the exercise training program at moderate intensity. Arterial stiffness and hemodynamic variables of the right common carotid artery were measured and computed during resting state and immediately following acute cycling intervention at weeks 0, 4, 8, and 12. Across the 12-week exercise training, carotid arterial stiffness was decreased at weeks 8 and 12 and hemodynamic variables were improved at week 12 during resting state. In response to acute cycling intervention, carotid arterial stiffness exhibited an acute increase foremost at 8 weeks, and arterial maximal and mean diameters showed acute decreases at weeks 0 and 4. Despite significant differences in arterial stiffness and hemodynamic variables between resting state and immediately after acute intervention for each time period, these differences presented a progressive decrease across the 12-week exercise training. In conclusion, long-term exercise training not only improved carotid arterial stiffness and hemodynamic alterations when at rest but also negated the acute responses of carotid arterial stiffness and hemodynamic variables to acute cycling intervention. Common carotid arteries are the main conduits that supply blood to the brain. The changes in the structure and function of common carotid arteries are relevant to the occurrence and development of atherosclerosis, coronary ischemia, and stroke . CarotidExercise training can modulate arterial stiffness and directly induce systemic and local arterial hemodynamic responses . Cross-sIt has been reported that increased arterial stiffness is observed after acute resistance training  and highTherefore, to better evaluate the effects of exercise training on arterial stiffness and hemodynamics, the same types of exercise modality and the same experimental subjects need to be chosen in one study, and intensive measurement time points should be set during long-term exercise training. Furthermore, due to the fact that the majority of investigations , 23 mentIn order to solve the above problems, 26 young healthy sedentary men were recruited, and four weekly measurements across a 12-week period of supervised exercise training were conducted. The stiffness and local hemodynamic forces of the right common carotid artery were obtained at rest and immediately after acute cycling exercise using the same workload for each lab visit. The present study will supply hemodynamic indicators induced by exercise training for mediating the arterial function and structure, furthermore achieving a better understanding of the arterial function and hemodynamic response to acute exercise interventions with the progression of exercise training.Ethical approval was obtained from the Ethics Committee of Dalian University of Technology, and the study was carried out in accordance with the Declaration of Helsinki (1964). Informed consent was acquired from the subjects prior to the onset of acute cycling intervention and exercise training.−2) completed the 12-week exercise training period, including acute cycling intervention and hemodynamic measurements.A total of 32 young male subjects were recruited from the community, and they consented to participate in the present study. The entry criteria included being a nonsmoker and having sedentary individuals who were not involved in any regular planned exercise program . None ofThe subjects visited the lab four times during the supervised exercise training during weeks 0, 4, 8, and 12. Subjects reported to the laboratory at the same time of day, 3 hours after a meal. For each measurement visit, subjects were asked to refrain from caffeine and exercise before each visit. During each visit, vascular ultrasound assessments were conducted after the subjects lay in a supine position for more than 15 min. Subsequently, the subjects performed an acute leg cycling exercise after a 5-minute warm-up of cycling. Finally, they resumed the supine position for an immediate vascular ultrasound measurement.A schematic presentation of acute cycling intervention and exercise training is provided in The supervised exercise training consisted of 60 min sessions three times a week during the 12-week period on a running track. Each session included a 10 min warm-up and 5 min of stretching, followed by 30 min of running at a moderate intensity of 60 to 70% of heart rate reserve , a 10 min cool down period, and 5 min additional stretching. During the supervised exercise training, the subjects wore a heart rate monitor .ps_mea), and diastolic pressure (pd_mea). Eventually, the same vascular ultrasound assessment procedure was performed immediately following the acute cycling exercise. All heart rate and blood pressure measurements were registered in triplet, and the average of three values was adopted for subsequent analysis.The subjects lay in a supine position for ≥15 minutes. Images of the right common carotid artery were obtained using Doppler ultrasound  and 7.5-10.0 MHz linear-array probe. Images were acquired 5-10 mm below the carotid bulb. The common carotid artery was assessed using a longitudinal view. The distance from the near wall to far wall lumen-intima interface was continuously traced using eTracking software and used to determine carotid artery diameters. Center-line velocity waveforms were measured using range gated spectral Doppler signals averaged along the Doppler beam. An insonation angle ⩽ 60° was maintained for all measures and sample volume was manually adjusted to encompass the entire vessel. Synchronously, a cuff-type manometer  positioned on the upper-left arm was used to record the heart rate, brachial systolic pressure  detected center-line velocity  and arte η or blood density ρ was determined as the same values for all subjects, i.e., η = 0.004 Pa·s and ρ = 1050 kg/m3, and the maximal harmonic number n= 20 was chosen for (Hemodynamic parameters were computed using –11) bel bel11) bosen for .pm and diastolic pressure pd to be approximately equal to the mean value of the brachial pressure pm_mea and diastolic pressure pd_mea, as done in the literature [pm) was calculated using the following approximate equation:There was a linear relationship between the changes in vessel diameter and the changes in blood pressure  within eterature . The mea pm and diastolic pressure pd (pd_mea). The maximal value of the carotid artery waveform was calculated and assumed to be the systolic pressure ps.Therefore, the carotid artery blood pressure waveform was calibrated using the brachial mean arterial R0 is the time-averaged value of the carotid artery radius during one cardiac cycle, y = r/R0, in which r is the radial coordinate, and u(y) meets [ n is the harmonic number, J0 is the 0th-order Bessel function of the first kind,  ρ is the density of blood, η is the blood viscosity, ωn = 2nπf is the angular frequency, f is the base frequency, and u is the n harmonic component of the measured center-line velocities and satisfiesThe FR was calculated fory) meets (3)uy,t=∑Qmax, Qmin, and Qmean are the maximal, minimal, and mean blood flow FR during one cardiac cycle.In addition, Ep) or Peterson modulus [The pressure-strain elastic modulus β=lnCS was determined through the following equation :(7)CS=Rsτw), which is the frictional force of blood flow acting upon the vascular endothelium [J1 is a first-order Bessel function of the first kind, and τw_max, τw_min, and τw_mean are the maximal, minimal, and mean WSS waveforms during a cardiac period.WSS τw=ηR0 OSI index represents the WSS acting in directions other than the direction of the temporal mean WSS vector and was provided by Ku et al. [T is the period of each cardiac cycle.Theu et al. .(9)OSI=1PR, resistance to the flow of blood, is determined by the vascular musculature and diameter of the blood vessels and is responsible for the blood pressure when coupled with the flow rate as follows:DR is defined as the relationship between changes in blood pressure and alterations in blood flow rate as P< 0.05. Data are presented for an at-rest state (●) and postcycling (□). The repeated ANOVA was used to assess the differences between the baseline and weeks 4, 8, and 12 during the 12-week exercise training period. An ANCOVA with a baseline score as a covariate was used for a statistical analysis to compare the differences between at-rest and postcycling intervention for each lab visit. Statistical analyses were conducted using SPSS 19.0  software.All data are presented as the mean (SD) and statistical significance was assumed to beEp) and arterial stiffness index (β) when at rest and immediately following cycling intervention at different points in time, i.e., weeks 0, 4, 8, and 12. It can clearly be seen from  Ep and β were significantly decreased at weeks 8 and 12, respectively, compared with week 0. In response to acute cycling intervention, Ep increased significantly at weeks 0 and 4, whereas β significantly increased at weeks 0, 4, and 8 in comparison with those during an at-rest state.The maximal, mean, and minimal diameters at rest and immediately following cycling intervention at different points in time  are shown in Vmean at week 12 and Qmin at weeks 4, 8, and 12 immediately after exercise, and Qmax and Qmean when at rest at week 12, were remarkably increased. In response to acute cycling intervention, the HR, Vmax, Vmean, Vmin, Qmax, and Qmean increased significantly at different time periods, with the exception ofQmean at week 12, whereas Qmin decreased significantly at the four measurement times.As indicated in As shown in  in vivo. The main results regarding arterial stiffness and hemodynamics at rest during the 12-week exercise training period can be summarized as follows. (1) The carotid arterial stiffness was decreased at week 8, whereas the arterial diameter was not changed at week 8 or 12 when at rest. (2) Carotid blood flow was improved at week 12, whereas a decrease in heart rate occurred at week 12. (3) Systolic pressure, diastolic pressure, dynamic resistance, and peripheral resistance were decreased, whereas the maximal WSS and mean WSS were increased, at week 12. In addition, arterial stiffness and hemodynamic responses to acute cycling intervention across the 12-week exercise training program are as follows. (1) Arterial stiffness was increased and the diameters were decreased the most at week 8 of exercise training, with no significant changes in arterial stiffness and diameters shown in week 12. (2) Despite the significant differences in carotid blood flow and the hemodynamic forces  between periods of at-rest and postcycling intervention for each time period, a progressive decrease was presented across the 12-week exercise training period.Acute exercise intervention is an effective method for investigating the cardiovascular functional response to external stimuli, whereas repeated episodic bouts of long-term exercise training induce chronic functional adaptation, and ultimately structural arterial remodeling . HoweverRegular aerobic-endurance exercise, such as running or cycling, has a positive effect on arterial stiffness , whereasOn the other hand, hemodynamic alterations induced through exercise training have been confirmed to play a role in cardiovascular disease reduction . The carAcute exercise intervention induces immediate changes in artery function and structure. A novel aspect of the current study is detecting arterial stiffness and hemodynamic responses to acute cycling interventions at different times across the 12-week supervised exercise training period. Considerable research efforts  have demBeneficial alterations have been observed following aerobic exercise training, whereas potentially detrimental effects occur with high-intensity acute exercise training. An acute bout of high-intensity cycling was shown to result in a significant increase in fluctuations of blood pressure and blood flow . High flThere are some limitations in the present study worth addressing. First, participant recruitment may be misleading for including only males and not including females. Literature , 49 and In the present study, a 12-week exercise training period at moderate intensity decreased arterial stiffness and altered the hemodynamic forces at different periods of time. In response to acute exercise intervention, arterial stiffness increased and the diameters decreased the most at week 8 of the exercise training, whereas arterial stiffness and diameters showed no significant responses at week 12. In addition, the hemodynamic responses presented a progressive decrease across the 12-week exercise training period. In summary, long-term exercise not only improves arterial stiffness and hemodynamic alterations at rest state but also negates the detrimental arterial stiffness and hemodynamic responses to acute exercise intervention.


It is meaningful to expand the available population information on forensic medicine and to investigate the genetic characteristics of Han population from Jilin Province, Northeast China.TM Y24 amplification kit. Additionally, we performed the population comparison between the Jilin population and the other nine reported populations based on the Y‐STR genotyping haplotypes.In this study, we investigated the genetic characteristics of 24 Y‐chromosomal short tandem repeat (STR) loci in 1,088 unrelated Chinese Han male individuals from Jilin Province, using DNATyperA total of 1,067 different haplotypes were found from 1,088 unrelated individuals, of which 1,046 were unique and 21 were shared by two individuals. The gene diversity values of 22 loci ranged from 0.3870 (DYS391) to 0.9668 (DYS385ab). The random match probability was 0.0010 with the discrimination capacity of 0.9807. Population comparison showed that there were minor differences compared to Beijing Chinese Han, China Manchu, Gansu Chinese Han, and Jiangsu Chinese Han, but major differences with respect to the populations of Guangdong Chinese Han, Yunnan Chinese Han, China Hui, China Korean, and China Tibetan.Our results showed that the 24 Y‐STR loci in Jilin Han population are valuable for forensic application and human genetics. Subsequently, separation and detection were performed using an Applied Biosystems™ 3500 Series Genetic Analyzer (Life Technologies). Finally, the raw data were analyzed using GeneMapper ID v4.1 software (Life Technologies). We strictly followed the recommendations of the DNA Commission of the International Society of Forensic Genetics on the analysis of Y‐STRs  between our data and the other published groups was generated by the analysis of molecular variance (AMOVA) and was visualized in two multidimensional scaling (MDS) plots of Rst using the YHRD online tools .PowerMarker v3.25 was used to calculate the allele frequencies and gene diversity of the 24 Y‐STR loci (Gusmao et al., 3Rst values for genetic distance showed that haplotypes of Jilin Han population had significant differences from those of the other nine populations (all p < .05, see Table A total of 1,067 different haplotypes were found from 1,088 unrelated individuals, of which 1,046 were unique and 21 were shared by two individuals (see Table 4We reported the 24 Y‐STR loci allelic frequencies, haplotype distribution of Jilin Han population, which exhibited the powerful discrimination capability and could be applied in forensic medicine. Additionally, the population comparison showed that Jilin Han population owned its unique structure, which could be used in ethnic tracing. In conclusion, the data provided a reference for Y‐STR database of Jilin Province and might be valuable for human genetics.None declared.XMX and JLZ participated in the design of the study and performed the statistical analysis. BJW and JY carried out the studies and drafted the manuscript. JY conceived the study and participated in its design and coordination. All authors contributed toward the drafting and revising of the final manuscript. Click here for additional data file. Click here for additional data file.


Fish protein hydrolysates are suggested to contain bioactive sequences capable of affecting metabolic pathways involved in the regulation of glucose metabolism and body weight when consumed in low doses. Modulation of the appetite-regulating hormone ghrelin may explain suppression of insulin secretion and weight loss observed in previous studies with fish protein hydrolysates.This study aimed to assess the effect of a single, low dose of cod protein hydrolysate (CPH) before a breakfast meal on postprandial acylated ghrelin concentration and sensations associated with appetite in healthy subjects.In this explorative trial with a crossover design, 41 healthy individuals  completed 2 study days separated by 4–7 days of washout. On both study days, a test drink containing 20 mg CPH or casein (control) per kg body weight was given immediately before a standardized breakfast meal. Acylated ghrelin concentrations were measured before test drink/breakfast (baseline) and at time 0, 20, 40, 80, and 180 min postprandially. Sensations associated with appetite were measured by a Visual Analog Scale (100 mm) at baseline and 0, 20, 40, and 180 min postprandially.P = 0.266), or between the total area under the curve (tAUC) for acylated ghrelin after CPH  and control . No differences were found between CPH and control for sensation of appetite, according to tAUC of postprandial scores for satiety (P = 0.794) and the feeling of fullness (P = 0.996).Statistically, no difference was observed between CPH and control for postprandial acylated ghrelin concentrations (mean difference geometric mean: 1.05 pg/mL, 95% confidence interval [CI]: 0.97–1.13, We did not find an effect of a single dose of CPH on postprandial concentrations of acylated ghrelin or sensations related to feeling of hunger, compared to control. Further studies should aim to evaluate the effect of a supplement with CPH given daily over a period of time. Ghrelin is an appetite-regulating hormone, with high concentrations before a meal and reduced concentrations after a meal. Compounds with the ability to suppress the action of ghrelin may be valuable for weight regulation.Fish protein hydrolysates are suggested to contain bioactive peptides capable of affecting glucose metabolism and body weight.In this study, no effect of a supplement with cod protein hydrolysate on postprandial ghrelin concentrations or sensations related to appetite was observed.Ghrelin is a gastric hormone, capable of stimulating hunger and influence energy homeostasis . It is aAcylated ghrelin is a natural ligand binding to the growth hormone secretagogue (GHS) receptor, leading to stimulation of the secretion of growth hormone (GH), reduction in insulin secretion and glucose tolerance –6. Furthper se, which is negligible compared to the normal recommended total daily dietary intake of a healthy individual (e.g. 65–80 g protein per day with body weight 80 kg)  . A possit 80 kg) . In the As metabolism and energy expenditure decrease with age, middle-aged individuals often experience weight gain. Thus, middle-aged individuals might benefit from an intervention targeting appetite and hunger regulation. Data on the specific effect of a dietary supplement with a fish protein hydrolysate on ghrelin concentrations and sensations associated with appetite have, to our knowledge, previously not been published. The present study aimed to assess the effect of a single, low dose of CPH before a breakfast meal on postprandial acylated ghrelin concentration and sensations associated with appetite in healthy, middle-aged to elderly subjects.Data on subjects and methods have been described in detail in a previous publication .The study was a double-blind crossover trial, which included two study visits for each subject, with 4–7 days of washout in between. The intervention included serving of a test drink containing 20 mg of CPH per kg body weight  or control (casein) in randomized order, immediately before a standardized breakfast meal was served. The CPH and casein powder were mixed with cold water and served as a drink. The primary outcome of the intervention  is reported in a previous publication . Here, wThis study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures were approved by the Regional Committees for Medical and Health Research Ethics of Central Norway (2017/1794). Written informed consent was obtained from all subjects. The trial was registered at clinicaltrials.gov as NCT03669796.Subjects were recruited at Haukeland University Hospital and Ålesund Hospital between October 2017 and February 2018. Potential subjects were interviewed for general eligibility and compliance with inclusion and exclusion criteria by telephone. Candidates were invited for a further screening visit.2. Exclusion criteria were fish allergy, pharmacologically treated diabetes mellitus, elevated blood pressure, chronic diseases that might affect the evaluation of the study endpoints, and acute infections.Inclusion criteria were as follows: age 40–65 years and body mass index (BMI) 20–30 kg/mThe screening visit included a clinical examination by a physician, biochemistry tests for safety purposes  and compliance with inclusion criteria, measuring of height, weight, and blood pressure, as well as assessment of the level of physical activity. The level of physical activity was assessed by asking the participants two questions regarding moderate physical activity and vigorous activity (self-reported). The participants were instructed not to change the diet composition or the level of physical activity during the study period. On the day preceding each study day, the participants received a standardized porridge evening meal to be eaten before 8:30 pm. After this, the subjects were instructed to fast until the next morning and were only allowed to drink water. On study days, the participants came to the research units in a fasting state between 08:00 am and 09:00 am. After the first blood sample, the subjects were served the test drink, before the breakfast meal was provided. Fifteen minutes after the breakfast was served, the first post-meal sample (0 min sample) was taken.ad libitum, but no coffee or tea was allowed. The subjects spent 4 h in the research units, and repeated sampling of blood was conducted before serving of the test drink and breakfast, and at time 0, 20, 40, 80, and 180 min postprandially.The standardized breakfast meal consisted of two slices of bread , 10 g margarine, 20 g strawberry jam, and 20 g white cheese. This provided a total of 355 kcal . The drink contained on average 35.9 g carbohydrate and 145 kcal (607 kJ). Thus, including the drink, the breakfast provided in total 500 kcal  and 77 g carbohydrate, equal at both study days. Water was given Assessment of medical history, measurement of biochemical variables and safety parameters were conducted before randomization. During the two study visits, acylated ghrelin was measured in plasma samples taken before serving of the test drink and breakfast (baseline), at time 0, 20, 40, 80, and 180 min postprandially. The subjects had 15 min to finish the breakfast before the 0 min sample was taken. Blood pressure was measured before intervention, after 40 and 180 min after the intervention, as a safety parameter.Appetite sensation was assessed on a Visual Analog Scale (VAS) of 100 mm in length, addressing the feeling of fullness and satiety. The VAS questionnaire also included three questions regarding adverse gastrointestinal symptoms . The VAS was filled out five times during the study visit, at baseline, time 0 and 20, 40, and 180 min after the breakfast meal. Additionally, a questionnaire validated for the evaluation of different gastrointestinal symptoms was filled out before the breakfast meal and at the end of each study day . The queThe test material was a lemon-flavored powder provided from the manufacturer  in standardized bottles to be added 150 mL cold water. The powder contained 4% protein  and 96% carbohydrate . Thorough laboratory tests assured that it was not possible to identify the active ingredient from the control, according to flavor or appearance. Each subject was given individually adjusted doses of 20 mg/kg body weight of CPH or casein. The drinks were made isonitrogenous to avoid bias due to difference in nitrogen content, and equal amounts of nitrogen in the form of casein were added to the control drink. Both drinks contained on average 1.6 g protein; thus it constituted only a small fraction of the total protein content of the standardized breakfast meal. Casein was chosen as the control as it has previously shown to not affect blood glucose or insulin sensitivity when compared with proteins from cod and soya . The casSamples of venous blood were repeatedly collected using an intravenous catheter from the antecubital vein. Samples were analyzed according to standard accredited methods at the Laboratory for Clinical Biochemistry, Haukeland University Hospital  and the Department of Medical Biochemistry, Ålesund Hospital .®, Greiner Bio One International GmbH, cat # 454261, Kremsmünster, Austria) at 1800 × g at 4°C for 10 min within 20 min after blood sampling. Plasma was stored frozen at −80°C prior to analysis. The ghrelin analyses were performed using Acylated Ghrelin-Easy Sampling Enzyme Immunoassay kit .Plasma ghrelin was obtained by centrifugation of full blood using 4 mL anticoagulant (EDTA-K3)/Aprotinin blood collecting tubes  and GraphPad Prism version 7.0  were used for statistical analysis. Shapiro–Wilk’s test was conducted to assess normal distribution of data. A multivariable, repeated-measures linear mixed-effects regression analysis (adjusted for BMI and gender) was conducted in SPSS in order to evaluate the difference between the concentrations of acylated ghrelin after CPH and control. The data for acylated ghrelin were non-normally distributed; thus, it was log-transformed before analysis and presented as log mean and back-transformed values (geometric means). Graphical work and total area under the curve (tAUC) analysis for acylated ghrelin concentrations and symptom VAS-scores were conducted in GraphPad. The difference in baseline and end-point scores for the additional questionnaire was evaluated by a paired The number of participants was not calculated according to a power analysis, due to lack of similar studies. Previous research reporting on effect of cod proteins in humans is based on whole fish  or long-2. Twenty-three participants had BMI ≤ 25 kg/m2, whereas 18 participants had BMI > 25 kg/m2. Baseline characteristics and comparison of gender distribution are presented in Seventy-eight subjects were screened for inclusion and exclusion criteria over telephone, of which 47 were enrolled to a screening visit. Six participants withdrew before the first study visit, and 41 participants completed the trial, of which 15 males and 26 females. The inclusion process has previously been described and illustrated . The meaP > 0.999). Suppression of acylated ghrelin (Cmin) was greatest: 80 min postprandially after CPH and 20 min postprandially after control, with a Cmin mean difference from baseline of −14.0 ± 21.4 after CPH and −5.3 ± 22.8 after control, at these times, respectively (P = 0.681).Mean fasting acylated ghrelin levels were higher before the CPH intervention than before the control intervention , but the difference was not significant (P = 0.266) (P = 0.991).Statistically, no differences were observed between CPH and control for postprandial acylated ghrelin concentration in a mixed-effects regression analysis  . Additior = 0.118, P = 0.463). When adjusting for BMI and gender in the mixed-effects regression analysis, no differences were observed between subjects with BMI ≤ 25 kg/m2 compared to those with BMI > 25 kg/m2 (P = 0.681), or between genders (P = 0.627).No correlation was observed between body weight (kg) and baseline concentration of acylated ghrelin  (P = 0.997). No difference was observed between the tAUC for the postprandial satiety scores after CPH  and control . Data are presented in Baseline scores for satiety were numerically the same before each intervention . No difference was observed between the tAUC for the postprandial feeling of fullness scores after CPH  and control . Data are presented in Baseline scores for the feeling of fullness were numerically the same before each intervention . Baseline scores did not differ before each intervention .The questionnaire addressing hunger did not reveal any differences in the feeling of satiety and hunger between CPH (3.4 ± 1.8) and control (3.4 ± 2.3) 180 min after the breakfast  during the exposure for either CPH or control.This study revealed no differences in postprandial concentrations of acylated ghrelin after a meal supplemented with CPH compared to control. Thus, we were unable to confirm our hypothesis that a single dose of CPH supplementation before a meal would suppress ghrelin concentrations postprandially, and thereby cause reduced feeling of hunger. Moreover, we found no differences between control and CPH drink in the feeling of satiety or feeling of fullness, as measured by the implemented questionnaires.In a previous publication, we reported that supplementation with 20 mg CPH per kg body weight before a breakfast meal reduced the postprandial concentrations of insulin compared to control in healthy individuals . AlthougOur results are partly in line with previous similar single-dose studies; however, few studies are comparable in test material. Most studies investigating the relationship between ghrelin levels after meals with different macronutrient composition have found that a high-protein meal causes the ghrelin levels to be suppressed for a longer time than after the intake of a meal high in carbohydrates –31. Furtin vitro and in vivo models, showing that the fish protein hydrolysate was capable of enhancing the secretion of CCK and GLP-1, both hormones contributing to the regulation of energy intake  is quite recently suggested to be an orexigenic hormone influencing appetite and regulation of food intake . Thus, fIn conclusion, we did not find any effect of a single dose of CPH on postprandial concentrations of acylated ghrelin, or sensations-related appetite in healthy individuals, when compared to control after a standardized breakfast meal.


BMJ 2019;364:l223, doi:10.1136/bmj.l223, published 13 February 2019), the authors identified the following errors after reanalysis of the dataset:In this paper by Taylor and colleagues .”In the results paragraph of the abstract, the last sentence should read: “There was a deprivation gap in median survival of 0.5 years between people who were least deprived and those who were most deprived .”In the results section, the second sentence of the socioeconomic inequalities paragraph should read: “Overall, there was a deprivation gap of 0.5 years in median survival between the least deprived and most deprived groups , indicated that the risk of death increased by 6% with level of deprivation .”In the discussion section, the last sentence of the principal findings paragraph should read: “The deprivation gap in survival of 0.5 years between the least deprived and most deprived groups suggests socioeconomic inequalities in heart failure care.”The article will be updated in due course.


This study proposed to access the clinical outcome of avulsion fractures around joints of extremities using the hook plate.A total of 60 patients with avulsion fractures of joints admitted in our hospital between January 2011 and June 2016 were performed the surgery of hook plate fixation. Functional recovery was evaluated using the Lysholm knee score, Kaikkonen ankle injury score, Mayo elbow and wrist function score, and Neer shoulder function score.p < 0.05).All the patients were healed within 3 months after surgery with stage I healing incision without vascular or nerve injuries. The average follow-up period was 18.1 months. At the last follow-up, no instability of joints, looseness of internal fixation or traumatic arthritis was observed. Mild joint fibrosis occurred in 5 cases. A total of 57 patients were well recovered with the excellent and good rate of 95%. Three patients with humeral avulsion fracture of the greater tuberosity had shoulder joint adhesion and peri humeral inflammation at the last follow-up due to the poor cooperation for early rehabilitation exercise. In the last follow-ups, the functional score of the affected limb was markedly greater than that in the 3-month follow-ups (Hook plate fixation has the therapeutic effect on treating avulsion fractures around joints of extremities with the advantages of reliable fixation, early rehabilitation after operation, high recovery rates of joint function, wide indications, and convenient uses. Joints are surrounded by articular capsule, ligaments and tendons. Avulsion fractures occur when the capsule, ligaments or tendons are avulsed from the bone due to unexpected contraction of the muscles or mechanical force . As repoRecently, accumulating evidence has strongly implied that the hook plate fixation has the advantages of more stable fixation of the fragments regardless of the bone quality and high accurate reduction in fracture with small fragments in the treatment of avulsion fractures . For exaThe purpose of the present study was to summarize clinical data of patients with avulsion fractures around joints of the extremities treated with hook plate fixation. More importantly, it was our goal to assess the necessity of surgery, the flaw of the mainstream internal fixation as well as the specific characteristics and therapeutic outcomes of hook plate.Inclusion criteria: (1) Avulsion fractures that were fit for the treatment of hook plate fixation, including the fractures of the humeral greater tuberosity, humeral condyle, ulna olecranon, ulnar styloid, ankle joint, and tibial condyle. (2) The width of the avulsed fracture bone is greater than 10 mm, which is larger than the opening of the hook plate. (3) The fracture separative shift shown in CT scan or X-ray is more than 5 mm. (4) Follow-up data were completed. Exclusion criteria: (1) Patients with concurrent severe fractures or vascular and nerve injury at the same joint, (2) The follow-up time was less than 12 months, (3) less than 18 years old.A total of 60 patients who met the criteria were recruited for this study from January 2011 to June 2016. n = 24), sports related injury (n = 17), falling injury (n = 11), and traffic injury (n = 8). Of the 60 patients, 19 cases were avulsion fractures of posterior cruciate ligament (PCL) at the tibial insertion, 6 cases were avulsion fracture of the humeral medial epicondyle, 8 cases were avulsion fractures of the humeral greater tuberosity, 6 cases were lateral malleolus avulsion fractures, 17 cases were ulna olecranon avulsion fractures, and 4 cases were ulnar styloid avulsion fractures. All fractures were fresh and unstable fractures. All patients presented to the clinic between 2 h to 19 days with the mean day of 6.6 days after injury. To confirm the diagnosis of avulsion fractures, patients received X-ray, computed tomography (CT) or/and Magnetic resonance imaging (MRI) examination. The general information of the patients was shown in Table There were 35 males and 25 females aged between 20 and 73 years (mean age 43.7). The causes of fracture included fall injury  or 1/3 titanium plates (n = 39) with two symmetric sharp teeth hooks, as shown in Fig. The operation was performed in all patients under general anesthesia. The hook plates used in this study were AO hook plates n = 2 or 1/3 tThe limbs were treated with the assistance of continuous pressure by elastic bandages within 2 days post operation. Functional rehabilitation training began after anesthesia failure. One week postoperatively, patients with avulsion fractures of lower limb ligaments were encouraged to do some gentle exercise in the case of wearing the orthosis. From the 8th week, a free motion and weight bearing were allowed without orthosis. However, on the third day after surgery, patients with avulsion fractures of upper limbs were allowed to do recovery exercise without orthosis. All of the patients were reviewed and examined monthly after the operation and every 2–3 months after fracture union. One year later, the review period was changed to every 6 months. At the final follow-up, the functional recovery was assessed using the Lysholm knee scores , Kaikkonp values of < 0.05 were considered significant. Data were presented as mean ± standard deviation (SD), and were analyzed using SPSS software .Statistical significance levels were determined by two-tailed t test or one-way analysis of variance (ANOVA). The p < 0.05). No fractures with dislocation or delayed union were found in all patients. In the last follow-up, 3 cases (average age 55) with avulsion fractures of the humeral greater tuberosity had shoulder joint adhesion and peri humeral inflammation, and the functional recovery of shoulder joint was poor. Other patients were scored excellent or good of the functional recovery results. The total excellent and good rate was 95%. In addition, no instability of joints, looseness of internal fixation or traumatic arthritis was observed. Mild joint fibrosis occurred in 5 cases. In the last follow-ups, the functional score of the affected limb was markedly greater than that in the 3-month follow-ups (Table p < 0.05). Internal fixation was removed after one year of surgery in 35 patients. The follow-up data for functional scores at each site was shown in Table All the patients were healed within 3 months after surgery with stage I healing incision without vascular or nerve injuries. The mean duration of follow-up was 18.1 months (range from 12 to 24 months). The functional score of the affected limb was significantly improved 3 months after surgery . However, the former was superior to the control group in terms of functional recovery and range of joint activity, and the difference was statistically significant (P < 0.05) [The hook plate used in surgical treatment of avulsion fractures had several advantages as follows , 13, 21  < 0.05) .In this study, 3 cases with avulsion fractures of greater tuberosity of humerus had shoulder joint adhesion and perihumeral inflammation, and the functional recovery of shoulder was poor. One reason is due to the high age of the patients (average age was 55). In addition, the patients did not actively cooperate with the treatment for early rehabilitation exercises because of their low requirements for shoulder joint function after the operation. Meanwhile, the hook is located above the greater tubercle, which may impact the acromion and affect shoulder joint activity. Therefore, anchor suture and fixation should be the first choice for avulsion fracture of greater tubercle of humerus. Our study was designed to evaluate the outcomes of the hook plate fixation for 60 patients with avulsion fractures around joints. All the patients performed early rehabilitation exercises and the incision healed well without complications, such as screw loosening, instability of joints and arthritis. The excellent and good rate of joint function recovery was about 95%, which was higher than traditional internal fixation . NeverthIn conclusion, hook plate fixation has the therapeutic effect on treating avulsion fractures around joints with the advantages of reliable fixation, early rehabilitation after operation, high recovery rates of joint function, wide indications, and convenient uses.


A unique population structure using principal component analysis illustrated clear distinctions between Duroc, Yorkshire, Hampshire, breeds of Chinese origin, and feral Pacific Island populations were identified. Five Y chromosome haplotypes were evaluated and demonstrated migration patterns from European, central Asia, and potentially Polynesian waves of gene flow. Quantifying diversity and potential origin of Pacific populations provides insight for future uses, and the need for preservation. Viewing gene bank holdings in context of diversity measures we found a lack of inbreeding within breeds, suggesting the collection represents a wide sampling of individual breeds.Human migration and trade facilitated domesticated livestock movement, gene flow and development of diverse populations upon which agriculture is based. In addition, varying USA ecological conditions has led to a diverse set of livestock populations to utilize. Quantifying genetic diversity of these populations is incomplete. This paper quantifies genetic diversity captured by the National Animal Germplasm Program and explores genetic structure and differences among 19 pig populations  using 70,231 SNP from 500 animal samples. Among continental US breeds F Specifically the assessment provides insights to the genetic variability captured in the USDA’s swine germplasm collection6. The study evaluated major breeds, often termed international commercial breeds, rare breeds, Chinese breeds imported into the U.S. during the late 1980’s, and feral populations from the Pacific islands of Hawaii, Kauai, and Guam. It has been hypothesized that such feral populations may be harbingers of genetic diversity not found in other US populations given the potential for either Asian or Polynesian ancestry7.The objective of this study was to quantify the genetic diversity of a substantial number of pig breeds found in the United States (US) as previous assessments tend to be focused on Europe and Asia8 and additional domestic pigs gone feral upon the chain of Hawaiian Islands. McCann et al.8 suggested animals sampled in the Hawaiian Islands originated from Europe, China, Papua-New Guinea, Vanuatu, and shared haplotypes with other domestic-feral pigs and S. verrucosus (from Java). Larson et al.9 suggest Hawaiian Islands pigs as members of the Pacific clade. No analysis has been performed on Guam’s feral population. But as part of the Mariana island chain, feral pigs might belong to an East Asia clade9.Current feral pig populations on the Hawaii and Kauai are the product of a migration wave associated with Polynesian settlers followed by European explorers during the 1700’s10: England, China, and the U.S. Obviously, U.S. breeds were imported or derived from other parts of the world at varying points in time; however, the imports have been used to maintain the original breed or construct new breeds like Duroc, Hereford, Ossabaw Island, Spots/Poland China, and Chester White. The Duroc in particular is an interesting example as its foundation breeds have not been shown to have strong genetic linkages with other European breeds3. These authors have suggested that the breed may have been comprised, in part, of Guinea Hog from Africa (among other breeds) due to its red color.Breed development occurred in three geographic areasA better understanding of the genetic diversity among these U.S. populations has a number of uses, including: general management of genetic diversity by public and private sectors, targeting future germplasm collections, and providing input into policies concerning feral pig populations on Pacific islands.Basic marker statistics of the GGP Porcine HD v1  have shown that more than half of the markers were polymorphic for feral and rare breeds/genetic groups Table  suggestiThe calculated genetic diversity parameters Table  were perin-situ population captured in the repository by the germplasm conservation program. The feral population on HI had the highest inbreeding level (0.081) which is higher than the inbreeding levels for KI (−0.017) and GI (0.011), which are geographically smaller islands.Overall, the levels of inbreeding in most of the populations evaluated were low indicating the high genetic diversity levels among the ST, was non-significant for only three comparisons MA vs LB, MA vs OI, and MA vs GH  accounted for 39.6% of the variation Fig. . The pri11. However, the YK subpopulations are likely due to imported animals during the 1980’s which were widely used within that breed.ADMIXTURE was run with K = 2 to 21 and those results were in agreement with PCA. Cross validation error was used to assess which K provided insights into population structure. That said from K = 7 to K = 21 the cross validation curve was asymptotic with an approximate 1.5% change per K. Therefore we selected K of 2, 3, 7, 8, and 10 for evaluation. At K = 2 the breeds were partitioned between Meishan (ME), originating in China, and those from the continental US. Admixed were the populations from GI, HI, KI, MI, and FE  within breed/population were low suggesting boars sampled for the germplasm collection were lowly related and a broad range of genetic variability has been captured in the gene bank. Furthermore, FIS values were lower than the pedigree based inbreeding levels previously computed20 and Canadian values15.Based upon the population parameters evaluated US breeds, as represented by germplasm samples in the repository, are genetically robust with regard to heterozygosity levels. Genetic diversity of repository samples were similar to European, Asian and previous US valuesST suggested the breeds evaluated are distinct; similar to21, using microsatellite markers (FST = 0.10 between DU and YK). As reported in the literature15, the estimates of FST within European and within Asian origin populations are smaller than among Asian and European breeds and are in agreement with ADMIXTURE analysis. Paired combinations of Fst between feral populations of the Pacific and breeds originating in China were similar and confirm the PC analysis.Among populations, pairwise F22. On the other hand, the low number of markers should not be an issue since these markers did not suggest the presence of bottlenecks (data not shown); conversely, they highlight the high genetic diversity of these populations (high Ho and low FIS).The number of polymorphic SNP in the larger panel Table  and with23. Typical of PCA analysis a relatively small proportion of genetic variance was accounted for, however previous reports for pigs display similar proportions24. Our results underscore the divergence of Duroc which usually appears as a main and separate cluster from other breeds26.The PCA provides a unique view of the relationship among the 19 populations evaluated, in terms of the placement of US mainland breeds (and selection efforts which have differentiated these populations), Pacific islands, and breeds from China  pair-wise FST values were similar to the DU – HS value. The three maternal white breeds had significant but smaller pair-wise FST values, the PC analysis indicated the YK was distinct from the LA and CW  and HI (H1 and H2) were present among continental US populations, suggesting admixture with later importation of European derived pigs. The PCA and ADMIXTURE results appear to support the haplotype analysis among the Pacific island populations. In Fig. Y-chromosome results provide insights into the migration of these swine populations. Interestingly GI’s population had haplotypes from China (H4) and an unknown origin (H5). We hypothesize that H5 may be part of the Pacific clade as reported earlier and HI H and H2 wth century. However, because of its presence in the Chinese MI breed H2 could have originated during the domestication of pigs in China and imported into Europe during the 18th century.Haplotypes H1 and H2 are common in the continental US and the Hawaiian Islands. Haplotype-2 was the most cosmopolitan of haplotypes appearing in China, Hawaiian Islands and US. Additionally its internal position in the network analysis Fig.  suggests27.Haplotype H3 was observed in 11 of the 19 populations but it was only found in the continental US. Also, it was the only haplotype among the prominent breeds: DU, HS and YK; and minor breeds: TA, Hereford and GH. Given that the Chinese and Pacific populations lack the H3 haplotype suggests a European origin, and may have as an original source wild boars found west of the Caucus Mountains14 assessing Canadian DU, YK, and LA using LD. This study’s results are also in agreement with the Ne computed with pedigree information19 for DU, YK, LA, HS, and BE. In all instances Ne was >50 exceeding the minimum Ne suggested by FAO28. By pooling study populations we noted substantial variation, as suggested by an Ne = 236 which resides in the gene bank and can be used to advance breeding goals, similar to that reported with sheep29.A major aspect of this study was to provide a first assessment of the within and among breed genetic diversity captured in the national gene bank collection. Effective population sizes for commercial breeds were in agreement with work28. Results for breeds with less than 15 animals’ are viewed as preliminary. That said, genetic parameters for SP, HE and TA were similar to breeds with larger sample size. Up to K = 10 in the ADMIXTURE analysis the SP and TA were admixed, at K = 10 the HF were placed in a unique cluster, but TA was not. Those early admixture patterns for low-medium K values reflect clusters of breeds that share recent admixture events, higher genetic diversity and/or were not undergoing selection nor experienced a recent bottleneck15.For breeds with more than 20 animals genotyped the results tended to be homogeneous, in that, levels of heterozygosity and inbreeding were similar and at levels that suggest sufficient genetic variability exists among the populations captured in the repositoryThe samples from Guam, Kauai, Hawaii, and US/Chinese ME provide interesting models for evaluating small isolated populations. The ME included randomly bred lines; while the island populations are all feral but confined to the island sampled. Genetic parameters Table  for GI aLarge and substantial genetic differences in pig populations dispersed across the continental US, Pacific Islands and Chinese breeds were found. Results suggest that feral populations from Pacific islands have a combination of European, East Asian and potentially South East Asian origin. Identifying the diverse origins of feral pigs on the Pacific islands contributes to the policy dialog about the control/eradication and/or conservation of these populations. While there may be environmental issues, the populations in question are unique and may have cultural significance for persons of Polynesian ancestry. Exploring feral genomes further may reveal other genetic attributes of interest for the research community and industry.in-situ populations, our results suggest no lack of genetic variability. Some rare breeds showed a lack of genetic distinction in PC analysis and were admixed which was likely due to limited sample size. But, HE and TA were distinct  and were shown to have measures of genetic variation in the same ranges as the commercial breeds. As the collection of swine genetic resources in the gene bank continues to grow it will be a resource to further explore various aspects of genetic diversity or gene function.This study’s evaluation of commercial breeds acquired for gene banking purposes is encouraging. Samples from commercial breeds suggest a range of genetic variability has been captured by the gene bank. As an indicator of genetic variability among No samples were collected for this study; rather they were collected as part of other studies or program activities not associated with this study. Semen samples were acquired from swine artificial insemination centers by the National Animal Germplasm Program as part of efforts to conserve genetic resources. Blood samples from Pacific island populations were collected by USDA Wildlife Services in their routine efforts to manage feral populations and then provided to the National Animal Germplasm Program.http://genomics.neogen.com/en/). All samples and genotypes are deposited with the USDA ARS National Animal Germplasm Program (https://nrrc.ars.usda.gov/A-GRIN/database_collaboration_page_dev).This study focused on assessing populations found upon the continental US and its Pacific islands. We were particularly interested in evaluating feral Pacific populations’ origin and relationship to rare and commercial breeds. Samples and data originated from 19 pig breeds , Meishan – U (randomly mated control herd maintained at Iowa State University) and Meishan – MARC (randomly mated control herd maintained by USDA-ARS).The Meishan was composed of three subpopulations2; Kauai, 1,440 km2; and Hawaii, 10,432 km2. Guam’s samples were from the same area of that island.Substantial distances exist between the geographic areas sampled. From continental U.S. to the Hawaiian Islands is approximately 3,900 km and Hawaiian Islands to Guam is approximately 6,000 km. The distance between the Hawaiian Islands of Hawaii and Kauai is approximately 500 km. Island size varies greatly: Guam, 544 km. Due to the use of feral and rare breeds we first evaluated the polymorphism content of the BeadChip transferable to these breeds. SNP with call rate <0.95 and located at sexual chromosomes were pruned from the initial dataset, leading to 57,668 SNP. Basic marker statistics as number of polymorphic SNP, average call rate and average number of alleles were calculated for each breed , using GGP Porcine HD v1 . Final Reports generated with Genome Studio Software  were imported into SNP & Variation Suite v8.6 ed Table .2 > 0.3. The final data set had 8,764 SNP used in all autosomal analysis.The next step was remove markers not mapped in the Sscrofa10.2 assembly . The 54,994 remaining SNP were filtered using the following genetic parameters: Hardy-Weinberg Equilibrium (HWE) p < 0.0001, and Linkage Disequilibrium (LD) r30. Principal Component Analysis (PCA) using SNP & Variation Suite v8.6 (SVS)  was performed to evaluate population stratification. The SVS software implements the EIGENSTRAT algorithm31. The results were represented in a 3D scatterplot of the first three principal components using SigmaPlot 13 (https://systatsoftware.com/products/sigmaplot/).Within breeds genetic diversity parameters were estimated with 8,764 SNP using Arlequin 3.5.1.331 which is based on maximum likelihood. The inference of number of populations or clusters (K) present in the analysis was estimated by the cross-validation procedure., where the smallest cross-validation error rate indicates the most probable K value for the data set33. The default termination criteria for the ADMIXTURE analysis was assumed when the log-likelihood increases by less than ϵ = 10−4 between interactions. The termination criteria of is ϵ = 10−5 between interactions was also tested, without any difference on the cross-validation error values. The routine analysis was carried out for 22 clusters (Ks) with 10 replicates for each one. A second ADMIXTURE analysis with nine populations consisting of minor and rare breeds, and feral populations (12 clusters tested with 10 replicates each) was performed. The graphic representations of the proportions of assignment of each individual within population were visualized using CLUMPAK (http://clumpak.tau.ac.il/)34.Population structure analysis was carried out using ADMIXTURE 1.2.230 and the relationship between haplotypes was investigated by constructing median networks using Network 5 (http://www.fluxus-engineering.com/).The GGP Porcine HD v1 contains 14 SNP within the Chromosome Y. The analysis of the Y chromosome in mammals reflects the paternal history of migration and evolution of breeds and populations. For this analysis all female samples were eliminated, therefore 475 male samples were used. The 14 markers were pruned using a call rate <0.95. Of the remaining 11 SNP, two were monomorphic, and three more presented heterozygosity ≠ 0. Ultimately, six SNP were used to estimate the number of different haplotypes, haplotypes frequencies and to reconstruct a network among populations. The haplotype estimates and frequencies were calculated by the software Arlequin 3.5.1.334 was used with default conditions. Prior to using SNeP, SNP filtering was redone dropping the LD aspects. As a result 16,021 SNP were used in this analysis. Due to small sample sizes for some breeds the LD analysis was performed on 10 groups indicated by the population structure identified when ADMIXTURE analysis used K = 10. The 10 populations were: BE, CW, DU, a Chinese group , a Pacific island group , a minor breed group , HS, HE, LA, and YK35.Effective Population Size: To establish baselines and trends of Ne the SNeP softwareUSDA, Agricultural Research Service, is an equal opportunity/affirmative action employer. Mention of companies or commercial products does not imply recommendation or endorsement by the USDA over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names mentioned solely to report factually on available data and provide specific information.Table S1 – Overall characteristics of the 18 populations genotyped with the SNP Beadchip after initial quality filter with a total of 57,668 SNPs.