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Abstract Background The relationship between liver fibrosis and left atrial (LA) remodeling in atrial fibrillation (AF) remains uncertain. We examined the associations between the fibrosis‐4 (FIB4) index, an indicator of liver fibrosis, and both LA low‐voltage areas (LVAs) on electroanatomic mapping and AF recurrence postablation. Methods We recruited 343 patients who underwent radiofrequency catheter ablation (RFCA) or cryoballoon ablation (CBA) for AF. First, the association between the FIB4 index and LA LVAs (5 cm2) compared to those without. Additionally, the quantitative size of LVAs showed a positive correlation with the FIB4 index (R = .642, p

Abstract Cause of Kawasaki disease (KD) is unknown. KD is often resistant to treatment with intravenous immunoglobulin (IVIG). Sano’s score, which is derived from total bilirubin (TBIL), aspartate aminotransferase (AST) and C-reactive protein (CRP), is predictive of IVIG resistance in Japan. A recent study reported that Torquetenovirus (TTV), especially TTV7, was present at a high viral load in the patients with KD. We used PCR to quantify TTV load and amplicon next generation sequencing to detect individual TTV species. We used serum samples that were collected between 2002 and 2005 from 57 Japanese KD patients before IVIG treatment. Correlations between TTV load and Sano’s score, the biomarkers that constitute this score, and IVIG resistance were examined. TTV load was positively correlated with Sano’s score (P = 0.0248), TBIL (P = 0.0004), and AST (P = 0.0385), but not with CRP (P = 0.6178). TTV load was marginally correlated with IVIG resistance (P = 0.1544). Presence of TTV7 was correlated with total TTV load significantly (P = 0.0231). The correlations between biomarkers for KD and TTV load suggested that TTV may play a role in the pathophysiology of KD. We hypothesize that TTV7 may be associated with a higher total viral load in KD.

Abstract DNA damage contributes to atherosclerosis. However, causative links between DNA double-strand breaks (DSBs) and atherosclerosis have yet to be established. Here, we investigated the role of DSBs in atherosclerosis using mice and vascular cells deficient in Ku80, a DSB repair protein. After 4 weeks of a high-fat diet, Ku80-deficient apolipoprotein E knockout mice (Ku80 +/− ApoE −/−) displayed increased plaque size and DSBs in the aorta compared to those of ApoE −/− control. In the preatherosclerotic stages (two-week high-fat diet), the plaque size was similar in both the Ku80 +/− ApoE −/− and ApoE −/− control mice, but the number of DSBs and mRNA levels of inflammatory cytokines such as IL-6 and MCP-1 were significantly increased in the Ku80 +/− ApoE −/− aortas. We further investigated molecular links between DSBs and inflammatory responses using vascular smooth muscle cells isolated from Ku80 wild-type and Ku80 +/− mice. The Ku80 +/− cells displayed senescent features and elevated levels of inflammatory cytokine mRNAs. Moreover, the cytosolic DNA-sensing cGAS-STING pathway was activated in the Ku80 +/− cells. Inhibiting the cGAS-STING pathway reduced IL-6 mRNA level. Notably, interferon regulatory factor 3 (IRF3), a downstream effector of the cGAS-STING pathway, was activated, and the depletion of IRF3 also reduced IL-6 mRNA levels in the Ku80 +/− cells. Finally, DSBs accumulation in normal cells also activated the cGAS-STING-IRF3 pathway. In addition, cGAS inhibition attenuated DNA damage-induced IL-6 expression and cellular senescence in these cells. These results suggest that DSBs accumulation promoted atherosclerosis by upregulating proinflammatory responses and cellular senescence via the cGAS-STING (-IRF3) pathway.

Abstract Purpose The purpose of this study was to investigate the safety and efficacy of high-power short-duration (HP-SD) ablation compared to conventional ablation in patients with atrial fibrillation (AF). Methods We enrolled consecutive 158 drug-refractory symptomatic AF patients (119 males, mean age 63 ± 10 years) who had undergone first radiofrequency pulmonary vein isolation (PVI). PVI was performed using the conventional setting (20–35 W) in 73 patients (Conventional group) and using the HP-SD setting (45–50 W) in 85 patients (HP-SD group). The rate of first pass isolation, remaining gaps after circumferential ablation, dormant conduction, and the radiofrequency application time in each pulmonary vein (PV) were compared between the groups. Results The first pass isolation ratio was significantly higher in the HP-SD group than in the Conventional group (81% vs. 65%, P = 0.027) in the right PV, but did not differ in the left PV. The remaining gaps were fewer in the right superior PV (4% vs. 21%, P = 0.001) and left inferior PV (1% vs. 8%, P = 0.032) areas, and the radiofrequency application time in each PV was shorter (right PV, 12.0 ± 8.9 min vs. 34.0 ± 31.7 min, P

Pulmonary hypertension is characterized by increased pulmonary arterial pressure, driven in part by inflammatory infiltrates. Here, the authors show that in mice, transgenic expression of mutant JAK2 leads to clonal hematopoiesis and lung accumulation of elastase- and cytokine-expressing neutrophils, and that the phenotype can be reversed by ALK1 inhibition.

Abstract Background Homo sapiens have experienced admixture many times in the last few thousand years. To examine how admixture affects local adaptation, we investigated genomes of modern Polynesians, who are shaped through admixture between Austronesian-speaking people from Southeast Asia (Asian-related ancestors) and indigenous people in Near Oceania (Papuan-related ancestors). Methods In this study local ancestry was estimated across the genome in Polynesians (23 Tongan subjects) to find the candidate regions of admixture-enabled selection contributed by Papuan-related ancestors. Results The mean proportion of Papuan-related ancestry across the Polynesian genome was estimated as 24.6% (SD = 8.63%), and two genomic regions, the extended major histocompatibility complex (xMHC) region on chromosome 6 and the ATP-binding cassette transporter sub-family C member 11 (ABCC11) gene on chromosome 16, showed proportions of Papuan-related ancestry more than 5 SD greater than the mean (> 67.8%). The coalescent simulation under the assumption of selective neutrality suggested that such signals of Papuan-related ancestry enrichment were caused by positive selection after admixture (false discovery rate = 0.045). The ABCC11 harbors a nonsynonymous SNP, rs17822931, which affects apocrine secretory cell function. The approximate Bayesian computation indicated that, in Polynesian ancestors, a strong positive selection (s = 0.0217) acted on the ancestral allele of rs17822931 derived from Papuan-related ancestors. Conclusions Our results suggest that admixture with Papuan-related ancestors contributed to the rapid local adaptation of Polynesian ancestors. Considering frequent admixture events in human evolution history, the acceleration of local adaptation through admixture should be a common event in humans.

A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman’s R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.

Domain wall skyrmions have been proposed but the experimental observation has been difficult. Here, the authors report experimental discovery of domain wall bimerons due to the interplay between magnetic anisotropy and Dzyaloshinskii-Moriya interaction in chiral magnet Co-ZnMn(110) thin films.

Objective: To validate Indonesian versions of two social/cultural psychological scales: the Self-Construal Scale (SCS) that measures independent and interdependent cultural values, and the Behavioral Inhibition (Avoidance) System and Behavioral Approach System (BIS/BAS) that measures motivation focus. We also explored the cultural background for the rising prevalence of depression in Indonesia. Design: Case (hospital)–control (population) study. Setting: Hasanuddin University Hospital (cases) and Makassar city region (controls), Indonesia. Participants: Participants (N = 369) were 165 patients with depression recruited from a university hospital, and 204 healthy controls without a history of mental disorders recruited from locations within a 30-minute walk from the hospital. Outcome measures: Depression was diagnosed by psychiatrists with reference to Indonesian mental disorder guidelines (Pedoman Penggolongan dan Diagnosa Gangguan Jiwa edisi 3). Participants’ independent and interdependent cultural values, and neural motivational systems were measured with the SCS and BIS/BAS. Results: Exploratory and confirmatory factor analyses showed that our revised 12-item SCS and the 13-item, three-factor BIS/BAS had a good model fit for the Indonesian population. MANCOVA showed that the SCS Independent subscale and the BAS subscales were significantly associated with depression after adjustment for age, sex, religion, education, and occupation. Conclusion: These findings may guide provision of appropriate treatment for patients based on their social and cultural environment. In addition, this study contributes to understanding underlying reasons for the increasing prevalence of depression in Indonesia, where society is changing from traditional collectivism to global individualism.

Abstract Background To investigate the clinical implication of the temporal difference in atrial fibrillation (AF)‐onset in acute decompensated heart failure (ADHF) and its impact on post‐discharge prognosis. Methods 336 new‐onset ADHF patients without any history of AF before admission were enrolled (201 males, 63 ± 16 year‐old) and classified into two groups based on their history of AF: the Control group (No AF was detected during hospitalization, n = 278), and the In‐hos‐AF group (AF occurred during hospitalization, n = 58). Post discharge prognosis including rehospitalization due to worsening HF, cardiac death, all‐cause death and cerebrovascular event were compared. Results Kaplan‐Meier analysis demonstrated that the incidence of rehospitalization due to HF, cardiac death, all‐cause death and cerebrovascular event in the In‐hos‐AF group was not significantly different from that in the Control group (P > 0.05 respectively). However, when AF recurred in the In‐hos‐AF group patients (n = 24, 41%) after discharge, the incidence of rehospitalization due to HF and cardiac deaths were higher than those without AF recurrence (P = 0.018 and P = 0.027 respectively). Cox proportional analysis revealed that AF developing after discharge was proven to be an independent risk factor for rehospitalization due to HF (HR 1.845, P = 0.043), cardiac death (HR 3.562, P = 0.013) and all‐cause deaths (HR 2.138, P = 0.020). Conclusion Clinical outcomes of new‐onset in‐hospital AF patients were as good as those without AF history until AF recurrence. However, AF recurrence led to worse prognosis. Therefore, treatment for new‐onset in‐hospital AF in ADHF patients might be postponed until AF recurrence.

Background: Exposure to certain carcinogens together with host genetic predisposition likely has an influence on cervical carcinogenesis. Objective: Our aim was to evaluate synergistic effects of glutathione S-transferase (GST) polymorphisms and risk behaviors (i.e., smoking and contraceptive use) on squamous cell cervical cancer (SCCA) development in northeastern Thailand. Methods: Subjects were 198 (SCCA) patients and 198 age-matched healthy controls. Multiplex PCR and PCR-RFLP were used to determine GSTT1 and GSTA1 gene polymorphism, respectively. Results: Interaction between the four polymorphic loci of GSTs (GSTM1, GSTT1, GSTP1 and GSTA1) and increased risk for cervical cancer was not observed. The three genotypes of GSTM1 consistently showed significant risks of smoking with a lower OR for the Null individuals (1.741) at around one -fourth of wild type homozygous individuals (8.000). The effects of GST polymorphisms on cervical cancer risk under the use of hormonal contraceptives apparently did not occur. Conclusions: The predisposition of smoking risk is related to the GST genotype. It is suggested that knowing one’s own genotype data will contribute to the prevention of SCCA by controlling risk habits.

Abstract Background Lubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs). Methods We induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses. Results Gene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor. Conclusion These results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.

Abstract Background The clinical significance of premature ventricular complexes (PVCs) in heart failure (HF) remains unclear. We aimed to clarify the associations of PVC burden with re‐hospitalization and cardiac death in HF patients. Methods We studied 435 HF patients (271 men, mean age 65 years). All patients were hospitalized for worsening HF. After optimal medications, echocardiography, 24 hours Holter monitoring and cardiopulmonary exercise testing were performed before discharge. The clinical characteristics and outcomes of the HF patients were investigated. Results During a median follow‐up period of 2.3 years, there were 125 (28.7%) cardiac events (re‐hospitalization due to worsening HF, fatal arrhythmias, or cardiac death). The patients with cardiac events had higher PVC burden compared to those without (median 0.374%/d [interquartile range 0.013‐1.510] vs median 0.026%/d [interquartile range 0.000‐0.534], P 0.145%/d) to be a predictive factor of cardiac events (area under the curve: 0.64). Kaplan‐Meier analysis demonstrated that cardiac events were more frequent in patients with high‐PVC burden (>0.145%/d, n = 194) compared to those with low‐PVC burden (≤0.145%/d, n = 241). Furthermore, the high‐PVC burden patients had left ventricular (LV) and atrial dilatation, reduced LV ejection fraction, and impaired exercise capacity, compared to the low‐PVC burden patients. In Cox proportional hazards analysis, high‐PVC burden was significantly associated with cardiac events with a hazard ratio of 2.028 (95% confidence interval: 1.418‐2.901, P

BackgroundThe Patterns of non-Adherence to Anti-Platelet Regimen in Stented Patients (PARIS) thrombotic risk score has been proposed to estimate the risk of stent thrombotic events after percutaneous coronary intervention (PCI). However, the prognostic value of the PARIS thrombotic risk score for long term all-cause and cardiac mortalities, as well as hospitalization due to heart failure, has not yet been evaluated. Therefore, the aim of the present study was to evaluate the prognostic value of the PARIS thrombotic risk score for all-cause and cardiac mortalities and hospitalization due to heart failure following PCI.Methods and resultsConsecutive 1,061 patients who underwent PCI were divided into three groups based on PARIS thrombotic risk score; low- (n = 320), intermediate- (n = 469) and high-risk (n = 272) groups. We followed up on all three groups for all-cause mortality, cardiac mortality and hospitalization due to heart failure. Kaplan-Meier analysis showed that all outcomes were highest in the high-risk group (P < 0.001, P = 0.022 and P < 0.001, respectively). Multivariate Cox proportional hazard analysis, adjusted for confounding factors, showed that the risk of all-cause mortality and hospitalization due to heart failure of the high-risk group were higher than those of the low-risk group (hazard ratios 1.76 and 2.14, P = 0.005 and P = 0.017, respectively).ConclusionThe PARIS thrombotic risk score is a significant prognostic indicator for all-cause mortality and hospitalization due to heart failure in patients after PCI.

BackgroundD-dimer is a sensitive biomarker for cancer-associated thrombosis, but little is known about its significance on cancer therapeutics-related cardiac dysfunction (CTRCD).MethodsConsecutive 169 patients planned for cardiotoxic chemotherapy were enrolled and followed up for 12 months. All patients underwent echocardiography and blood test at baseline and at 3-, 6-, and 12 months.ResultsThe patients were divided into two groups based on the level of D-dimer (>1.65 μg/ml or ≦ 1.65 μg/ml) at baseline before chemotherapy: high D-dimer group (n = 37) and low D-dimer group (n = 132). Left ventricular ejection fraction (LVEF) decreased at 3- and 6 months after chemotherapy in high D-dimer group [baseline, 65.2% (62.8–71.4%); 3 months, 62.9% (59.0–67.7%); 6 months, 63.1% (60.0–67.1%); 12 months, 63.3% (58.8–66.0%), p = 0.03], but no change was observed in low D-dimer group. The occurrence of CTRCD within the 12-month follow-up period was higher in the high D-dimer group than in the low D-dimer group (16.2 vs. 4.5%, p = 0.0146). Multivariable logistic regression analysis revealed that high D-dimer level at baseline was an independent predictor of the development of CTRCD [odds ratio 3.93, 95% CI (1.00–15.82), p = 0.047].ConclusionWe should pay more attention to elevated D-dimer levels not only as a sign of cancer-associated thrombosis but also the future occurrence of CTRCD.

Gibbons (Family Hylobatidae) are a suitable model for exploring hybridization in pair-living primates as several species form hybrid zones. In Khao Yai National Park, Thailand, white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) are distributed parapatrically and hybridize in a narrow zone. Their phenotypic characteristics suggest limited inter-species gene flow, although this has never been assessed. To uncover the history and degree of gene flow between the two species, we studied the genetic structure of gibbons in the hybrid zone by analyzing fecal DNA samples, phenotypic characteristics, vocalizations and individuals' social status. We determined eight autosomal single nucleotide variant (SNV) loci, and mitochondrial DNA (mtDNA) and Y-chromosomal haplotypes of 72 gibbons. We compared these markers with reference types of wild pureblood white-handed gibbons (n = 12) in Kaeng Krachan National Park and pureblood pileated gibbons (n = 4) in Khao Soi Dao Wildlife Sanctuary. Autosomal genotypic analyses confirmed the various levels of mixed ancestry for several adult gibbons with or without atypical phenotypic traits in Khao Yai National Park. In some other adult gibbons, the mixed ancestry was not detected in either autosomal SNVs or their phenotypic traits but the mtDNA. Both male and female adult hybrids formed reproductive units mainly with a phenotypic pureblood partner and many of them produced offspring. Taken together, our results suggest that once hybridization occurs, white-handed-pileated-gibbon hybrids can reproduce with either parental species and that the backcrossing and thus introgression may occur in successive generations, with no drastic changes in phenotypic appearance.

In this study, we developed a one-step method for fabricating hydrophobic surfaces on copper (Cu) substrates. Cuprous oxide (Cu2O) with low free energy was successfully formed after low-fluence laser direct irradiation. The formation of Cu2O enhanced the hydrophobicity of the Cu substrate surface, and the contact angle linearly increased with the proportion of Cu2O. The Cu2O fabricated by low-fluence laser treatment showed the same crystal plane orientation as the pristine Cu substrate, implying an epitaxial growth of Cu2O on a Cu substrate.

Abstract Background Patients with some mutations in the lamin A/C (LMNA) gene are characterized by the presence of dilated cardiomyopathy (DCM), conduction abnormalities, ventricular tachyarrhythmias (VT), and sudden cardiac death (SCD). Various clinical features have been observed among patients who have the same LMNA mutation. Here, we show a family with cardiac laminopathy with a c.475G > T, p.E159* LMNA mutation, and a family history of conduction disorder, DCM, VT, and SCD. Case presentation A proband (female) with atrial fibrillation and bradycardia was implanted with a pacemaker in her fifties. Twenty years later, she experienced a loss of consciousness due to polymorphic VT. She had a serious family history; her mother and elder sister died suddenly in their fifties and sixties, respectively, and her nephew and son were diagnosed as having DCM. Genetic screening of the proband, her son, and nephew identified a nonsense mutation (c.475G > T, p.E159*) in the LMNA gene. Although the proband’s left ventricular ejection fraction remained relatively preserved, her son and nephew’s left ventricular ejection fraction were reduced, resulting in cardiac resynchronization therapy by implantation of a defibrillator. Conclusions In this family with cardiac laminopathy with a c.475G > T, p.E159* LMNA mutation, DCM, SCD, and malignant VT occurred. Clinical manifestation of various atrial and ventricular arrhythmias and heart failure with reduced ejection fraction occurred in an age-dependent manner in all family members who had the nonsense mutation. It appears highly likely that the E159* LMNA mutation is related to various cardiac problems in the family of the current report.

Objectives This study explored the health problems of inhabitants of small South Pacific Islands under the influence of climate change, focusing on three communities in the Solomon Islands.Design Cross-sectional study of the Solomon Islands’ populations.Setting A field survey was conducted in Taro Island, a small, urbanised island with a whole-community relocation plan; Manuopo community of Reef Islands, a small remote island on an atoll environment and Sasamungga, an intermediately urbanised community on a larger island. The Sasamungga community was used for comparison.Participants Each community’s participants were recruited through local health authorities, and 113, 155 and 116 adults (aged 18+ years) from Taro, Manuopo and Sasamungga, respectively, participated voluntarily.Methods Each participant’s body height, weight and body mass index were measured. A drop of blood was sampled for malaria testing; glycated haemoglobin and C reactive protein levels, measured from another drop of blood, were markers for diabetes and inflammation, respectively. The Primary Care Screening Questionnaire for Depression measured depressive mental states.Primary and secondary outcome measures Regarding health status, the dependent variables—communicable diseases, non-communicable diseases and mental state—and independent variables—differences in communities and socioeconomic status—were measured through health check-ups and interviews of individual participants.Results Taro Island inhabitants had a higher risk of obesity (OR 1.13, 95% CI 1.02 to 1.27, p=0.0189), and Manuopo inhabitants had a higher risk of depression (1.25, 95% CI 1.08 to 1.44, p=0.0026) than Sasamungga inhabitants. Manuopo inhabitants recognised more serious problems of food security, livelihood, place to live and other aspects of daily living than other communities’ inhabitants.Conclusions The three small island communities’ observation identified different health problems: the urbanised community and remote community had a high risk of non-communicable diseases and mental disorders, respectively. These health problems should be monitored continuously during future climate-related changes.

Abstract Background Takayasu arteritis is a rare systemic vasculitis, which affects the aorta and its major branches, especially in young females. Diagnosis and treatment for Takayasu arteritis with coronary stenosis are important to prevent fatal complications. Immunosuppressive treatment such as corticosteroid is a common treatment for this condition. However, the effects of immunosuppressive treatment on inflammatory coronary stenosis caused by Takayasu arteritis remains unknown. Case presentation An 18-year-old female had chest oppression on effort and was referred to our hospital due to ST-segment depression in I, aVL, and V2–4 on electrocardiogram. Coronary angiography showed severe stenosis in the ostium of both the left main trunk and the right coronary artery. 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography showed isolated inflammation of the aortic root. She was diagnosed with Takayasu arteritis and treated with combined immunosuppressive treatment with corticosteroid and tocilizumab, which decreased the FDG uptake in the aortic root. Four months after initiation of the immunosuppressive treatment, coronary angiography showed regression of the coronary ostial stenosis. Coronary artery bypass surgery was considered, but the patient rejected invasive revascularization for coronary artery disease. She did not have chest oppression or ST-segment depression after the immunosuppressive treatment. She had no cardiac events for 6 months after discharge. Conclusions We described regressed coronary ostial stenosis in a young female patient with Takayasu arteritis. Immunosuppressive treatment might have a favorable effect on coronary ostial stenosis in Takayasu arteritis.

Objective Autophagy is a bulk degradation system for intracellular proteins which contributes to skeletal muscle homeostasis, according to previous studies in humans and rodents. However, there is a lack of information on the physiological role of autophagy in the skeletal muscle of meat animals. This study was planned as a pilot study to investigate changes in expression of two major autophagy-related genes, microtubule-associated protein 1 light chain 3β (MAP1LC3B) and autophagy related 7 (ATG7) in fattening beef cattle, and to compare them with skeletal muscle growth. Methods Six castrated Japanese Black cattle (initial body weight: 503±20 kg) were enrolled in this study and fattened for 7 months. Three skeletal muscles, M. longissimus, M. gluteus medius, and M. semimembranosus, were collected by needle biopsy three times during the observation period, and mRNA levels of MAP1LC3B and ATG7 were determined by quantitative reverse-transcription polymerase chain reaction. The expression levels of genes associated with the ubiquitin-proteasome system, another proteolytic mechanism, were also analyzed for comparison with autophagy-related genes. In addition, ultrasonic scanning was repeatedly performed to measure M. longissimus area as an index of muscle growth. Results Our results showed that both MAP1LC3B and ATG7 expression increased over the observation period in all three skeletal muscles. Interestingly, the increase in expression of these two genes in M. longissimus was highly correlated with ultrasonic M. longissimus area and body weight. On the other hand, the expression of genes associated with the ubiquitin-proteasome system was unchanged during the same period. Conclusion These findings suggest that autophagy plays an important role in the growth of skeletal muscle of fattening beef cattle and imply that autophagic activity affects meat productivity.

JAK2V617F is the most frequent driver mutation in myeloproliferative neoplasms (MPNs) and is associated with vascular complications. However, the impact of hematopoietic JAK2V617F on the aortic aneurysms (AAs) remains unknown. Our cross-sectional study indicated that 9 (23%) out of 39 MPN patients with JAK2V617F exhibited the presence of AAs. Next, to clarify whether the hematopoietic JAK2V617F contributes to the AAs, we applied a bone marrow transplantation (BMT) with the donor cells from Jak2V617F transgenic (JAK2V617F) mice or control wild-type (WT) mice into lethally irradiated apolipoprotein E-deficient mice. Five weeks after BMT, the JAK2V617F-BMT mice and WT-BMT mice were subjected to continuous angiotensin II infusion to induce AA formation. Four weeks after angiotensin II infusion, the abdominal aorta diameter in JAK2V617F-BMT mice was significantly enlarged compared to that in the WT-BMT mice. Additionally, the abdominal AA-free survival rate was significantly lower in the JAK2V617F-BMT mice. Hematopoietic JAK2V617F accelerated aortic elastic lamina degradation as well as activation of matrix metalloproteinase (MMP)-2 and MMP-9 in the abdominal aorta. The numbers of infiltrated macrophages were significantly upregulated in the abdominal aorta of the JAK2V617F-BMT mice accompanied by STAT3 phosphorylation. The accumulation of BM-derived hematopoietic cells carrying JAK2V617F in the abdominal aorta was confirmed by use of reporter GFP-transgene. BM-derived macrophages carrying JAK2V617F showed increases in mRNA expression levels of Mmp2, Mmp9, and Mmp13. Ruxolitinib decreased the abdominal aorta diameter and the incidence of abdominal AA in the JAK2V617F-BMT mice. Our findings provide a novel feature of vascular complications of AAs in MPNs with JAK2V617F.

Background: Red blood cell distribution width (RDW) is associated with prognosis in widespread cardiovascular fields, but little is known about relationship with the onset of cancer therapeutics-related cardiac dysfunction (CTRCD).Objectives: The purpose of this study was to assess whether RDW could predict the onset of CTRCD by anthracycline.Methods: Consequential 202 cancer patients planed for anthracycline treatment were enrolled and followed up for 12 months. The patients were divided into 2 groups based on the median value of baseline RDW before chemotherapy [low RDW group, n = 98, 13.0 [12.6–13.2]; high RDW group, n = 104, 14.9 [13.9–17.0]]. Cardiac function was assessed serially by echocardiography at baseline (before chemotherapy), as well as at 3, 6, and 12 months after chemotherapy with anthracycline.Results: Baseline left ventricular end systolic volume index and ejection fraction (EF) were similar between two groups. After chemotherapy, EF decreased at 3- and 6-month in the high RDW group [baseline, 64.5% [61.9–68.9%]; 3-month, 62.6% [60.4–66.9%]; 6-month, 63.9% [60.0–67.9%]; 12-month, 64.7% [60.8–67.0%], P = 0.04], but no change was observed in low RDW group. The occurrence of CTRCD was higher in high RDW group than in low RDW group (11.5 vs. 2.0%, P = 0.008). When we set the cut-off value of RDW at 13.8, sensitivity and specificity to predict CTRCD were 84.6 and 62.0%, respectively. Multivariable logistic regression analysis revealed that baseline RDW value was an independent predictor of the development of CTRCD [odds ratio 1.390, 95% CI [1.09–1.78], P = 0.008]. The value of net reclassification index (NRI) and integrated discrimination improvement (IDI) for detecting CTRCD reached statistical significance when baseline RDW value was added to the regression model including known risk factors such as cumulative anthracycline dose, EF, albumin, and the presence of hypertension; 0.9252 (95%CI 0.4103–1.4402, P < 0.001) for NRI and 0.1125 (95%CI 0.0078–0.2171, P = 0.035) for IDI.Conclusions: Baseline RDW is a novel parameter to predict anthracycline-induced CTRCD.

Background The prognostic impact of benzodiazepines has been unclear in patients with heart failure (HF). Methods and Results This was a historical observational cohort study. A total of 826 patients who had been hospitalized for HF and were being treated for insomnia with either benzodiazepines or Z‐drugs (zolpidem, zopiclone, or eszopiclone), were enrolled and divided on the basis of their hypnotics: benzodiazepine group (n=488 [59.1%]) and Z group (n=338 [40.9%]). We compared the patient characteristics and postdischarge prognosis between the groups. The primary end points were rehospitalization for HF and cardiac death. The benzodiazepine group was older (age, 72.0 versus 69.0 years; P=0.010), had a higher prevalence of depression (17.4% versus 8.9%; P

The association of socio-economic-demographic (SED; e.g., income-related) factors with depression is widely confirmed in the literature. We conducted a hospital-based case-control study of 160 patients with psychiatrist-diagnosed clinical depression. The control group comprised 160 participants recruited from local communities. We used a questionnaire to collect SED data from all participants. We replaced missing values using multiple imputation analyses and further analyzed the pooled data of five imputations. We also recorded the results from the original analysis and each imputation. Univariate analyses showed income was associated with depression. Multiple logistic regression analyses revealed that, among all SED variables, high income (odds ratio = 2.088 [95% confidence interval = 1.178-3.700]; p = 0.012), middle-level (completed junior or senior high school) education (1.688 [1.042-2.734]; p = 0.033) and cohabitating with four or more family members (1.632 [1.025-2.597]; p = 0.039) were significant predictors for the case group. We conclude that cash income is a determinant of depression in hospital outpatients in Indonesia. This study suggests health policy implications toward better hospital access and service for people with depression in middle- or low-income households, and recommends considering high income as correlated with a high risk of depression, owing to socio-cultural changes.

Background: Acetoacetate is used as an alternative energy source in the heart, and has the potential to improve cardiac function. However, the prognostic impact of acetoacetate has not been investigated in heart failure. Methods: This study enrolled consecutive 615 hospitalized patients with heart failure. We investigated the associations between circulating acetoacetate and clinical characteristics or prognosis in HF patients. Results: We divided the patients into two groups based on circulating acetoacetate levels (high group: acetoacetate ≥35 µmoL/L, n = 313; and low group: acetoacetate

Background: A missense variant (rs373863828:G > A; p.Arg457Gln) of the CREBRF gene is strongly associated with a higher body mass index (BMI; kg/m2) in Polynesian populations. This variant has also been reported to be associated with lower total cholesterol in Samoans. Aim: The aim of this study is to examine the association of rs373863828:G > A with levels of serum lipids in four Pacific populations. Methods: A total of 613 adult subjects were recruited from Tonga (Polynesians) and the Solomon Islands (Melanesians and Micronesians). Multiple regression analyses adjusted for age and sex were performed to examine the association of rs373863828 with levels of serum lipids in each population. Results: A significant association of rs373863828:G > A with lower level of HDL-cholesterol was detected in the Tonga population (β = –3.32 and p-value = 0.030). The expected change in HDL-cholesterol with respect to a single copy of the rs373863828-A allele was 3.32 mg/dL. However, the association between rs373863828-A and lower levels of HDL-cholesterol was not significant after further adjustment for BMI in the Tonga population (β = –2.32 and p-value = 0.13). Conclusions: The rs373863828-A allele may not directly affect the level of serum HDL-cholesterol independent of BMI. To confirm the present findings, association studies with large sample sizes and functional analyses are required.

Abstract Background Breath acetone is a noninvasive biomarker of heart failure; however, its significance in heart failure patients with diabetes mellitus has yet to be clarified. The objective of this study is to investigate whether exhaled acetone concentration is a noninvasive biomarker in heart failure patients with diabetes mellitus. Methods This study prospectively included 35 diabetic patients with stage C heart failure and 20 diabetic patients with or at risk of heart failure (stage A or B). Exhaled breath was collected after an overnight fast. Results The stage C group had significantly higher brain natriuretic peptide levels, larger left ventricular diameter, lower left ventricular ejection fraction, and more frequent use of β-blocker, compared with the stage A or B group. The stage C group had higher exhaled acetone concentrations than the stage A or B group (p = 0.013). Exhaled acetone concentration was correlated with total ketone bodies (r = 0.588, p

Background The Get With the Guidelines–Heart Failure (GWTG‐HF) risk score was developed using American Heart Association GWTG‐HF program data and predicts in‐hospital mortality in patients with acute heart failure (HF). We aimed to clarify the prognostic impacts of the GWTG‐HF risk score in patients with HF after discharge. Methods and Results We examined the GWTG‐HF score in 1452 patients with HF, who were admitted to our hospital and discharged after treatment, by calculating 7 predetermined variables. We divided all subjects into 3 groups according to the GWTG‐HF risk score (low, moderate, and high score groups). The plasma B‐type natriuretic peptide level significantly increased with increasing GWTG‐HF risk score severity (median values of B‐type natriuretic peptide: 167.0 in low, 260.7 in moderate, and 418.2 pg/mL in high score groups). We followed up all subjects after discharge, and there were 347 (23.9%) all‐cause deaths and 407 (28.0%) cardiac events in follow‐up periods. A Kaplan‐Meier survival curve demonstrated that event rates of all‐cause death and cardiovascular events, including worsening HF and cardiac death, significantly increased with increasing GWTG‐HF risk score severity in all subjects, and also in 749 patients with HF with preserved ejection fraction (ejection fraction ≥50%) and 703 patients with HF with reduced ejection fraction (ejection fraction

Kawasaki disease (KD), first identified in 1967, is a pediatric vasculitis of unknown etiology that has an increasing incidence in Japan and many other countries. KD can cause coronary artery aneurysms. Its epidemiological characteristics, such as seasonality and clinical picture of acute systemic inflammation with prodromal intestinal/respiratory symptoms, suggest an infectious etiology for KD. Interestingly, multiple host genotypes have been identified as predisposing factors for KD. To explore experimental methodology for identifying etiological agent(s) for KD and to optimize epidemiological study design (particularly the sample size) for future studies, we conducted a pilot study. For a 1-year period, we prospectively enrolled 11 patients with KD. To each KD patient, we assigned two control individuals (one with diarrhea and the other with respiratory infections), matched for age, sex, and season of diagnosis. During the acute phase of disease, we collected peripheral blood, nasopharyngeal aspirate, and feces. We also determined genotypes, to identify those that confer susceptibility to KD. There was no statistically significant difference in the frequency of the risk genotypes between KD patients and control subjects. We also used unbiased metagenomic sequencing to analyze these samples. Metagenomic sequencing and PCR detected torque teno virus 7 (TTV7) in two patients with KD (18%), but not in control subjects (P = 0.111). Sanger sequencing revealed that the TTV7 found in the two KD patients contained almost identical variants in nucleotide and identical changes in resulting amino acid, relative to the reference sequence. Additionally, we estimated the sample size that would be required to demonstrate a statistical correlation between TTV7 and KD. Future larger scale studies with carefully optimized metagenomic sequencing experiments and adequate sample size are warranted to further examine the association between KD and potential pathogens, including TTV7.

BACKGROUND:Pulmonary hypertension (PH) is a common complication of chronic respiratory disease. Recent studies have reported diabetes mellitus (DM) to be a poor prognostic factor in patients with chronic respiratory disease, including chronic obstructive pulmonary disease or interstitial pneumoniae. However, the association between DM and PH in chronic respiratory disease remains unclear. In this study, we aimed to investigate whether DM is a predictor of PH in patients with chronic respiratory disease. METHODS:We prospectively analyzed 386 patients in our hospital with chronic respiratory disease. An echocardiographic pressure gradient between the right atrium and the right ventricle of ≥ 40 mmHg was defined as PH. We compared the clinical characteristics and impact of DM between chronic respiratory disease patients with and those without PH. RESULTS:Of the 386 patients, 42 (10.9%) were diagnosed as having PH. The PH group had higher modified medical research council (mMRC) grade and complication rate of DM, but not hypertension and hyperlipidemia, when compared to the non-PH group. Multivariable logistic regression analysis revealed that mMRC scale (odds ratio 1.702, 95% confidence interval, 1.297 to 2.232, P < 0.001) and presence of DM (odd ratio 2.935, 95% confidence interval, 1.505 to 5.725, P = 0.002) were associated with PH in chronic respiratory disease patients. CONCLUSION:DM is potentially associated with PH and is an independent factor for prediction of PH in patients with chronic respiratory disease.

Omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), likely prevent cardiovascular disease, however their mechanisms remain unclear. Recently, the role of DNA damage in atherogenesis has been receiving considerable attention. Here, we investigated the effects of EPA and DHA on DNA damage in vascular endothelial cells to clarify their antiatherogenic mechanisms.We determined the effect of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells. Immunofluorescence staining showed that γ-H2AX foci formation, a prominent marker of DNA damage, was significantly reduced in the cells treated with EPA and DHA (by 47% and 48%, respectively). H2O2-induced activation of ATM, a major kinase orchestrating DNA damage response, was significantly reduced with EPA and DHA treatment (by 31% and 33%, respectively). These results indicated EPA and DHA attenuated DNA damage independently of the DNA damage response. Thus the effects of EPA and DHA on a source of DNA damage were examined. EPA and DHA significantly reduced intracellular reactive oxygen species under both basal condition and H2O2 stimulation. In addition, the mRNA levels of antioxidant molecules, such as heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain and manganese superoxide dismutase, were significantly increased with EPA and DHA. Silencing nuclear factor erythroid 2-related factor 2 (NRF2) remarkably abrogated the increases in mRNA levels of antioxidant molecules and the decrease in intracellular reactive oxygen species. Furthermore, EPA and DHA significantly reduced H2O2-induced senescence-associated β-galactosidase activity in the cells (by 31% and 22%, respectively), which was revoked by NRF2 silencing.Our results suggested that EPA and DHA attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of NRF2-mediated antioxidant response. Therefore omega-3 fatty acids likely help prevent cardiovascular disease, at least in part, by their genome protective properties.

The people of the Solomon Islands represent an Austronesian (AN)-speaking population's adaptation to a humid tropical environment and subsistence of tuberous crops. Genome-wide association studies (GWASs) of other populations (e.g. the Human Genome Diversity Project [HGDP]) have suggested the existence of genotypes adaptive to ecoregion, diet, and subsistence, and that those genotypes are also associated with metabolic and cardiovascular diseases. Recently, the incidence of non-communicable diseases has been increasing in the Solomon Islands. In the present study, we explored the association of genotypes adaptive to a tropical environment and tuberous crop diet with metabolic and cardiovascular conditions in rural and urban AN-speaking Melanesian and Micronesian populations of the Solomon Islands. A total of 561 participants were genotyped for single nucleotide polymorphisms (SNPs) potentially associated with a tropical environment (rs174570 and rs2237892) and a tuberous crop diet (rs162036, rs185819, and rs2722425). The results showed that the allele frequencies of the Solomon Islands populations adopted patterns similar to those in populations from other hot, tropical areas with a tuberous crop diet in previous studies. Furthermore, rs162036, rs185819, rs2237892, and rs2722425 were all strongly associated with one or more metabolic and cardiovascular conditions. The derived allele of rs2722425 (i.e. rs2722425-G) was significantly associated with an elevated LDL level (P = 0.000264) even after the significance level was adjusted for multiple testing (i.e., α = 0.0005). Our results suggest that the inhabitants of the Solomon Islands exhibit the effects of the tropical environment and tuberous crop diet on their allele frequencies, and that their susceptibility to metabolic and cardiovascular diseases is therefore considered to be associated with their environment and diet.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture and subsequent high mortality. Establishment of medical therapies for the prevention of AAAs requires further understanding of the molecular pathogenesis of this condition. This report details the possible involvement of Osteoprotegerin (OPG) in the prevention of AAAs through inhibition of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In CaCl2-induced AAA models, both internal and external diameters were significantly increased with destruction of elastic fibers in the media in Opg knockout (KO) mice, as compared to wild-type mice. Moreover, up-regulation of TRAIL expression was observed in the media by immunohistochemical analyses. Using a culture system, both the TRAIL-induced expression of matrix metalloproteinase-9 in smooth muscle cells (SMCs) and the chemoattractive effect of TRAIL on SMCs were inhibited by OPG. These data suggest that Opg may play a preventive role in the development of AAA through its antagonistic effect on Trail.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture with a high mortality rate. Although eicosapentaenoic acid (EPA) has been reported to prevent AAA formation, the mechanism by which EPA works on vascular smooth muscle cells is unknown. This study aimed to investigate the mechanism by which orally-administered EPA prevents the formation of severe AAAs that develop in Osteoprotegerin (Opg) knockout (KO) mice. In the CaCl2-induced AAA model, EPA attenuated the enhanced progression of AAAs in Opg-KO mice, including the increase in aortic diameter with destruction of elastic fibers in the media. Immunohistochemical analyses showed that EPA reduced the phosphorylation of transforming growth factor beta-activated kinase-1/Map3k7 (Tak-1) and c-Jun NH2-terminal kinase (JNK), as well as the expression of Matrix metalloproteinase-9 (Mmp-9) in the media of the aorta. In smooth muscle cell cultures, rh-TRAIL-induced activation of the Tak-1-JNK pathway and increase in Mmp-9 expression were inhibited by EPA. Moreover, GW9508, a specific ligand for G-protein coupled receptor (Gpr)-120/Free fatty acid receptor (Ffar)-4, mimicked the effects of EPA. The effects of EPA were abrogated by knockdown of the Gpr-120/Ffar-4 receptor gene. Our data demonstrate that the Trail-Tak-1-JNK-Mmp-9 pathway is responsible for the enhancement of AAAs in Opg-KO mice, and that EPA inhibits the Tak-1-JNK pathway by activating Gpr-120/Ffar-4, which results in the attenuation of AAA development.

An outbreak of hepatitis E virus occurred in an outdoor monkey breeding facility in Japan during 2004–2006. Phylogenetic analysis indicated that this virus was genotype 3. This virus was experimentally transmitted to a cynomolgus monkey. Precautions should be taken by facility personnel who work with monkeys to prevent infection.

OBJECTIVE:Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs), the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs). APPROACH AND RESULTS:Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX). Young healthy active smokers (n = 15) showed increased γ-H2AX foci number when compared with non-smokers (n = 12) (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3'-end of an AluSx element, flanked by target site duplications (5'-ACTRCCAR-3'or 5'-ACTRCCARTTA-3'), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.

Objective: To investigate whether the real-time polymerase chain reaction (R-PCR) assay with SYBR green I and melting curve analysis could be used for glutathione S-transferase M1 gene (GSTM1) polymorphism detection in Thai nasopharyngeal carcinoma (NPC) patients by comparing the results of this assay with the conventional PCR (C-PCR) assay. Methods: DNA samples from peripheral blood leukocytes of 60 Thai NPC patients were investigated in this study. GSTM1 polymorphism [GSTM1 normal genotype (GSTM1+) and GSTM1 null genotype (GSTM1-)] were examined by using the R-PCR assay with SYBR green I and melting curve analysis and the C-PCR assay. Results: The results of GSTM1 polymorphism detection by the R-PCR assay were in concordance with the C-PCR assay (κ= 1.0). Twenty-six individuals with GSTM1+ in the R-PCR assay showed 2 peaks of melting point at 82.5o C and 87.5o C that correlated with the appearance of 2 DNA bands of GSTM1 [215 base pair (bp)] and β-globin (268 bp) in the C-PCR assay, respectively. In addition, thirty-four individuals with GSTM1- in the R-PCR assay showed only 1 peak of melting point at 87.5o C that correlated with the appearance of 1 DNA band of β-globin (268 bp) in the C-PCR assay. Moreover, we found that the R-PCR assay was a faster and safer method for detection of GSTM1 polymorphism than the C-PCR assay. Conclusion: The present study suggests that the R-PCR assay with SYBR Green I and melting curve analysis may be a useful screening tool for more convenient, rapid, reliable, and safer detection of GSTM1 polymorphism in Thai NPC as compared to the C-PCR assay.

Contemporary hunter-gatherer groups are often thought to serve as models of an ancient lifestyle that was typical of human populations prior to the development of agriculture. Patterns of genetic variation in hunter-gatherer groups such as the Kung and African Pygmies are consistent with this view, as they exhibit low genetic diversity coupled with high frequencies of divergent mtDNA types not found in surrounding agricultural groups, suggesting long-term isolation and small population sizes. We report here genetic evidence concerning the origins of the Mlabri, an enigmatic hunter-gatherer group from northern Thailand. The Mlabri have no mtDNA diversity, and the genetic diversity at Y-chromosome and autosomal loci are also extraordinarily reduced in the Mlabri. Genetic, linguistic, and cultural data all suggest that the Mlabri were recently founded, 500-800 y ago, from a very small number of individuals. Moreover, the Mlabri appear to have originated from an agricultural group and then adopted a hunting-gathering subsistence mode. This example of cultural reversion from agriculture to a hunting-gathering lifestyle indicates that contemporary hunter-gatherer groups do not necessarily reflect a pre-agricultural lifestyle.