Perinatal group A streptococcal infection is a rare but life‐threatening condition. Few reports have focused on its clinical characteristics and how to prevent deterioration. We report our experience with two antenatal fatal cases and reviewed 96 cases in the literature to assess the clinical characteristics of group A streptococcal infection.English‐language clinical reports of antenatal and postnatal group A streptococcal infection in 1974–2019 were retrieved and examined. Relationships between clinical characteristics and maternal outcomes were assessed.P = 0.025) and a quick sepsis‐related organ‐failure assessment score  of ≥2  were significantly related to maternal death.Univariate analysis revealed that antenatal group A streptococcal infection was significantly associated with an age of ≤19 or ≥ 35 years, cesarean section, sore throat as an initial symptom, positive throat culture, maternal death and fetal death. Multivariate analysis revealed that antenatal onset (odds ratio = 7.922, 95% confidence interval = 1.297–48.374; Per our findings, antenatal group A streptococcal infection was significantly associated with maternal and fetal death. Further, the antenatal infection was revealed as a more critical risk factor. We suggest that the presence of any sign related to the qSOFA is a potential clue suspecting perinatal group A streptococcal infection in primary obstetric facilities. Perinatal group A streptococcal (GAS) infection is a rare disease, occurring in six of every 100 000 births according to a North American report.Patient: A 26‐year‐old woman.Past obstetric history: Gravida 2, para 1. Three years prior, the patient was transported to our hospital due to the rupture of membranes and labor with no prenatal care. An emergency cesarean section due to breech presentation was performed and a 2214 g healthy male was delivered.Past medical history: Nothing remarkable.History of present illness: At 13 weeks' gestation, the patient presented to a clinic with abdominal pain. She was diagnosed with a potential threat of miscarriage because of vaginal bleeding. Despite the offer of hospitalization, she refused and returned home. Six hours later, she experienced a rupture of the membranes and spontaneous abortion at home and thus returned to the clinic. The transvaginal ultrasound revealed a retained placenta; she decided to be admitted and undergo surgical treatment. However, she experienced sudden shortness of breath on admission, followed by altered mental status. Since her systolic blood pressure dropped to 60 mmHg, and peripheral capillary oxygen saturation (SpO2) also dropped to 60%, she was provided artificial ventilation. Despite the initial treatment, she went into cardiopulmonary arrest. She was transported to our tertiary medical center while being resuscitated. On arrival, she was still under cardiopulmonary arrest, and the resuscitation was continued. Her laboratory findings showed the following: white blood cell (WBC) count, 35 600 cells/μL; hemoglobin, 9.0 g/dL; platelet count, 113 000 cells/μL; D‐dimer, 32.0 μg/mL; fibrinogen, <70 mg/dL; blood urea nitrogen, 26.0 mg/dL; creatinine, 1.62 mg/dL; aspartate aminotransferase, 3993 U/L; alanine aminotransferase, 3041 U/L; C‐reactive protein, 8.4 mg/dL and procalcitonin, 9.5 ng/mL. GAS was later detected from a culture of a vaginal sample taken at this time. Transthoracic echocardiography revealed no right ventricular dilatation or pericardial effusion. Because she did not respond to resuscitation, percutaneous cardiopulmonary support was started. An enhanced computed tomography scan revealed no pulmonary embolism but diffuse infiltration in the lungs. She died 11 h and 36 min after the initial examination at the clinic.Patient: A 37‐year‐old woman.Past obstetric history: Gravida 1, para 0.Past medical history: Acute hepatitis at 24 years of age.History of present illness: The patient had regular prenatal check‐ups at a clinic. At 21 weeks' gestation, she presented with a fever of 38.0°C. Because she was suspected of having an influenza viral infection, she was prescribed oseltamivir and went home. Hypogastric pain appeared, and she returned to the clinic 6 h later. Upon arrival, her body temperature was 39°C, blood pressure was 91/62 mmHg and heart rate was 107 beats per minute. Intrauterine fetal death was diagnosed with transabdominal ultrasound. As the influenza point‐of‐care test result was negative and her WBC count elevated to 13 600/μL, intravenous flomoxef was initiated. Labor started after that, and she had a spontaneous abortion. The placenta was delivered simultaneously, revealing bloody amniotic fluid. Directly after the delivery, significant bleeding was noted, and the patient appeared to be drowsy. At this time, her blood pressure was 83/56 mmHg, heart rate was 124 beats per minute and postnatal hemorrhage had reached 1272 g. Therefore, she was transferred to a secondary medical center. Laboratory findings on arrival showed the following: WBC count, 32 800 cells/μL; hemoglobin, 8.1 g/dL; platelet count, 67 000 cells/μL; D‐dimer, 907.3 μg/mL; fibrinogen, <25 mg/dL; blood urea nitrogen, 31.0 mg/dL; creatinine, 2.28 mg/dL; aspartate aminotransferase, 146 U/L; alanine aminotransferase, 22 U/L and C‐reactive protein, 9.3 mg/dL. GAS was later identified from vaginal discharge taken at this hospital. She went into cardiopulmonary arrest, and cardiopulmonary resuscitation was provided. She restored spontaneous circulation 2 min later and was transferred to our tertiary medical center. She again went into cardiopulmonary arrest during the transfer. After arrival, she did not respond to continued attempts of cardiopulmonary resuscitation. She died 21 h and 46 min after the initial visit to the clinic.et al. reported the largest number of cases  from the literature review.Our two cases suspected of perinatal GAS infection had severe maternal outcomes with an astonishingly acute clinical course. Several studies have addressed the clinical characteristics of perinatal GAS infection. Of these, Hamilton streptococcus' as search terms.English‐language clinical reports describing antenatal and postnatal GAS infection between 1974 and 2019 were examined according to a PubMed database search using 'pregnancy' and 'group A Because data were incomplete in some of the published reports, we used the following definitions: Data were defined as a valid 'negative' finding when at least one finding, whether positive or negative, was reported for another item in the same category. Conversely, data were defined as 'missing' when no findings were reported for the category as a whole. For example, if abdominal pain was not reported as an initial symptom in a clinical report, abdominal pain was defined as 'negative' if at least one other initial symptom was reported, while abdominal pain was defined as 'missing' if no initial symptoms were reported. Missing data were not included in the analysis. Among the data collected, quick sepsis‐related organ failure assessment (qSOFA) scoreThe above data were analyzed with IBM SPSS Statistics for Windows, version 23.0 (IBM Corp.). The following tests were used for comparison: Shapiro‐Wilks test for the distribution of variables, Student t‐test for normally distributed variables, Mann–Whitney U test for non‐normally distributed variables; two‐tailed Pearson's chi‐square test and one‐tailed Fisher's exact test for categorical variables; multivariate logistic regression analysis for regression analysis. The level of significance adopted was 5% (α = 0.05).This study was approved by the ethical board at Saitama Medical Center (registration number: 2233).We included a total of 98 patients reported over a long time period (45 years) Table 60, 61P = 0.025, and OR = 6.166, 95% CI = 1.066–35.670; P = 0.042, respectively).Univariate analysis was used to test the relationship between the maternal outcome and several clinical characteristics assumed to be potential risk factors Table . Five clet al. reported the largest number of cases of perinatal GAS infections  and noted that GAS infection during the third trimester was associated with maternal and fetal death,et al. examined the qSOFA score in maternal death cases.Antenatal onset and a qSOFA score of ≥2 were significantly associated with maternal death in our study. In the literature, Hamilton Why was antenatal GAS infection associated with poor maternal outcomes? Compared with postnatal cases, antenatal infections had initial symptoms outside of the hospital and the initiation of treatment might have been delayed. Generally, potentially invasive surgical treatment often makes physicians hesitant to operate upon pregnant patients.Second, we assume there may be several types of perinatal GAS infection depending on the infection route, as well as the initial infection site. However, as is mentioned in previous article,We next investigated potential indicators of risk that could be used to suspect a perinatal GAS infection in pregnant women. Although the CENTOR criteria is a diagnostic criterion for GAS pharyngitis and the qSOFA score is that for sepsis, we assumed that these criteria can be used for the detection of perinatal GAS infection. However, because of the limited data availability, we were not able to examine the sensitivity in this study. In antenatal cases, all the criteria in the qSOFA score  were available in 15 cases. Of these, 13 cases (86.7%) satisfied one or more criteria in qSOFA. Thus, we suggest that medical resources should be provided earlier to pregnant women who feel ill, and medical providers should triage using the qSOFA score. If a patient is positive for any of the qSOFA criteria, she should be immediately referred to a critical care medical center. Further study should be done because of the limited number of cases in this study.P = 0.363, Mann Whitney U test). Since most cases did not fully describe all the data we required  for the study, we were not able to exclude the possibility of selection bias. In addition, our two cases are not definite cases, but probable cases according to the definition of Streptococcal toxic shock syndrome.There were some other limitations to our study that should be considered. Because patients from a wide time period were included, patients from a more recent chronological timeframe may have experienced better outcomes than those from an earlier chronological period. However, the maternal outcome was not related to the published year of the case report (In conclusion, antenatal GAS infection is more critical than postnatal GAS infection, and a qSOFA score of ≥2 is also potentially critical. We suggest that the presence of any of the qSOFA signs may represent a useful clinical marker for antenatal GAS infection among patients presenting at primary obstetric facilities.The authors report no conflict of interest.


Soybean plants were grown in a growth chamber with natural solar radiation under four treatments, that is, normal , N + Fr, Low (L) + Fr, and L light.Shading includes low light intensity and varying quality. However, a low red/far-red (R/Fr) ratio of light is a signal that affects plant growth in intercropping and close- planting systems. Thus, the low R/Fr ratio uncoupling from shading conditions was assessed to identify the effect of light quality on photosynthesis and COP < 0.05). Proteomic analysis of soybean leaves under different treatments was performed to quantify the changes in photosynthesis and CO2 assimilation in the chloroplast. Among the 7834 proteins quantified, 12 showed a > 1.3-fold change in abundance, of which 1 was related to porphyrin and chlorophyll metabolism, 2 were involved in photosystem I (PS I), 4 were associated with PS II, 3 proteins participated in photosynthetic electron transport, and 2 were involved in starch and sucrose metabolism. The dynamic change in these proteins indicates that photosynthesis and CO2 assimilation were maintained in the L treatment by up-regulating the component protein levels compared with those in N treatment. Although low R/Fr ratio increased the photosynthetic CO2 assimilation parameters, the differences in most protein expression levels in N + Fr and L + Fr treatments compared with those in N treatment were insignificant. Similar trends were found in gene expression through quantitative reverse transcription polymerase chain reaction excluding the gene expression of sucrose synthase possible because light environment is one of the factors affecting carbon assimilation.Low R/Fr ratio significantly increased the total biomass, leaf area, starch and sucrose contents, chlorophyll content, net photosynthetic rate, and quantum efficiency of the photosystem II compared with normal R/Fr ratio under the same light level (2 assimilation in the same light intensity by improving the photosynthetic efficiency of the photosystems.Low R/Fr ratio (high Fr light) can increase the photosynthetic CO Light is an essential factor for crop growth and development in agricultural production . Intra- Plant shading reduces the amount of photosynthetically active radiation (PAR) and changes the spectral composition of light , 7. PlanPhytochromes play a key role in the perception of the R/Fr ratio signal and regulation of the plant photomorphogenesis via gene expression and physiological processes . PhytochPn) under the same light intensity condition [A low R/Fr ratio indicates high enrichment of the Fr light spectrum in the plant canopy. Plant leaves absorb less amount of Fr light (λ > 700 nm); therefore, it contributes less towards the quantum yield of photosynthesis . Howeverondition , resultiondition , 9. Typiondition . The revondition , 18. The2 assimilation.Photosystems I and II  operate in series to carry out the primary photochemical reactions of photosynthesis . These pGlycine max (L.) Merr.) is one of the most important cultivated crops of protein and oil worldwide [Pn by up-regulating the gene expression levels of differential proteins compared with low light [2 assimilation by increasing the photosynthetic capacity. Therefore, this work aims to analyze the soybean morphology, carbohydrate, and photosynthesis in response to low R/Fr ratio in different light intensities and reveal the effect of low R/Fr ratio on soybean photosynthesis using the iTRAQ technique in different light intensities.Soybean  were significantly lower than those in low light (L and L + Fr treatment)  Fig. . The tot− 2 s− 1. The maximum values of photosynthetic rate (Pmax) and light saturation point (LSP) appeared in N + Fr treatment compared with those in other treatments. Pmax and LSP under N treatment were 14.9 and 47.4% lower than the corresponding values under N + Fr treatment, respectively. Similarly, Pmax and LSP under L treatment were 23.8 and 38.2% lower than those under L + Fr treatment, respectively. The minimum values of Pmax and LSP appeared in L treatment compared with those in other treatments, were 5.8 μmol CO2 m− 2 s− 1 in the 318.2 μmol m− 2 s− 1, respectively. In this study, the quantum yields of PSII in treatments N and N + Fr was noted significantly higher than those in L and L + Fr treatments. However, treatment L + Fr enhanced the quantum yields of PSII by 15.2% as compared to those in treatment L.The light response curves of the assimilation rate vs. the photosynthetic photon flux density (PPFD) and the quantum yield of PSII in four treatments were shown in Fig. P < 0.05), among the quantified proteins, we observed that 15 and 41 proteins were up-regulated and down-regulated, respectively in N + Fr vs. N; 102 and 548 were up-regulated and down-regulated, respectively under L + Fr vs. N, 180 and 183 proteins were up-regulated and down-regulated, respectively in L vs. N  enrichment analysis, differentially accumulated proteins were categorized into three main groups, including biological process, molecular function, and cellular components Fig. b. Biolog2 assimilation. The proteins involved in photosynthesis in N + Fr and L + Fr treatments were down-accumulated compared with those in N treatment, whereas proteins in L treatment were up-accumulated.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways visualized as heat map through a two-tailed Fisher’s exact test were used to identify the differences in protein abundance among L + Fr, N + Fr, N, and L treatments. Thirteen different functional categories were selected for analysis. As illustrated in Fig. 2 assimilation were detected by iTRAQ analysis under N + Fr, L + Fr, and L treatments compared with those detected in N treatment. Among these differentially expressed proteins, one protein was related to porphyrin and chlorophyll metabolism, two proteins were involved in PS I, four proteins were associated with PS II, three proteins participated in photosynthetic electron transport, and two proteins were involved in starch and sucrose metabolism  were significantly higher than those in N and L treatments, respectively. Although low light intensity significantly decreased the Chl content (Chl a and b) compared with normal light condition, a low R/Fr ratio significantly increased the Chl content per unit area under the same light level [Pn were consistent with the total biomass in different treatments  of Chl contents [Proteomics is an approach for the systematic identification of all proteins expressed in a cell or tissue . In thiscontents , 34.The PsaD subunit of PS I is a peripheral protein that provides a docking site for ferredoxin and interacts with other PS I subunits . PsaD isThe light-harvesting antenna (LHCII) is the major pigment- protein complex associated with PS II, thereby forming the PS II-LHCII supercomplex in which several LHCIIs surround the reaction center core complex . The LhcPn decreased compared with other treatments  in the same light intensity can increase the photosynthetic CONandou 12, which is a major soybean cultivar that is closely planted or intercropped with other crops in agriculture production in Southwestern China, was selected as the experimental material. Soybean seeds were provided by the Nanchong Institute of Agricultural Sciences, Sichuan Province, China. All soybean seeds were covered with wet filter paper for 24 h at 30 °C. After that, the germinated soybean seeds were grown in containers with a seedling spacing of 10 cm. The size of each container was 15 cm in height, 40 cm in length, and 20 cm in width, and filled with humidified organic soil.The experiment was divided into two parts. One part was used for morphology measurement. The containers including germinated seeds were directly placed under different light environment treatments, and each treatment included three containers.The other part was used for physiological and proteomic analyses. The difference in soybean growth period under the various light environments was avoided by growing soybean under normal ambient condition, until the complete development of the first trifoliate leaf. Then, plants were subjected to four different light treatments just after the appearance of the second trifoliate. Furthermore, after 15 days of treatments, all samples were collected at 10 am for the analysis of physiological parameters and differentially expressed proteins. Plants were grown in growth chamber with natural solar radiation, with the temperature maintained at 25 °Cin the day and 20 °C during the night. Additionally, the humidity was maintained at 60%, and plants were irrigated with nutrients solution (0.2% Hoagland’s solution) after every couple of days .− 2·s− 1 under dark condition. The PAR and spectral irradiance of soybean canopy were measured at noon on a sunny day, every measurement was replicated five times. The following four treatments were used (Table The black nylon gauze and Far Red (Fr)-LED was used according to our previous knowledge , to cont2) was measured by Image J 1.45 s. Biomass samples were over-dried at 105 °C for 0.5 h to stop metabolic processes of tissues and then dried at 80 °C for 72 h to a constant weight [The plant height from the soil surface to the growing point of soybean. The biomass and leaf area of five soybean seedlings were measured every 14 days after 14 days of sowing under four treatments. Leaves were scanned using a flatbed scanner , and the leaf area  with some changes , soluble2 concentration of 400 μmol mol− 1 from 9:00 to 11:00. Eleven light intensity levels  were imposed. On a light response curve, PPFD was located on the horizontal axis and Pn was on the vertical axis (Pn-PPFD curve). The Pmax and LSP were then estimated using the method proposed by Yang et al. [− 2 s− 1) was switched on, and saturating pulses were applied at 20 s intervals for 15 min. From each of these, the maximum fluorescence (F′m) and the steady-state fluorescence (Fs) were determined in the light condition. The quantum efficiency of the photosystem II was calculated according to the formula (F′m- Fs)/ F′m [As described by Yang et al. , the secAfter that, four-leaf disks of 15 mm diameter were obtained from the center of each leaf and then cut into pieces of 3 mm. These leaf-disks were placed in 10 ml of 80% acetone in the dark at 20 °C for 24 h. The chlorophyll contents were determined by following a previously published procedure . TriplicAfter 2 weeks of treatment, the second leaf of soybean plants was ground using liquid nitrogen. Following the method of Yang et al. , the powAs previously reported , high PHP < 0.05, respectively. GO annotation was used to annotate the proteins [MaxQuant, with an integrated Andromeda search engine (v.1.5.2.8), was used to process the MS/MS data. iTRAQ 8-plex was used for the quantification, and the default values of all the other parameters in MaxQuant were selected, as reported by Yang et al. . To idenproteins . The difproteins , 48.Proteomics reliability was confirmed by the help of qRT-PCR assay. RNA isolation was done according to the protocol of Yuan et al. . In our P < 0.05.Significance was calculated using one-way ANOVA by computer-based program SPSS (version 16.0). Data are given as mean ± standard deviation from three replicates. Statistical significance was recognized at Additional file 1: Figure S1. Light response curves of net photosynthetic rate (A), light saturation point (B), and quantum yield of PSII (C) of soybean leaves under different treatments. N, N + Fr, L + Fr, and L denote normal light , normal light plus far-red light , low light plus far-red light (low PAR and low R/Fr ratio), and low light , respectively. Pmax and LSP represent the maximum photosynthetic rate and the light saturation point. Data are expressed as the means ± SD of triplicates. Means followed by different letters are significantly different at P = 0.05.Additional file 2: Table S1. Differentially expressed statistics of unique proteins.Additional file 3: Table S2. List of primers for characterizing Glycine max genes.


The Ministry of Health has reported that in Japan, the Shiga prefecture has the highest life expectancy. Subjective health perception is a predictive indicator of mortality. For this study, we examined the association between subjective health perception and multiple lifestyle factors.Data were obtained from the 2015 Health and Nutrition Survey in Shiga prefecture. The analytic sample comprised 6057 adults aged 20 or older. Information on subjective health perception and lifestyle behaviors was obtained from a self-administered questionnaire. As for subjective health perception, participants were divided into 2 groups: (1) Excellent or Good and (2) Average, Poor, or Very Poor. A 1-day dietary survey was also administered. The health behaviors score (HBS) was calculated based on 5 factors: consuming a healthy diet, never smoking, low-risk alcohol drinking, regular exercise, and moderate sleep duration. HBS scores ranged from 0 to 5. Multiple logistic regression was used to calculate the sex-, age- BMI- and energy intake-adjusted odds ratios (ORs) of poor subjective health across HBS, with 0 points as the reference.p for trend: < 0.001). Additional analyses revealed that the combinations including regular exercise were particularly associated with a lower risk of subjective average/poor health.Among all participants, 2397 (39.6%) individuals were classified into the good subjective health group. Participants with an HBS of 3 , 4  or 5  had a lower OR of rating themselves as being average/poor health compared with those having zero. The association with a higher HBS was remarkable (This study showed that the higher the number of healthy lifestyle factors, the lower risk of subjective average/poor health. Combinations of healthy lifestyle factors, especially those involving exercise, suggest good subjective health for individuals living in the Shiga prefecture. Life expectancy in Japan is higher than in other developed countries . The MinThere are reports that in Japan, increased life expectancy is due to decreased mortality from cardiovascular diseases and neoplasms or cancers . In factStudies have revealed that lifestyle factors are independently associated with the risks of all-cause and cause-specific mortality. A meta-analysis has reported that current smoking is a risk factor of all-cause and lung cancer death  On the oSubjective health perception often used in an overall measure of health status, consists of one simple question. This indicator has been found predictive of all-cause and cause-specific mortality. A large body of evidence has shown that those with a poor subjective health perception had a higher risk of mortality than those with a good subjective health perception –20. As fSome study have revealed that subjective health perception is associated with lifestyle factors –27. An RThe Health and Nutrition Survey of Shiga prefecture (HNSS) is a survey that covers approximately 4000 households, once every 5 years, in randomly selected census units defined by the Shiga prefecture. It provides the largest prefecturally representative sample available by which to monitor dietary intake and lifestyle factors of the people in the Shiga prefecture. The present study was approved by the Shiga prefecture in Japan. The most recent data in 2015, covering 4229 households, were analyzed in the study. The HNSS was conducted on one day in November 2015.2) and energy intakes . Age were significantly higher (55.9 ± 18.5 years) and subjective health perception were poor (“excellent” and “good” = 37.2%); however, the difference between the groups was not large.Data were the HNSS obtained from, Japan, in 2015. Figure The present analysis was based on a secondary analysis of observational survey data. No ethical review was sought based on the Ethical Guidelines for Medical and Health Research Involving Human Subjects, given this study used only information that had already been anonymized. The HNSS, conducted by the government of Shiga prefecture, has stringent protocols and procedures that ensure confidentiality and protect individual participants from being identified. The Shiga prefecture government anonymized all individual-level data before providing the authors with the datasets for this study.We used a single item to measure general health: “How is your current health? (1) Excellent, (2) Good, (3) Average, (4) Poor, (5) Very Poor.” Those who answered excellent or good were classified into the good subjective health group, and those who answered average, poor or very poor were classified into the average or poor subjective health group .1, vitamin B2, niacin, vitamin B6, vitamin B12, folic acid, vitamin C, calcium, magnesium, iron, zinc, copper, dietary fiber, and saturated fatty acid. For each of the 18 nutrients, a score of 1 was allocated if the nutrient met or exceeded the recommended daily allowance (RDA) or tentative dietary goal (DG) given in DRIs-J and a score of 0 if it did not meet RDA or DG. DG was defined as the current goals for Japanese individuals to reach the average daily intake of nutrients for the prevention of lifestyle-related diseases in the DRIs-J 2015. It was determined based on the results of many nutritional epidemiology studies. Additionally, the nutrients having a tolerable upper intake level (UL) were given a score of 1 if the nutrient was below UL, and 0 if it was above the UL. For the 18 nutrients included, we used the RDA for 14 nutrients  and the DG for 4 nutrients . Energy intake was assessed using the Estimated Energy Requirement (EER). EER = basal metabolic reference value  × body weight (kg) × physical activity level (PAL). Basal metabolic reference values and PAL were given in DRIs-J 2015. Body weight of each individual were used. A score of 1 was allocated if an individual’s energy intake was ≥ EER with PAL = 1 and ≤ EER with PAL = 3, and 0 if it deviated from the range. The range of PAL values were 1.50–2.00 for participants aged 20–69 years and 1.45–1.95 for participants aged ≥70 years. In DRIs-J 2015, PALs of 1, 2, and 3 were considered low, moderate, and high, respectively, and can be characterized as a sedentary lifestyle, sedentary work including housework and light-intensity sports, and work with high-intensity physical activity or leisure-time physical activity, respectively. The total DRIs-J score was constructed by summing the scores of the 18 nutrients and energy intake. The DRIs-J scores ranged from 0 to 19. Each participant was given 1 point for a DRIs-J score greater than or equal to the median score by sex in the participants (the median score was 7.0 for both sexes).The nutrient and food intake were estimated by a 1-day household dietary record. The record included the approximate proportions the dish was divided into among the family members to estimate the energy, nutrient, and food intakes of each individual. The energy and nutrient intakes of each individual were calculated using the Japanese Standard Food Composition Table . Energy 2). Smoking was assessed with three options: current, former, or never smoker. Participants who never smoked were given 1 point. Current and former smokers were scored 0 to avoid misclassification for those who stopped smoking recently. Information on alcohol drinking was obtained regarding the frequency and quantity by using 2 questions. The frequency of alcohol drinking was reported with seven options: “never”, “in the past”, “occasionally”, “1–2 times per week”, “3–4 times per week”, “5–6 times per week”, and “every day”. If participants answered “never”, “in the past”, or “occasionally” for the first question, they were scored 1. If participants consumed alcoholic beverages 1–2 times per week or more, a second question was asked about the average quantity consumed per day. Alcohol consumption ≥40 g/d for men or ≥ 20 g/d for women was scored 0. Alcohol consumption < 40 g/d for men or < 20 g/d for women was scored 1. Excessive amounts of alcohol have been reported to be associated with a higher risk of lifestyle-related diseases [Information on height, body weight, smoking, exercise, sleeping, alcohol drinking was obtained from the self-administered questionnaire. BMI was calculated as body weight (kg) divided by height squared  hour per week has been reported to be associated with a low risk of all-cause and cancer mortality , 42 and Sleep duration was assessed using the question, “How many hours do you usually sleep per day?” The response categories were: < 5 h/d, ≥ 5 h/d and < 6 h/d, ≥ 6 h/d and < 7 h/d, ≥ 7 h/d and < 8 h/d, ≥ 8 h/d and < 9 h/d, ≥ 9 h/d. Those who answered ≥7 h/d and < 8 h/d or ≥ 8 h/d and < 9 h/d were given 1 point. The rest were scored 0. This duration has been suggested to have an association with a low risk of all-cause mortality .We established health behaviors score (HBS) referring to previous studies –12. Tabl2 test were used for mean values and SD for continuous variables, and percentages for categorical variables. Multiple logistic regression was used to investigate the association between subjective health perception  and the HBS. The odds ratios (ORs) and 95% confidence intervals (CIs) for average/poor subjective health perception were calculated with the HBS as the exposure variable. Next, to examine the specific patterns of health behaviors, variables representing possible combinations of the HBS were created. Some variables were not included in the analysis because they were rarely observed in this population. These models were adjusted for sex, age (continuous) and BMI (continuous) based on previous studies that examined the relationship between subjective health perception, death and lifestyle factors [p <  0.001); the higher the dietary score, the higher the energy intake (Pearson’s correlation coefficient: r = 0.632). We tested for interactions by introducing a multiplicative term into the main effect models; however, there was no change in the statistical significance of the results. For all analyses, statistical significance was defined as a two-tailed p-value of < 0.05. All statistical analyses were performed using SAS software .To compare the characteristics of the individuals according to their subjective health perception, t tests and a χ factors , 15, 25,A total of 6057 individuals were included in the analysis. Of them, 39.6% reported their subjective health perception as “Excellent” or “Good”. Table p for trend < 0.001; OR: 0.38, 95% CI 0.22–0.67, p for trend < 0.001 respectively). After adjusting for sex, age, BMI and energy intake, compared with those with an HBS of 0, those with an HBS of 1 and 2 did not have significantly lower odds of rating themselves as being in average/poor health. However, those with an HBS of 3, 4, or 5 had a lower OR of rating themselves as being average/poor health . This association became stronger as the HBS increased (p for trend < 0.001).Table p for trend < 0.001). Among those with an HBS of 4, two combinations included exercise: exercise, better diet, low-risk alcohol drinking and never smoking and exercise, low-risk alcohol drinking, never smoking and moderate sleep duration were associated with a lower risk of average/poor subjective health . However, the association between exercise, better diet, low-risk alcohol drinking and moderate sleep duration and a risk of subjective average/poor health was not significant . The results of investigating for individual health behaviors were shown in Additional file p <  0.001).To examine which combinations of the HBS were associated with a lower risk of average/poor subjective health perception, we conducted further analyses of those with an HBS of 3 or higher. Table The present study of adults revealed that the higher the number of healthy lifestyle factors, which comprised regular exercise, dietary adherence to DRIs-J, no excessive alcohol drinking, moderate sleep duration, and never smoking, the lower the risk of an average/poor subjective health perception. The combinations including exercise behavior were particularly likely to decrease the OR of subjective average/poor health perception.We found that maintaining 3 or more healthy lifestyle behaviors was associated with lower risk of subjective average/poor health perception. Previous studies have shown that individual lifestyle-related behaviors are associated with subjective health perception. A longitudinal study reported that adults who smoked 10 or more cigarettes per day had a higher risk of rating themselves as having poor health compared with those who had never smoked after adjusting for potential confounders, including the participants’ disease conditions . A largeIn the HBS used in our study, the combinations including exercise were strongly associated with a lower risk of subjective average/poor health perceptions. Furthermore, those with an exercise score of 1 had lower odds of rating themselves as having average/poor health compared with those with an exercise score of 0, which was the strongest association among the five health behaviors. In an overview examining epidemiological evidence for an association between physical activity and mental health, the potential anti-inflammatory effects of exercise, involving serotonin and noradrenaline, might be relevant at the neurobiological level as antidepressants and anxiolytics . Double-On the other hand, in our study, the association between exercise, better diet, low-risk alcohol drinking and moderate sleep duration and a risk of subjective average/poor health was not significant. Of those engaged these 4 behaviors, 77.4% aged 65 or older, although the other individuals with an HBS of 4 included exercise did not have differences between the rate of 65 or older and under 65 years. Older people were more likely to perceive their subjective health poor than younger people . In termThere are some limitations to the study. First, because it was a cross-sectional study, we could not show a causal relationship between subjective health perception and the HBS. Second, the dietary record was conducted for only 1 day; thus, the dietary data might not reflect habitual intake. However, the nutrient intake of the individuals in our study was similar to Japanese habitual nutrient intake . Third, This study also has several strengths. The HBS consisted of the factors based on significant scientific evidence of an association with mortality risk. Subjective health perception is a useful measure for public health and nutrition assessment using a single survey question, and it has been validated as a tool to predict mortality in populations. To our knowledge, this is the first study of combinations of HBS and subjective health perception.The present study showed that 3 or more healthy lifestyle factors including regular exercise, high adherence to a healthy diet, moderate sleep duration, nonexcessive alcohol drinking, and never smoking were associated with a low risk of subjective average/poor health. The combinations of healthy lifestyle factors, particularly those that include exercise, indicate a good subjective health perception for individuals in the Shiga prefecture in Japan and widen the scope of choices available for people with poor subjective health.Additional file 1. The association between the individual health behaviors and an average/poor subjective health perception. OR: Odds ratio, CI: Confidence interval. a Health behavior: Better Diet; DRIs-J score ≥ median by sex in the participants (7.0 for both sexes), never smoking, Low-risk Alcohol Drinking; consuming alcohol < 40 g/d for men or < 20 g/d for women, Exercise; engaging in 30 min of exercise at least twice a week and continuing for a year, and Moderate Sleep Duration; sleeping for ≥7 h/d and < 9 h/d. b Adjusted for sex, age, BMI, energy intake and the other health behaviors included in this table. * p <  0.05, **p <  0.01, ***p <  0.001.


This study examined racial/ethnic differences in health-related quality of life (HRQOL) among adults and identified variables associated with HRQOL by race/ethnicity.This study was conducted under a cross-sectional design. We used the 2011–2016 Hawaii Behavioral Risk Factor Surveillance System data. HRQOL were assessed by four measures: self-rated general health, physically unhealthy days, mentally unhealthy days, and days with activity limitation. Distress was defined as fair/poor for general health and 14 days or more for each of the other three HRQOL measures. We conducted multivariable logistic regressions with variables guided by Anderson’s behavioral model on each distress measure by race/ethnicity.Among Hawaii adults, 30.4% were White, 20.9% Japanese, 16.8% Filipino, 14.6% Native Hawaiian and Pacific Islander (NHPI), 5.9% Chinese, 5.2% Hispanics, and 6.2% Other. We found significant racial/ethnic differences in the HRQOL measures. Compared to Whites, Filipinos, Japanese, NHPIs, and Hispanics showed higher distress rates in general health, while Filipinos and Japanese showed lower distress rates in the other HRQOL measures. Although no variables were consistently associated with all four HRQOL measures across all racial/ethnic groups, history of diabetes were significantly associated with general health across all racial/ethnic groups and history of depression was associated with at least three of the HRQOL measure across all racial/ethnic groups.This study contributes to the literature on disparities in HRQOL and its association with other variables among diverse racial/ethnic subgroups. Knowing the common factors for HRQOL across different racial/ethnic groups and factors specific to different racial/ethnic groups will provide valuable information for identifying future public health priorities to improve quality of life and reduce health disparities. Health-related quality of life (HRQOL) measures a person’s subjective perception of his/her life with a multi-dimensional concept encompassing physical and mental health, emotional well-being, and social functioning . HRQOL hA growing body of literature (or research) has revealed racial/ethnic differences in HRQOL. Most of the studies have focused on Blacks and Hispanics and described poor HRQOL among these racial groups compared to Whites –7. HowevNumerous studies have been conducted to identify predictors of HRQOL and social and clinical determinants for HRQOL. For example, a study conducted among older adults using the National Health and Nutrition Examination Survey (NHANES) data found significant association between HRQOL and education, depression, and activities of daily living . A studyPredisposing variables are social and cultural characteristics that exist prior to individuals’ disease including demographic and social structure . Need variables refer to conditions that laypeople or health care providers recognize as requiring medical treatment . Enabling variables show logistical aspects of getting care which comprise medical personnel, health facilities, and patient’s personal means to get access to healthcare including financing factors and organizational factors .To fill this gap, we explored important variables associated with HRQOL for these disaggregated and detailed racial/ethnic categories. Andersen’s behavioral model guided determination of candidate variables. The model has been used to delineate a comprehensive view of multiple influences from three groups of variables to HRQOL – predisposing, need, and enabling variables , 14. PreWe examined racial/ethnic differences in HRQOL among adults who live in Hawaii and identify important variables associated with HRQOL among six major racial/ethnic groups in Hawaii – White, Chinese, Filipino, Japanese, NHPI, and Hispanic. We hypothesized that race/ethnicity is significantly associated with HRQOL and the significance, magnitude and direction of variables guided by Anderson’s model on HRQOL differ by race/ethnicity.http://www.cdc.gov/BRFSS). For Hawaii BRFSS, the Hawaii state department of health (DOH) collects and distributes the BRFSS. We received detailed disaggregated race/ethnicity information from the Hawaii DOH and linked to the publicly available BRFSS data through the participants’ IDs. This study included 45,230 respondents after excluding 313 who did not provide their racial/ethnic information. The study received exempt certification from the University of Hawaii Internal Review Board (IRB).We used the 2011–2016 data from the Hawaii Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is an annual cross-sectional ongoing state-based health survey for US non-institutionalized adults aged 18 years or older. The BRFSS collects data on sociodemographic variables, chronic illness, health behaviors, access to healthcare, and other health-related information in all 50 states in the US and the District of Columbia, Puerto Rico, and the US Virgin Islands. Further information about survey design and sampling method can be found on the BRFSS website (http://www.cdc.gov/HRQOL/methods.htm): (1) General Health: “Would you say that in general your health is? ”; (2) Physical Health: “For how many days during the past 30 days was your physical health not good?”; (3) Mental Health: “For how many days during the past 30 days was your mental health not good?”; and (4) Activity Limitation: “During the past 30 days, for about how many days did poor physical or mental health keep you from doing your usual activities, such as self-care, work, or recreation?” General health was further categorized as fair/poor (1) versus excellent/very good/good (0). We dichotomized the other HRQOL measures into ≥ 14 days or more (1) and < 14 days (0), as previously used in other studies [The HRQOL was assessed by the following four core questions developed by the Center for Disease Control and Prevention  refused to report or did not know their income levels, we included a missing category for income. We defined housing status as owning vs other (renting or other arrangement). Residential area was categorized as Oahu (considered urban) vs other islands . Two healthcare access variables were 1) having any health care coverage (yes vs. no) and 2) medical cost affordability defined by the question “Couldn’t see a doctor because of medical cost” (yes vs. no).The need variables in this study belong to two major groups—behavioral and clinical. Behavioral variables were exercise, heavy drinking, and smoking status . We defined exercise for a positive response on the following question: “During the past month, other than your regular job, did you participate in any physical activities or exercises such as running, calisthenics, golf, gardening, or walking for exercise?” We defined heavy drinking as having more than 14 drinks per week for men and more than 7 drinks per week for women (yes vs. no).2 (yes vs. no).Clinical variables were obese/overweight status and history of eleven major chronic diseases (yes vs. no): heart attack, coronary heart diseases (CHD), stroke, asthma, cancer, chronic obstructive pulmonary disease (COPD), arthritis, depression, kidney disease, and diabetes—for simplicity, we excluded ‘history of’ in front of the chronic diseases in this manuscript. Obese/overweight was defined as body mass index greater than 25.0 kg/msurvey package [P value less than 0.01 was considered statistically significant.All analyses were implemented in R version 3.6.1 using the  package , incorpoThe study data comprised 30.4% White, 20.9% Japanese, 16.8% Filipino, 14.6% NHPI, 5.9% Chinese, 5.2% Hispanic, and 6.2% Other in Hawaii , three from enabling variables  and six from need variables . Those aged 45–64 years had 1.48 times higher odds of reporting fair/poor general health compared to those aged 18–44 years. Higher education level was negatively associated with distress in general health. People with at least some college level education were less likely to report fair/poor health compared to those with high school graduate or less education. Employed people or those with higher income were less likely to be distressed compared to their counterparts. We also explored bivariate association between education and employment status. Higher education was significantly associated with higher employment rate (results are not shown). Thus, these variables might independently affect general health. Those who couldn’t see a doctor due to medical cost were 1.62 times more likely to be distressed than those who can afford.Specific to Chinese, three need variables were significant: cancer, COPD, and kidney disease. Those with these diseases were more likely to be distressed than those without it. Across the racial/ethnic groups, the effect of COPD was the highest among Chinese (OR = 3.86).Specific to Filipino, four need variables were significant: smoking, CHD, cancer, and kidney disease. Smokers are more likely to report distress in general health than non-smokers. CHD, cancer, and kidney disease were risk factors for distress in general health; the effect of kidney disease was the highest for Filipino among the racial/ethnic groups (OR = 3.96).Predictors specific to Japanese were two from predisposing variables , two from enabling variables , and four from need variables . Those aged 45–64 years have 1.81 times higher odds of reporting fair/poor general health compared to those aged 18–44 years. People with college graduate level education were less likely to report distress in general health compared to those with high school graduate or less education. Those having ≥ $50,000 were less likely to be distressed compared to those having < $50,000. Different from the other racial/ethnic groups, Japanese who refused to disclose their income were 1.54 times more likely to be distressed in general health than those having < $50,000. Those who couldn’t see a doctor due to medical cost were 2.43 times more likely to be distressed than those who can. Obese or overweight people had a 1.43 times higher distress rate than people having normal or underweight.Specific to NHPI, four need variables were significant: smoking, obese/overweight COPD, and kidney disease. Smokers are more likely to report distress in general health than non-smokers. The odds of being distressed in general health among obese or overweight people were 1.45 times higher than people having normal or underweight.Predictors specific to Hispanic were age (predisposing), residential area (enabling), and heavy drinking (need). Those aged 45–64 years and 65 years or older have 2.78 and 2.70 times higher odds of reporting fair/poor general health compared to those aged 18–44 years, respectively. Oahu residents are 1.98 times more likely to be distressed in general health compared to people who live in the other islands.The relative importance of variables for physical health differs by race/ethnicity Table . None waPredictors specific to White were one from predisposing variables, one from enabling variables, and five from need variables: employment (OR = 0.57), medical cost affordability (OR = 1.75), exercise (OR = 0.34), stroke (OR = 1.97), cancer (OR = 1.37), COPD (OR = 2.27), arthritis (OR = 2.25), and depression (OR = 2.23). Medical cost affordability was associated with physical health only for White and those who couldn’t see a doctor due to medical cost were 1.75 times more likely to be distressed in physical health than those who can afford.Specific to Chinese, one from enabling variables and five from need variables were significantly associated with physical health: employment (OR = 0.44), exercise (OR = 0.39), asthma (OR = 2.77), COPD (OR = 3.72), arthritis (OR = 2.03), and kidney disease (OR = 3.98). The effects of COPD and kidney disease were the highest among all racial/ethnic groups, and asthma was significant only for Chinese. In addition, the effect of stroke, though not significant at 0.01 (P = 0.034), but was the highest for Chinese among the racial/ethnic groups (OR = 4.37).Specific to Filipino, one from predisposing variables and four from need variables were significant: age, stroke (OR = 2.52), arthritis (OR = 2.69), depression (OR = 3.49), and kidney disease (OR = 3.14). People aged 45–64 years had 1.96 times higher odds of distress compared to those aged 18–44 years. The effects of arthritis and depression were the highest among the racial/ethnic groups.Predictors specific to Japanese were one from predisposing variables, two from enabling variables, and five from need variables: age, employment (OR = 0.54), housing status, exercise (OR = 0.55), cancer (OR = 1.74), COPD (OR = 1.97), arthritis (OR = 2.53), depression (OR = 2.33), and diabetes (OR = 1.44). Those aged 45–64 years have 2.01 times higher odds of distress in physical health compared to those aged 18–44 years. Housing status and diabetes were significant only for Japanese. Those who rent or live in other arrangements were 1.51 times more likely to be distressed than those who own their home.Predictors specific to NHPI were two from predisposing variables, one from enabling variables, and four from need variables: sex (OR = 1.67), marital status (OR = 0.57), employment (OR = 0.64), exercise (OR = 0.43), COPD (OR = 3.09), arthritis (OR = 1.89), and depression (OR = 2.05). Sex and marital status were significantly associated with physical health only for Japanese. Men were 1.67 times more likely to report distress in physical health than women, and single people were less likely to be distressed in physical health than married people.Specific to Hispanic, only one need variable, depression, appeared to be significant. People with depression were 2.68 times more likely to report distress in physical health than people without it.Again, race/ethnicity plays an important role for mental health Table . DepressSpecific to White, two from predisposing variables, one from enabling variables, and three from need variables were significant: age, education, medical cost affordability, exercise, smoking, and heart attack. People aged 65 years or older are less likely to be distressed compared to those aged 18–44 years (OR = 0.33). People with at least some college level education were less likely to be distressed in mental health compared to those with high school graduate or less education (OR = 0.67). Those who couldn’t see a doctor due to medical cost were 2.14 times more likely to be mentally distressed than those who can. Those who exercise were less likely to report distress in mental health than those who do not (OR = 0.56). The odds of distress among people who do not smoke are lower by a factor of 0.71 than current smokers. Education, exercise, and smoking were significantly associated with distress in mental health only for White.P = 0.012) but the odds of distress in mental health among non-smokers was the smallest among all racial/ethnic groups compared to smokers (OR = 0.44). Similarly, having health coverage did not meet the significance level at 0.01 (P = 0.016) but the OR was the smallest among the racial/ethnic groups (OR = 0.43).Specific to Chinese, beside depression, only one enabling variable, medical cost affordability, was significant. People who couldn’t see a doctor due to medical cost were 4.36 times more likely to be mentally distressed than those who can. Smoking did not meet the significance level at 0.01 . Those who couldn’t see a doctor due to medical cost were 2.59 times more likely to be distressed than those who can. Heavy drinking was positively associated with mental health only for Japanese (OR = 1.91).Specific to NHPI, one from predisposing variables, one from enabling variables, and two from need variables were significant: age, medical cost affordability, COPD (OR = 2.07) and arthritis (OR = 1.69). People aged 65 years or older are less likely to be distressed, compared to those aged 18–44 years (OR = 0.34). Those who couldn’t see a doctor due to medical cost were 1.97 times more likely to be distressed than those who can.A predictor specific to Hispanic was a need variable, asthma. It was associated with mental health only for Hispanic, and people with asthma was 2.53 times more likely to be mentally distressed than those without.Race/ethnicity affects the importance and significance of variables Table . DepressPredictors specific to White were one from predisposing variables, three from enabling variables, and four from need variables: age, employment (OR = 0.30), income, medical cost affordability, exercise (OR = 0.39), heart attack (OR = 1.74), stroke (OR = 2.07), and arthritis (OR = 2.06). People aged 65 years or older are less likely to be distressed compared to those aged 18–44 years (OR = 0.44). Income was associated with distress in activity limitation only for White and those having ≥ $50,000 were less likely to be distressed compared to those having < $50,000 (OR = 0.61). Those who couldn’t see a doctor due to medical cost were 1.26 times more likely to be distressed than those who can.For Chinese, the common variable, depression, was only significantly associated with distress in activity limitation. People with depression was 5.82 times more likely to be distressed than those without.Predictors specific to Filipino were one from enabling variables and one from need variables: employment (OR = 0.45) and arthritis (OR = 3.36). In addition, although stroke was not significant at 0.01 (P = 0.024), its effect was the highest among all racial/ethnic groups (OR = 3.21).Specific to Japanese, two from enabling variables and three from need variables were significant: employment (OR = 0.21), housing status, exercise (OR = 0.28), CHD (OR = 4.71) and arthritis (OR = 2.83). Housing status was significant only for Japanese. People who rent or live in other arrangements were 2.37 times more likely to be distressed than those who own their home.P = 0.013), its effect was the highest among the racial/ethnic groups (OR = 2.63).Specific to NHPI, one from predisposing variables, two from need variables, and one from need variables were significantly associated with distress in activity limitation: age, employment (OR = 0.27), medical cost affordability and exercise (OR = 0.38). Those aged 65 years or older are less likely to be distressed compared to those aged 18–44 years (OR = 0.20). People who couldn’t see a doctor due to medical cost were 2.15 times more likely to be distressed than those who can. Although COPD was not significant at 0.01 . Because of hormone changes, the prevalence rate of chronic conditions and obesity increases so people in menopause feel and see their health differs from what it used-to-be. The distress rate in general health decreased after this age group, in which resilience may play an important role with age. Resilience refers to “successful adaptation and swift recovery after experiencing life adversities” . Older aEmployment and medical cost affordability were associated with at least one of the HRQOL measures except for Hispanic and Hispanic/Filipino, respectively. This indicates that HRQOL can be improved by increasing support for medical expenditure for some of the economically disadvantaged. As Chaffee et al. suggested , there iOur study provides some insight with clinical and public health implications in Hawaii, a state with diverse racial/ethnic subpopulations. Racial/ethnic differences emphasize the importance of tailoring interventions for specific racial/ethnic groups to improve individual’s health outcomes and quality of life. Although some progress has been made toward elimination of racial/ethnic health disparities, significant disparities persist, particularly for NHPI , 43. ThiWe want to point out several limitations in interpreting the study results. First, we could not account for other potential predictors (e.g., activities of daily living, adverse childhood experiences , and visDespite these limitations, our study has several strengths. The use of a representative dataset makes it possible to generalize our findings of HRQOL perceptions to the Hawaii adult population. The use of the detailed racial information allows us to identify differences in the HRQOL among NHPI and various Asian ethnic groups. Finally, investigating the four HRQOL measures together is an additional strength of the current study.This study contributes to the literature on disparities in HRQOL and predictors of HRQOL among diverse racial/ethnic subgroups. The factors associated with HRQOL by different racial/ethnic groups will provide valuable information for identifying future public health priorities to improve quality of life, and will help state governments and healthcare professionals remove the potential barriers to increase healthcare utilization.


Drought is a major cause of losses in crop yield. Under field conditions, plants exposed to drought are usually also experiencing rapid changes in light intensity. Accordingly, plants need to acclimate to both, drought and light stress. Two crucial mechanisms in plant acclimation to changes in light conditions comprise thylakoid protein phosphorylation and dissipation of light energy as heat by non‐photochemical quenching (NPQ). Here, we analyzed the acclimation efficacy of two different wheat varieties, by applying fluctuating light for analysis of plants, which had been subjected to a slowly developing drought stress as it usually occurs in the field. This novel approach allowed us to distinguish four drought phases, which are critical for grain yield, and to discover acclimatory responses which are independent of photodamage. In short‐term, under fluctuating light, the slowdown of NPQ relaxation adjusts the photosynthetic activity to the reduced metabolic capacity. In long‐term, the photosynthetic machinery acquires a drought‐specific configuration by changing the PSII‐LHCII phosphorylation pattern together with protein stoichiometry. Therefore, the fine‐tuning of NPQ relaxation and PSII‐LHCII phosphorylation pattern represent promising traits for future crop breeding strategies. Elucidating the regulation of photosynthesis under the combination of abiotic stress and fluctuating light is crucial for understanding the plant physiology in realistic conditions and for crop improvement. Wheat copes with drought by reconfiguring the photosynthetic machinery and by slowing down the NPQ relaxation in fluctuating light before photodamage becomes detectable. Hence, plants employ a number of different protection mechanisms to mediate the dissipation of excess energy from light‐harvesting under CO2‐limiting conditions  is crucial for crop productivity. The rate of NPQ relaxation can account for up to 15% of crop yield . First, plants were subjected to a slowly‐developing drought (similar to what may occur in field conditions) under natural light in greenhouse. Here the monitoring of water use and NPQ allowed distinguishing four drought phases. Subsequently, we applied a similar drought treatment in a climate chamber, where plants were grown under controlled light conditions until a phase of drought was achieved in which they did not yet show symptoms of long‐term molecular damage. At different time points during the drought treatment, we applied a method, denoted here as “FL analysis” , on single leaves for a few minutes, while simultaneously monitoring the photosynthetic activity. This method was used to challenge the photosynthetic regulatory mechanisms under a fluctuation of light intensity and at the same time to quantify consequent photodamage. In other words, the growth light level served as a baseline, on which we applied FL for a few minutes and evaluated the FL‐induced photodamage during drought progression. As our aim was to understand the basic acclimation processes, we chose two spring wheat cultivars originated from very different environments, with the purpose of identifying common response mechanisms implemented by both cultivars. FL analysis in the chamber experiments revealed the same drought phases as observed in the greenhouse experiment before. Moreover, the “FL analysis” uncovered also changes in the NPQ kinetics. This implies that stressed wheat plants employed drought‐dependent long‐term modifications that had effects also in the time scale of fast light changes. Drought‐stressed plants actually exhibited a reconfiguration of the photosynthetic machinery, consisting in the modification of the photosynthetic enzymes stoichiometry and in the acquisition of a long‐term PSII and Light Harvesting Complex II (LHCII) phosphorylation pattern. These structural and functional changes constitute active regulatory mechanisms because they were independent from both short‐term and long‐term drought‐induced damage. Finally, we integrated these results to develop a model of the photosynthetic acclimation of wheat under water deficit. Suggestions are put forward for developing new traits to be applied to crop breeding.The interaction between drought and FL effects is therefore very complex. In order to dissect the effects of drought and FL, we applied the following experimental setup to wheat ; temperature was 24°C in the day and 16°C at night. All measurements were carried out on leaves acclimated to the light phase of 450 μmol photons m−2 s−1. For in vitro analyses, all leaves were collected at 3 p.m. For in vivo analyses (applied on single leaves), leaves were chosen randomly.Seeds of two spring wheat cultivars were obtained from the IPK genebank, one originating from the United Kingdom, accession number TRI 5357 (here indicated as UK), the other from Iran, accession number TRI 5630 (indicated as IR). Seeds were sown directly to soil in cylindrical pots . Soil mixture: 3 parts of potting compost , 2 parts of sand, 1 part of Styromull . After the seedling establishment, we thinned out to one plant per pot. Drought stress was applied to wheat plants at the age of 4 weeks, when they reached a developmental stage comprised in BBCH 23–33 phases . In the chamber, plants were grown in controlled conditions: relative humidity was 55%; light was provided by metal halide lamps  at the intensity of 200 μmol photons m−2 s−1, additional light was supplied by halogen lamps (at 200 μmol photons m−2 s−1) until a maximum of 15 h light per day.The greenhouse experiment was carried out at IPK. Temperature remained in the range of 17–25°C during the day, and 17–19°C in the night; relative humidity in the range of 50–80%. Wheat plants were grown in 2 L pots. Watering and pot weighing were performed automatically by the phenotyping and imaging platform LemnaTec‐Scanalyzer 3D . When the internal sunlight intensity was lower than 200 μmol photons m2.2In both greenhouse and chamber experiments, water deficit was applied by complete water withdrawal until the soil water content approached a plateau value, that is, at the exhaustion of extractable water. In greenhouse, soil water content was monitored by weighing the pots and quantified as percentage of soil saturation (% of field capacity). Subsequently, soil was rewatered and maintained at the well‐watered level (90% of field capacity) until harvest. In the chamber, soil dehydration was monitored by measuring the volumetric soil water content  through ML3 sensors . Sensors were placed at the bottom of soil through a hole in the pot. When the soil water content reached the plateau value, leaves were collected and frozen in liquid nitrogen for further analysis. Leaf water content was estimated by the formula (FW − DW)/(TW − DW) × 100, where FW is the leaf fresh weigh, DW is the leaf dry weight, and TW is the leaf turgid weight. Leaves were cut at the basal portion, and the cut edge of leaf section was placed in distilled water (until about 2 mm depth) overnight at 4°C for allowing full leaf rehydration and the determination of TW. Subsequently, leaves were dried at 60°C for 4 days for the determination of DW. Stomata conductance was measured by PWMR‐4 porometer  between 11 a.m. and 2 p.m.2.3All leaves from every plant were collected. In order to exclude leaf extremities, only the central portion (approximately one‐third of leaf length) was collected and frozen in liquid nitrogen for biochemical analyses. Chlorophyll content per leaf area was determined on leaf discs (4 mm diameter) according to the method described in  and solvent B (100% ethyl acetate), and pigments were detected at 440 nm using a Dionex UVD 340U detector. Pigment standards for HPLC analysis were obtained from Extrasynthese S.A.S. FR  for β‐carotene (Cat. No. 0303‐S), zeaxanthin (Cat. No. 0307‐S), and lutein (Cat. No. 0306‐S); and from DHI LAB Products , for violaxanthin (Cat. No. PPS‐VIOL). The plant hormone abscisic acid (ABA) was detected using the ELISA kit from Cusabio (www.cusabio.com/ELISA-Kit/Plant-hormone-abscisic-acidABA-ELISA-Kit-62434.html). Briefly, 50 mg lyophilized leaf material where extracted with 700 μl sample extraction buffer (provided with kit) over night at 4°C, and the ABA content was measured photometrically at 450 nm using a standard dilution curve according to the manufacturer's instructions. Malondialdehyde (MDA) levels were quantified colorimetrically at 532 nm, using the thiobarbituric acid (TBA) method, which detects the MDA‐TB complex using a kit from abbexa (www.abbexa.com/lipid-peroxidation-mda-assay-kit). Therefore, 50 mg of lyophilized leaf material where extracted with 450 μl PBS buffer and colorimetrically quantified with thiobarbituric Acid (TBA) as MDA‐TB complex at 532 nm according to the manufacturer's protocol.Pigments were determined by HPLC analysis essentially according to , and the resulting suspension was filtered through Miracloth. Chloroplasts were collected by centrifugation , after which they were osmotically ruptured with shock buffer . The released thylakoid membranes were collected by centrifugation  and suspended in storage buffer . Chlorophyll concentration (Chl a + b) was determined according to Porra et al. .Fluorescence emission at the temperature of 77 K was recorded from isolated thylakoids with Ocean Optics S2000 spectrophotometer, using excitation wavelength of 480 nm. Thylakoid membranes were diluted to a concentration of 10 μg chl/ml with storage buffer  glycerol, 4.3% (wt/vol) SDS, and 10% β‐mercaptoethanol)  and separated with SDS‐PAGE with 15% acrylamide and 6 M urea. For immunoblotting, proteins were transferred on PVDF membrane (Millipore). From the membrane, phosphorylated threonine residues were recognized with P‐Thr antibody , while proteins D1, PSAB, LHCB2, and CP29 were detected with protein‐specific antibodies produced by Agrisera . In detection, horseradish peroxidase‐linked secondary antibody (Agrisera) and Amersham ECL Western blotting detection reagents  were used. The membranes were subsequently stained with 0.1% Coomassie Brilliant Blue diluted in 40% methanol and 10% acetic acid.2.8Total protein was extracted from wheat leaf extract and digested. Peptides were analyzed by LTQ‐Orbitrap Elite (Thermo). More information is available in 2.9−2 s−1 intensity) to allow the estimation of photosynthetic parameters . Before measurements, plants were acclimated to darkness for 15 min. In the greenhouse, NPQ was measured by MultispeQ v1.0  , followed by 10 min of 166 μmol photons m−2 s−1  and a subsequent “high light” phase (HL) for 3.5 min at 1466 μmol photons m−2 s−1, finally followed by a second low light (LL2) phase (at the same intensity as LL1), which was prolonged until a steady‐state NPQ value was reached . The light intensity of VLL had the purpose to induce a very low level of “photosynthetic control,” that is, the feedback downregulation propagated through the decrease of thylakoid lumenal pH  on the Imaging‐PAM data. The lifetime of qE could not be exactly determined, because the frequency of the measuring points (limited by the frequency of SPs) was too low (30 s against a lifetime range of about 20–30 s). A much higher SP frequency would alter chlorophyll fluorescence kinetics, because the SP application induces a transient perturbation of PET. The amplitudes of qE and qZ, on the contrary, could be accurately determined because of the very different order of magnitude in the lifetime of fast and slow components. The confidence limits of the fitted parameters and the p‐values associated to their estimation are indicative of optimal fitting  used for calculating linear electron flow (LEF) was estimated by SPAD measurements , based on the equation Abs = 89.2 − 56.8*exp(−0.0723*SPAD), published by  was measured by PEA fluorometer  after 30 min dark incubation.https://www.walz.com/downloads/manuals/dual-pam-100/515_535_module_01.pdf): the differential light transmittance at 550 and 515 nm, and the transmittance at 535 nm (I535nm). Plants were acclimated to darkness for 5 min in order to relax pmf, then illuminated with actinic light at the intensity of 166 μmol photons m−2 s−1 for 10 min . After this phase, actinic light was increased to the intensity of 1,287 μmol photons m−2 s−1 for 3.5 min . Subsequently, actinic light intensity was set again at the value of 166 μmol photons m−2 s−1 for 5 min (LL2 phase), followed by 30 s of darkness to determine the pmf, ΔpH and ΔΨ. The gH+ parameter was estimated at the end of LL1 and LL2. Absorbance changes at 535 nm are associated to a pool of zeaxanthin (J‐type zeaxanthin) and can be used to monitor the aggregation of LHCII trimers  showed a biphasic decrease: a first phase, relaxing in approximately one minute, and a second phase, showing a much slower (and approximately linear) decline .The proton conductivity through chloroplast ATP synthase (gH+) and the proton motive force (pmf) components (ΔpH and ΔΨ) were determined by the dark‐interval relaxation kinetic of electrochromic shift . In the measuring cuvette, air CO2.10https://github.com/BIONF/HaMStR) in the KEGG annotated protein sets of 30 organisms  groups , considering as factors “drought treatment” and “wheat genotype.” Only the significant differences attributed to the factor “drought treatment” are reported and discussed. For the sake of robustness, we consider only effects equally seen in both cultivars.All experimental results on UK and IR cultivars were evaluated via two‐way Analysis of variance (ANOVA) analysis, carried out by the 33.1In the greenhouse, 4‐week‐old spring wheat plants were subjected to drought stress by complete water withdrawal for 24 days. After drought, plants were rewatered until harvest in order to observe the effects of water deficit to final biomass and grain yield. Soil water content was maintained at an optimal range for plant growth (around 90% field capacity) in control (well‐watered) plants Figure . At the In summary, based on monitoring of photosynthesis and of transpiration and soil water content, we could distinguish four phases of drought acclimation in the greenhouse Figure :Drought Phase I: Soil water content decreased with no changes of the transpiration rate, NPQ, and PSII photodamage, as compared to the well‐watered control plants (days 1–7).Drought Phase II: The transpiration rate decreased with no change of NPQ and FL‐induced photodamage, as compared to the control (days 8–12).Drought Phase III: NPQ values increased with no increase of FL‐induced photodamage, as compared to the control (days 13–17).Drought Phase IV: NPQ further increased and FL induced more photodamage in stressed plants, as compared to the control (days 18–24).These phases will subsequently be used throughout the manuscript.A similar drought treatment was applied on the same wheat cultivars in a climatic chamber under controlled ambient conditions Figure . The soiIn the chamber experiment, water deficit induced clear modifications of NPQ kinetics. In drought‐stressed plants the lifetime of qZ in LL2 significantly increased compared to control on day 14, showing two phases in both cultivars: the first phase where values increased up to around 500 s and the second phase where lifetime steeply increased beyond 1,000 s Figure . The qZ 3.2p‐values equal to .048 and .0033, respectively; VLL‐LL1 comparison by two‐way ANOVA). However, in control, the average CEF/LEF at LL1 increased only 3.1% compared to VLL, while in stressed plants it increased 34.0% . Because CEF in VLL is presumably at the minimal value in all conditions, this observation indicated an increase of absolute CEF in LL1 and LL2 under drought stress compared to well‐watered condition. The values of PSII and PSI efficiencies are reported in Figure 535nm). Drought treatment induced a change of the variation from negative values to values around zero, revealing a slowdown of LHCII disaggregation in concomitance to qZ decay , although severely affected by water deficit, were still responsive to the increase of light intensity . The reduced slope of stress data (compared to well‐watered plants) indicates the increase of the portion of photosynthetic electron flow directed to alternative electron sinks .To better characterize Drought Phase III, a light curve with the simultaneous measurement of chlorophyll fluorescence and gas exchange was performed. Stomata conductivity, LEF and net COy Figure c‐e. In tt Figure f, the re−2 s−1) at day 14 of the chamber experiment (corresponding to Drought Phase III for the drought stressed plants). In both cultivars subjected to water deficit, the phosphorylation of D1, D2, and CP43 subunits of PSII was found to clearly increase, while LHCII (Lhcb1 and Lhcb2 proteins) phosphorylation decreased, as compared to the well‐watered control condition  to lower (30 μmol photons m−2 s−1) or higher light intensity  supplied by the Imaging‐PAM fluorometer. At the end of the illumination period, the illuminated leaf section was cut and immediately frozen in liquid nitrogen. In leaves exposed to high light, both well‐watered and drought‐stressed plants showed a typical response of PSII‐LHCII phosphorylation to short‐term exposure to high light , that is, PSII was highly phosphorylated, while LHCII showed a low phosphorylation level  . This analysis revealed that drought treatment induced a slight decrease of the fluorescence emitted by PSII  in comparison to PSI fluorescence  in both cultivars  Figure d.p‐value = .012 for stress against control comparison, two‐way ANOVA) . Also the amount of CaS increased. Finally, several components of the proteasome and ROS scavenging system significantly increased in drought‐stressed wheat plants . Only proteins detected in all samples were taken into consideration for this study (indicated as “full dataset” in Table ) Figure . Three p) Figure , was fou) Figure . No signd Figure . Also hed Figure . Among mts Table .a/b ratio (Chl a/b) did not differ between control and treated plants  are related to the capacity to acclimate to water deficit in both constant illumination and fast fluctuations of light intensity . In particular, our approach allowed us to discover two novel regulatory mechanisms for the acclimation of the photosynthetic machinery to drought in wheat: (a) the acquisition of a drought‐specific configuration of the photosynthetic machinery, consisting of changes in protein stoichiometry and PSII‐LHCII phosphorylation (active under growth light); (b) the slowdown of NPQ decay upon shift from high to low light (active under FL).4.2Pisum sativum), subjected to severe drought by water withdrawal showed an increase of phosphorylation in both PSII and LHCII , on the contrary, Liu and coworkers  but did not change LHCII phosphorylation , measured on well‐watered (WW) and drought‐stressed (DS) wheat plants in the greenhouse experiment.Click here for additional data file.Figure S2. Outdoor sunlight intensity during the greenhouse experiment described in Figure 1.Click here for additional data file.Figure S3. Additional data of the chamber experiment described in Figure 2.Click here for additional data file.Figure S4. Assessment of the quality of the NPQ decay fitting in FL analysis on UK and IR wheat cultivars  in the UK and IR wheat cultivars in Drought Phases III and IV of the chamber experiment, based on the data shown in Figure 2. WW = well‐watered; DS = drought stressed.Click here for additional data file.Figure S6. Example of raw data for the determination of the parameters described in Figure 3d‐g.Click here for additional data file.Figure S7. Additional data on short‐term changes of photosynthetic activity in UK and IR wheat cultivars in the chamber experiment in Drought Phase III.Click here for additional data file.Figure S8. Thylakoid protein phosphorylation and 77 K fluorescence emission spectra in UK and IR wheat cultivars in the Drought Phase IV of the chamber experiment.Click here for additional data file.Table S1. List of model organisms obtained from KEGG  and Iran (IR) wheat cultivars.Click here for additional data file.Table S3 Proteomics analysis on the United Kingdom (UK) and Iran (IR) wheat cultivars.Click here for additional data file.


Repair of obstructive azoospermia caused by childhood herniorrhaphy may be difficult. Therefore, intracytoplasmic sperm injection using testicular sperm is performed. However, vasovasostomy combined with laparoscopic surgery is challenging.A 42‐year‐old man underwent inguinal hernia repair at age 3. He had normal testicular size, azoospermia, normal hormone levels , absence of Y chromosome micro deletion, and karyotype:46XY, t(1:21)(p34.1:q22.3). He was diagnosed with obstructive azoospermia. Repeated intracytoplasmic sperm injections using testicular sperm resulted in miscarriages. Vasovasostomy combined with laparoscopic surgery was subsequently performed. Postoperative semen analysis result was almost normal. After intracytoplasmic sperm injection of ejaculated sperm, his wife got pregnant.Even if patients have chromosomal abnormalities, performing microsurgical re‐anastomosis first is recommended. To our knowledge, this is the first case of a laparoscopy‐assisted vasovasostomy for post‐herniorrhaphy vas deferens obstruction in Japan. ICSIintracytoplasmic sperm injectionIHinguinal herniorrhaphyMRmicrosurgical reconstructive proceduresOAobstructive azoospermiaTESAtesticular sperm aspirationVasvas deferensV‐VvasovasostomyWe report a case of laparoscopy‐assisted V‐V for OA after IH repair. Despite repeated attempts of ICSIs using testicular sperm, pregnancy did not occur. Laparoscopy‐assisted V‐V was subsequently performed. After ICSI of ejaculated sperm, the wife got pregnant and delivered a healthy boy.Vas obstruction caused by IH repair is a common cause of seminal tract obstruction.et al. reported a total patency rate of 65% and a pregnancy rate of 39% after MR.For vas obstruction treatment, Sheynkin Moreover, repair of OA caused by childhood herniorrhaphy may be difficult. Therefore, ICSI using testicular sperm is performed. However, V‐V combined with laparoscopic surgery is challenging, and no case has been reported in Japan. In our experience, inguinal vas obstruction was corrected by laparoscopic harvesting of the pelvic vas to be anastomosed microsurgically through the internal inguinal ring, thus bypassing the obstructed inguinal vas. This technique should provide enough length of the vas for a tension‐free anastomosis, and laparoscopy provides easier access without difficult dissection through the site of the previous hernia repair. Herein, we present our experience of laparoscopy‐assisted V‐V for post‐herniorrhaphy vas obstruction.A 42‐year‐old man underwent IH repair at age 3. His wife was 32 years old. He was diagnosed with OA. ICSI was performed repeatedly using TESA for infertility treatment at a hospital, but the wife did not become pregnant. Later, he decided to have V‐V. Testicular size was normal . The epididymis swelled slightly, and bilateral vas deferentia were dilated. Serum follicle‐stimulating hormone, luteinizing hormone, estrogen, and testosterone levels were 2.67 IU/L, 2.03 IU/L, 21 pg/mL, and 4.28 ng/mL respectively. Semen analysis revealed azoospermia. Chromosomal examination revealed 46XY, t(1:21)(p34.1:q22.3).6/mL, with progressive motility rate of 38.0%. However, natural pregnancy was not achieved at 12‐month post‐operation, so they chose ICSI with ejaculated sperm.First, testicular sperm extraction was performed for the left testis for sperm cryopreservation. Then, we exfoliated the spermatic duct toward the cranial side. We opened the left inguinal canal but could not identify the obstructed vas, so we performed laparoscopy with head‐down position. A 10‐mm port was placed at the inferior umbilical crease, and the laparoscope was inserted. The abdominal pressure was 10 mmHg. Two 5‐mm ports were placed between the anterior superior iliac spine and umbilicus Fig. . A 5 cm et al.Herniorrhaphy is one of the most common causes of iatrogenic vas obstruction.et al. emphasized that the benefits of surgery, natural pregnancy, and cost‐effectiveness in the MR group were higher than those in the ICSI group. Compared with ICSI, recent data suggest that MR done by a skillful microsurgeon is cost‐effective.There is controversy of whether we should proceed with MR or ICSI using TESAIn our case, the physician first performed ICSI because the husband’s chromosomal analysis revealed autosomal translocations. However, the wife had frequent miscarriages. Despite surgical success and normalized semen findings, natural pregnancy was not achieved for 1 year. ICSI with ejaculated sperm was selected; fortunately, the wife became pregnant and gave birth. The successful birth after MR cannot be clearly defined because chromosome examination of fertilized eggs was not performed, but we would like to interpret that the fertilized eggs had normal chromosomes or balanced reciprocal translocations, and the success of birth could be attributed to the benefits of the maturation process during the passage of the testicular sperm through the epididymis.Even if OA patients have chromosomal abnormalities, it may be acceptable to perform MR prior to ICSI.Consent was obtained from the patient.The authors declare no conflict of interest.


Heart failure with preserved ejection fraction (HFpEF) is the most common type of HF in older adults. Although no pharmacological therapy has yet improved survival in HFpEF, exercise training (ExT) has emerged as the most effective intervention to improving functional outcomes in this age‐related disease. The molecular mechanisms by which ExT induces its beneficial effects in HFpEF, however, remain largely unknown. Given the strong association between aging and HFpEF, we hypothesized that ExT might reverse cardiac aging phenotypes that contribute to HFpEF pathophysiology and additionally provide a platform for novel mechanistic and therapeutic discovery. Here, we show that aged (24–30 months) C57BL/6 male mice recapitulate many of the hallmark features of HFpEF, including preserved left ventricular ejection fraction, subclinical systolic dysfunction, diastolic dysfunction, impaired cardiac reserves, exercise intolerance, and pathologic cardiac hypertrophy. Similar to older humans, ExT in old mice improved exercise capacity, diastolic function, and contractile reserves, while reducing pulmonary congestion. Interestingly, RNAseq of explanted hearts showed that ExT did not significantly modulate biological pathways targeted by conventional HF medications. However, it reversed multiple age‐related pathways, including the global downregulation of cell cycle pathways seen in aged hearts, which was associated with increased capillary density, but no effects on cardiac mass or fibrosis. Taken together, these data demonstrate that the aged C57BL/6 male mouse is a valuable model for studying the role of aging biology in HFpEF pathophysiology, and provide a molecular framework for how ExT potentially reverses cardiac aging phenotypes in HFpEF. Heart failure with preserved ejection fraction (HFpEF) is the most common type of heart failure in older adults, but has no effective pharmacological therapies. This study shows that exercise training can reverse cardiac aging biology and phenotypes that contribute to HFpEF, and identifies cell cycle pathways as potential novel therapeutic targets for age‐related HFpEF. Althoug2.6To determine whether ExT potentially reverses cardiac aging biology associated with HFpEF, we next assessed how the cardiac transcriptome changes with normal aging to provide a comparison to the ExT‐induced transcriptome seen in the aged heart. Not surprisingly, RNAseq analyses identified a large number of genes that were differentially expressed in 30‐month‐old compared to 4‐month‐old hearts Figure . Gene seHAUS8, GADD45A, MAPRES2, MCM5, and FANCI. Increased expression trends of these genes were validated by QPCR in an independent cohort of old male mice that underwent eight weeks of voluntary wheel running  and ExT (30 mo sedentary vs. 30 mo ExT) cohorts identified 216 pathways, which were regulated in opposing directions by aging and ExT Tables  and S6. g Figure  and S6.3HFpEF is a clinical syndrome with high morbidity and mortality, most commonly seen in older adults . Systolic function was assessed by fractional shortening and radial systolic strain, while diastolic function was assessed by early diastolic strain rate. Refer to supplemental methods for details on echocardiographic image acquisition and analysis.4.3Mice were anesthetized with isoflurane and imaged using a 9.8‐T MRI system (Bruker Biospin). Refer to supplemental methods for details of the cardiac MRI protocol.4.4Mice were anesthetized with isoflurane and mechanically ventilated throughout the procedure. The LV was entered via the right carotid artery with a Scisence 1.2F high‐fidelity micromanometer catheter (Transonic Systems Inc.) to record pressure‐volume (PV) loops. PV loops were analyzed off‐line with LabScribe2 software (iWorx).4.5To measure exercise capacity and cardiac reserves, a stress echocardiography protocol was designed in which mice were run to exhaustion and then immediately imaged via echocardiography. Refer to the supplemental methods for details of the protocol.4.6Two aerobic exercise training protocols were used in this study. In the initial discovery cohort, moderate‐intensity treadmill running was performed five days per week for eight consecutive weeks. Treadmill running was done on an automated treadmill (Columbus Instruments) at a constant speed of 10 m/min at 10° incline. To ensure that the biological effects of ExT were not limited to a specific type of aerobic exercise, we also performed eight consecutive weeks of voluntary wheel running (STARR Life Sciences) in an independent validation cohort, using previously published methods , Masson's trichrome for fibrosis, and rabbit‐anti‐mouse CD31  for capillary density. Cardiomyocyte CSAs were measured in three to five random sections , which were averaged to represent a single data point for each heart. Capillary density was quantified by dividing the number of CD31+ cells by the area of randomly selected sections. Three to five sections were measured per heart and averaged to represent a single data point. Given the extensive variability in fibrosis distribution throughout the heart, BZ‐X Analyzer software (Keyence) was used to quantify fibrosis in full mid‐ventricular sections. Percent fibrosis was calculated as the ratio of fibrotic area to total tissue area. Measurements from two sections were averaged to represent a single data point for each heart. Quantitative histologic analyses were done in a blinded fashion.4.8Real‐time PCR products were carried out using SYBR‐green and standard amplification protocols. Expression levels were calculated using the ΔΔCt method. Primer sequences are listed in Table 4.92FC>+1 or downregulated by log2FC<−1 with an adjusted p value < .05 (using Benjamini‐Hochberg correction). Pathway analysis was performed with Gene Set Enrichment Analysis  using the Gene Ontology database. For all differentially expressed genes, a metric was computed as the product of logFC and −log10. A “running sum” statistic was calculated for each gene set in the pathway database based on the ranks of the members of the set relative to those of the nonmembers. Enrichment score (ES) was defined as the maximum sum of the running sum with the genes making up this maximum ES contributing to the core enrichment in that pathway. A normalized enrichment score (NES) was generated based on the gene set enrichment scores for all dataset permutations. Pathways with a false discovery rate (FDR) <0.25 were considered significant.RNA sequencing was performed by the MGH Sequencing Core. Libraries were constructed from polyA‐selected RNA using a NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) and sequenced on Illumina HiSeq2500 instrument. The R package DESeq2 was used for differential gene expression analysis. Genes were considered differentially expressed if upregulated by log4.10SEM with all individual data points displayed. For comparisons of two groups, unpaired Student's t tests were performed. For comparisons of ≥3 groups, one‐way ANOVA followed by post hoc Tukey's multiple comparison testing was done. Pearson method was used for correlation studies in Figure p value <.05 was considered statistically significant.RNAseq and GSEA data analyses were performed with R and DESeq2 software, as described in Section None declared.JDR, NH, and AR designed the study. JDR, NH, AYu, HL, CL, AV, FD, DZ, RS, and MJH performed the in vivo experiments. JDR, NH, AYu, BC, AYe, HL, RH, VC, CL, AV, CP, FD, DZ, RS, MJH, and RTL assisted with tissue analyses and/or data interpretation. JDR and AR wrote the manuscript with contributions from all authors.Supplementary MaterialClick here for additional data file.Table S1‐S7Click here for additional data file.