Increasing CRF was inversely associated with incidence and mortality of respiratory/thoracic cancers, HRs 0.78 (95%CI:0.67–0.90) and 0.73 (95%CI:0.63–0.84) respectively; and all cancer-cause incidence 0.92 (95%CI:0.86–0.98) and mortality 0.85 (95%CI:0.79–0.91). Physical activity (PA) was not associated with most outcomes. We found no evidence of interactions between CRF or PA and BMI on cancer risk. This evidence suggests that midlife CRF is associated with lowered risk of cancer incidence and mortality with no evidence of cancer risk modification by BMI.Little knowledge exists about the role of cardiorespiratory fitness (CRF) or its interaction with excess adiposity determined by body mass index (BMI) in cancer prevention. A total of 5,128 middle-aged men, without a history of cancer at baseline in 1970–71, were examined for subsequent incidence and mortality of several cancer types. Participants’ data were linked with cancer registration and mortality data to March 2017. During 47 years of follow-up, a total of 1,920 incident cases and 1,638 cancer-related deaths were ascertained. BMI, particularly obesity, was associated with (i) incidence and (ii) mortality from respiratory/thoracic cancers; and (iii) all cancer-cause mortality. The respective adjusted hazard ratios (HRs) were: (i) 0.51 (95%CI:0.32–0.79), (ii) 0.48 (95%CI:0.30–0.75) and (iii) 0.73 (95%CI:0.59–0.89) when compared obese men (BMI ≥30 kg/m Higher incidence rates were reported in OECD nations whereas higher mortality was documented in developing countries1. Globally, the highest incidence rate amongst men and women was reported in Denmark, age-standardised 338 per 100.000 people in 20122, with 1 in 3 cancers being potentially preventable thought modification of lifestyle risk factors3.Cancer is a prominent cause of morbidity and mortality worldwide, accounting for 14 million new cases and 8 million deaths in 20122 or greater, may increase cancer risk resulting in higher incidence and death4. The increased prevalence of overweight and obesity has virtually spread to every country. In 2014, 69.2% of Danish males were either overweight or obese5; this rise in excess body mass is a major concern as there is sufficient evidence for an associated increased risk for 13 types of cancer, including esophageal adenocarcinoma, gastric cardia, colorectum, liver, gallbladder and pancreatic cancers6.Lifestyle modifications are promising strategies to reduce cancer risk. High body mass, defined as having a body mass index (BMI) of 25 kg/m7. This lack of association may be hampered due to imprecise measurement of this complex and multifaceted behaviour8, which is usually determined through self-report in most epidemiological studies9. Cardiorespiratory fitness (CRF), an objective attribute of repetitive training activities and of genetics, is measured by the maximal oxygen uptake (VO2 max) required by the body during sustained physical exertion10. Thus, CRF provides the most accurate population measure of regular fitness11. However, little is known about its impact on cancer prevention, as very few studies have examined this objective measurement of PA with the most common incident cancers in men14. Furthermore, the possible pre-diagnostic role of CRF in cancer specific mortality has not been fully explored15.Conversely, physical activity (PA) has consistently been linked to a decreased risk of colon cancer, is probably associated with reduced risks of postmenopausal breast and endometrial cancer; and less consistent with other types of cancer16, the interaction between these two determinants and cancer outcomes has not been well investigated using objective measures of PA10. Disease risk modification has been documented in several epidemiological studies of all-cause mortality and cardio-metabolic outcomes; in those studies, obese individuals with high levels of PA or fitness had lower cardio-metabolic risk or better survival compared to inactive or unfit individuals with a healthy BMI17. This phenomenon is also known as “fat but fit”18. Nevertheless, cancer risk modification still remains to be investigated.Although PA and high body mass are considered independent risk factors for some types of cancerThe current prospective study examined main and interactive effects of PA, fitness and BMI on the incidence and mortality of different cancer groups in the Copenhagen Male Study.Of 5,245 participants included in the examination, 117 were excluded due to a history of cancer other than non-melanocytic skin cancer prior to recruitment (n = 78) or did not perform the exercise test (n = 39). After exclusions, a total of 5,128 participants remained for analysis. There were 391 incident cases of prostate cancer (PC), colorectal cancer (CRC) (n = 299), oral/digestive (n = 546), respiratory/intrathoracic (n = 455), genito-urinary (n = 571), other cancers (n = 348) and all-cancers combined . Additionally, there were 253 deaths due to PC, CRC (n = 218), oral/digestive (n = 446), respiratory/intrathoracic (n = 482), genito-urinary (n = 380), other cancers (n = 322) and all-cancers combined (n = 1638). Mean follow-up was 29.7 years, ranging from 0.3 to 44.1 years. The mean age at recruitment was 48.8 years (age range 39.0 to 61.0 years).2) compared to the lowest (<25 kg/m2), were more likely to be older, of higher social class, to report more units of alcohol per day, to have higher mean systolic and diastolic blood pressure; and less likely to be current smokers. Compared to participants who reported almost no PA, those who reported a lot were more likely to be of higher social class, less likely to drink large quantities of alcohol and to have lower mean diastolic blood pressure. Participants who had high fitness levels compared to those with low fitness, were more likely to be younger, current smokers and to consume more grams of tobacco, less likely to drink large amounts of alcohol and to have much lower mean systolic and diastolic blood pressure.Baseline socio-demographic and lifestyle characteristics are shown for BMI, PA and CRF in Table 2 had a 49% and 52% decreased risk of being diagnosed and dying from respiratory/thoracic cancers respectively, when compared to participants with a BMI <25 kg/m2. Additionally, participants categorised as obese had a 27% risk reduction of all cancer-cause mortality compared to participants with a healthy range BMI. Sensitivity analyses excluding underweight participants from the healthy BMI category did not appreciably change any effects of relative risk of neither incidence nor mortality .BMI was associated with incidence and mortality of respiratory/thoracic cancers and all cancer-cause mortality Tables  and 3. P2 max; the respective HRs were 0.78 (95%CI: 0.67–0.90) and 0.92 (95%CI:0.86–0.98). Besides, a 10 ml/kg/min increase in VO2 max was associated with a 27% and 15% decreased risk of respiratory and thoracic cancer mortality; and all cancer-cause mortality. Sensitivity analysis excluding the first ten years of follow-up did not substantially change any effects of relative risks for the different study variables .PA was only related to genitourinary cancer incidence in sensitivity analysis; those participants who reported a lot of PA had a 25% decreased risk. The respective HR was 0.75 (95%CI: 0.57–0.99). Additionally, CRF was associated with reduced incidence and mortality of respiratory/thoracic cancers; and all cancers combined Tables . Lower r2 and a high CRF (36–78 ml/kg/min). A p-value below 0.1 may be an acceptable cut-off for interactions19. Overall, no significant interactions were evident between BMI and CRF or BMI and PA on cancer risk. Figures In this cohort, 2.1% or 35 individuals were identified to be “fat but fit”, based on a BMI ≥ 30 kg/m2 compared to participants with a healthy BMI (<25 kg/m2). Increasing levels of CRF also lowered cancer risk incidence and mortality of respiratory/thoracic cancers; and all-cancers combined. Self-reported PA was not associated with most outcomes assessed. Additionally, we detected no evidence of interaction between the effects of BMI and PA or CRF on cancer risk. Testing the “fat but fit” concept was difficult in this cohort because obesity was not associated with higher risks of cancer. Obesity is usually a marker of social inequality in developed countries and these disparities are known to influence the burden of cancer20. However, in this early epidemiological study, obese participants were from high socioeconomic status (SES), and thus they might have different health-risk behaviour profiles, which may explain the reduced risk associated with obesity20. Additionally, confounding by smoking (smokers were thinner) could be another possible reason for the reduced risk observed for obesity21.In this Danish cohort, BMI and CRF were independent predictors of cancer risk in men. Reduced risks of respiratory/thoracic cancer incidence and mortality; and all cancer-cause mortality were noticed for participants with BMI ≥30 kg/m6. Additionally, this group reported limited evidence for fatal PC and inadequate evidence for lung cancer (LC)6. A reduced risk of LC has been reported with obesity in a fairly recent systematic review and meta-analysis21. In our study, we also observed that obesity conferred lower risk of respiratory/thoracic cancers. Possible explanations for this obesity paradox include (i) confounding by smoking status since this behaviour influences body weight and body composition; and (ii) storage, mobilisation, and metabolism of carcinogen-DNA adducts by the adipose tissue21.The International Agency for Research on Cancer (IARC) working group on body mass reported that there is sufficient evidence for a positive association between obesity and some gastrointestinal cancers, with the highest risk for esophageal adenocarcinoma and the lowest for colorectum or gallbladder22. Most studies on cancer-related death have observed an increased risk from all-cancer combined or CRC in men with obesity compared to their healthy BMI counterparts23. The elevated risk for all-cancers combined has been documented around 10% and 32% for CRC22. In a recent meta-analysis of nearly 4 million participants from different world regions, a BMI of 25 kg/m2 or greater was associated with increased all-cancer cause mortality in a log-linear manner which did not differ across each region24. The increased risk of cancer death was 10% for overweight, 31% for obesity grade I (BMI ≥30 to <35 kg/m2), 57% for obesity grade II (BMI ≥35 to <40 kg/m2) and 96% for obesity grade III (BMI ≥40 to <60 kg/m2)24. Mortality relationships with PC or other types of cancer are less consistent with conflicting results26.Ambivalence still persists in the association of BMI with cancer mortality27. The World Cancer Research Fund (WCRF) and IARC have described the association between PA and LC as suggestive7. Nevertheless, a recent review of the literature suggests that the majority of studies support a role of PA in reducing LC incidence by 20–50% in men28; and proposes a weak inverse association of PA and PC with an average risk reduction of 10%29. Studies assessing CRF and cancer incidence have reported lower risks of LC or CC in participants with high levels of CRF compared to the lowest; the respective reduced risk were 55% and 44%14. However, the association with PC has produced inconsistent findings; an earlier analysis from an American cohort study reported a reduction of 74%30 while subsequent analysis observed an increased risk which ranges from 22% to 74%14. Our data suggest that increasing CRF levels lower cancer risk incidence of respiratory/thoracic cancers; and all-cancers combined.Considerable evidence exists for a protective role of PA in lowering the risk of some types of cancer, particularly with colon cancer (CC)7. We did not find evidence for a beneficial effect of PA or fitness on mortality for other clinical cancer groups. Genetics and habitual PA behaviour are considered the main determinants of CRF. Although the reported correlation between PA and CRF ranges from 60 to 70%31, this study suggests that CRF may be a better predictor of regular vigorous activity than subjective self-report measures on cancer outcomes. Additionally, PA captured by self-report was broad-ranging and the generic question used in this analysis may have introduced measurement error mis-classification, attenuating the observed association with cancer outcomes32.Pre-diagnostic CRF was associated with a decreased risk of respiratory/thoracic cancer mortality; and all cancer-cause mortality even after adjusting for BMI levels; this shows that the apparent protection is not explain by adiposity as was previously suggested for several types of cancer1. Therefore, the elucidation of the interaction between body mass and levels of PA or CRF on cancer outcomes is of public health interest because these independent lifestyle factors contribute independently to the burden of cancer1. Very few studies have examined this interaction on cancer incidence, providing contradictory results38. Four observational studies have focused on CC36; of them, only two case-control studies detected a significant interaction, reporting that high levels of PA offset risks among those with the highest BMI36. Two studies appraised this interaction on LC risk38; of which, a case-control study reported a significant interaction37. The authors noticed lower risk in healthy BMI or overweight individuals with high levels of activity but the same observation was not detected in obese individuals. Regarding prostate cancer, one prospective study noted a significant interaction where obese individuals with moderate or high levels of CRF offset obesity risks10. Case-control studies were more likely to report significant interactions than other epidemiological designs. To our knowledge, the interaction between BMI and PA on all cancer mortality has been assessed in one study, finding no significant interaction39. We found no evidence of interaction between BMI and levels of PA or CRF on cancer incidence or mortality. Despite yielding significant p-values for CRC and genito-urinary cancer mortality, stratified effect sizes were not significant. These spurious statistical interactions may have emerged from categorization of CRF and BMI in accordance with established values, producing unequal observations across different stratum or inappropriate median splits41.In 2011, cancer as a single entity was the leading cause of death globally and 20 million new cancer cases are projected by 20252 max. However, this measurement is known to have a high correlation of 0.87 compared to more direct methods of estimating VO2 max42. Finally, confidence intervals of two-way interactions were wide within strata, perhaps suggesting limited statistical power to detect such interactions19.This analysis has several strengths and limitations. Among its strengths are the prospective nature of the study design, the linkages of questionnaire data to deaths records and cancer registry; the long follow-up period, which was sufficient to allow the ascertainment of a large number of cancer end points and the objective assessment of physical fitness (CRF) which is not usually feasible in large studies. A limitation is that changes in health-related fitness could not be assessed since CRF was only collected at baseline. Additionally, CRF was estimated using an indirect method of VOIn conclusion, findings from this study underline the importance of improving and maintaining high CRF, which can be achieved through a minimum of 150 minutes a week of moderate to vigorous intensity activity, to reduce cancer risk, but this factor does not interact with obesity.43. 6,125 eligible men aged between 39 to 61 years were invited to participate; of them, 5,245 provided informed consent to participate in the study and underwent a medical examination, which consisted of a short interview by a physician based on prior completion of a standardized questionnaire, measurement of blood pressure, height, weight and CRF43. Recruitment was conducted between 1970 and 1971 and the estimated response rate was 86 per cent. In 1985–1986, 3,260 men completed a questionnaire to update exposures, lifestyle and disease diagnosis. For the purpose of this analysis, we used data collected at baseline (1970–71) and second wave (1985–86) from the CMS study and record linkage data from the Danish Cancer Registry and the Danish Register of Causes of Death. This analysis was approved by the steering committee of the Copenhagen Male Study and was conducted in accordance with relevant guidelines and regulations. The datasets generated during and/or analysed during the current study are not publicly available due to ethical reasons but are available from the corresponding authors on reasonable request.The Copenhagen Male Study (CMS) is a prospective cohort study of middle-aged men employed in large private or public workplaces in Copenhagen, Denmark. This study was established to assess the relationship between PA or CRF and coronary heart disease in relatively healthy men. Details of the study design, sampling method, data collection and examination have been published elsewherend of March 2017. This cancer registry is population-based and contains records for all incident malignant neoplasms in the Danish population from 1943 onwards. Although reporting to the cancer registry has been mandatory since 1987, the prior voluntary system ensured completeness and high quality data based on multiple reports from different sources, including hospitals, treatment, follow-up of cancer patients and death certificates44. Mortality data were obtained from the Danish Register of Causes of Death, which includes individual data on all deaths among Danish, Greenlanders and Faroese residents dying in Denmark, Greenland or the Faroe Islands45. Cancer incidence and mortality were coded to 3 digits using any of the International Classification of Diseases (ICD) 8–10; the following codes were used for PC 185/C61, CRC 153-154.1/C18-20, oral/digestive 140-159/C00-26, respiratory/intrathoracic 160-163/C30-39, genito-urinary 185-189/C60-68, other cancers 170-184,190-209/C40-58,C69-96; and all cancers combined except melanoma and other malignant neoplasms of skin 140-209/C00-96. Skin cancers were excluded as the association with PA might be confounded by ultraviolet (UV) sun exposure and increased risk of sunburn46.For the individual cancer types examined in this analysis, incident cancers were identified and dates of diagnoses obtained through linkage to data from the Danish Cancer Registry for all cancer registrations until the 2242.Questionnaires collected self-reported information on age, occupation, parental history of coronary heart disease, hypertension, diabetes mellitus, personal medical history and health behaviours, including: PA, daily alcohol intake and smoking habits. SES was derived from Svalastoga’s system which is based on educational attainment and job profile2). Height and weight were ascertained with the subject wearing light clothing and shoes; 2 centimetres were deducted from the height and 2 kilograms from weight43. Participants with extreme measures of BMI (<15 kg/m2 or >50 kg/m2) were excluded from the analysis to reduce the probability of measurement error47. All remaining participants were categorised into the recommended BMI categories by WHO; those with a BMI <25 kg/m2 , ≥25 kg/m2 to <30 kg/m2 (Overweight) and ≥30 kg/m2 (Obese) as time-varying exposures. Missing values of BMI were imputed using the method of Last Observation Carry Forward (LOCF)48 since baseline BMI and a re-measurement years later are highly correlated 0.9022. Underweight participants (BMI <18.5 kg/m2) were combined with the healthy group as this number was too small to influence observed associations (n = 27). The category corresponding to the lowest BMI was used as the reference group.Body mass index was calculated at baseline and second wave from measured weight and height, dividing weight in kilograms by the square of height in meters , some or a lot as time varying exposure. This question was developed by Finn Gyntelberg as there was no reliable or valid PA question at the time of inception or follow-up43. The lowest group of PA was used as the reference.Physical activity was assessed at baseline and second wave with a closed-ended question in the respective questionnaires as “How much physical activity do you believe you do” 2 max. This approach relies on heart rate, work load from a bicycle ergometer and the Åstrand nomogram43. Heart rate was measured during a submaximal bicycle work in a steady state with the aid of a stethoscope and a stopwatch. The loads used were 100, 150 and 200 watts. One, two or in a few cases three different loads were used. The chosen load for each case was determined from weight and age of the subject or heart rate during the first minute of the test. The examination was supervised by an experienced physician with the assistance of trained nurses43. The effect of CRF in statistical analysis was obtained per 10 ml/kg/min increase in VO2 max so as to obtain reasonable-sized risk estimates.Cardiorespiratory fitness was determined only at baseline, using an indirect method of VOPotential confounders for cancer incidence and mortality included: birth cohort obtained from date of birth , smoking status  and grams of tobacco a day , alcohol consumption , diabetes (yes or no), systolic and diastolic blood pressure, history of acute myocardial infarction (AMI) (yes or no) and SES . Most of the selected confounders were incorporated as time-varying risk factors with the exception of AMI and SES.Analyses were conducted separately for PC, CRC and cancer groupings. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated for cancer incidence and cause-specific mortality, using Cox regression with age as the underlying time scale. Time-varying risk factor analysis was conducted for BMI and self-reported PA and fixed baseline risk factor for CRF. For cancer incidence, participants were censored if they died, were diagnosed with other cancers or were alive at the end of follow up period, whichever came first. For cancer mortality, participants were censored if they died from other causes or were alive at the end of follow-up.49. Furthermore, the proportional hazard assumptions of the Cox regression models were assessed by Wald tests of covariates through log-time interactions. Sensitivity analyses were also performed by excluding: (i) underweight (BMI <18.5 kg/m2) participants from the lowest BMI category and (ii) the first ten years of follow-up to reduce the potential impact of reverse causality. Statistical analyses were performed using R version 3.4.0.We examined potential two-way interactions between (i) BMI and PA, and (ii) BMI and CRF, on cancer outcomes by adding appropriate interaction terms into the respective models. Based on the nature of the interaction, the multiplicative scale was used as presence of interaction on this scale will also be present on the additive scaleSupplementary material


Palaemonetes sinensis, was determined through Illumina sequencing, and the basic characteristics and gene arrangement were analyzed. The mitogenome of P. sinensis was 15955 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and one control region, with tightly packed. 33 of these genes were encoded on the heavy strand, and the remainders encoded on the light strand. The composition of P. sinensis mitogenome presented a strong A + T bias, which account for 66.7%. All PCGs were initiated by a canonical ATN codon, except nad5, which was initiated by GTG. The termination codons of the PCGs were TAA, TAG and T–. The secondary structures of 22 tRNAs of P. sinensis had the typical clover structure, except of trnS1 owing to the lack of dihydroxyuridine (DHU) arm. Gene order comparison of P. sinensis and previously-sequenced Palaemoninae revealed a unique translocation between trnT and trnP in Macrobrachium. The phylogenetic analyses showed that three Exopalaemon species formed a monophyletic group and then clustered with two Palaemon species and P. sinensis successively whereas Macrobrachium clustered with Palaemon capensis in the other clade.The mitogenome of Chinese grass shrimp,  Palaemonidae are divided into two subfamilies: Pontoniinae Kingsley, 1879  and Palaemoninae Rafinesque, 1815 . Despite of the numerical dominance of Pontoniinae, researchers have done more works on Palaemoninae due to its wide distribution, economic value and ecological importance. Nevertheless, the phylogenetic relationship within this subfamily is still disputed because the current classification system failed to describe their underlying evolutionary relationship4. For example, Pereira pointed out the paraphyly on generic level based on his cladistic analysis of morphological characteristics3. Murphy & Austin found species belonging to three different genera, Macrobrachium intermedium, Palaemon serenus, and Palaemonetes australis formed a monophyletic assemblage instead of with their congeneric species4. The topology given by Cuesta et al. showed that species from Palaemon and Palaemonetes clustered according to global geographical distribution and by genera with the exception of the Australian Palaemonid shrimps, which demonstrated the dichotomy between Palaemon and Palaemonetes genera  was phylogenetically questionable5. Ashelby et al. suggested the reevaluation of morphological traits to separate the genus of Palaemon, Palaemonetes, Exopalaemon and Coutierella because some species from those genera present monophyly6. However, most molecular studies on Palaemoninae are based on the analysis of partial sequences of 16S rRNA and fragment of the nuclear genes histone3 (H3)7. Certainly, analysis of other gene sequences (both from mitogenome and nuclear genome) are necessary to improve the understanding of phylogenetic relationship amongst Palaemonid shrimps.Palaemonidae, as the second most species-rich family in Caridean, including 134 genera and 934 extant speciesrrnL & rrnS), 22 transfer RNA (tRNA) genes, and one control region (CR)9. It has been widely accepted that mitogenome has rapid evolutionary rate and lack of genetic recombination8.Animal mitochondrial DNAs are typically circular molecules, approximately between 14 and 18 kb in length, normally containing 13 protein-coding genes (PCGs), two ribosomal RNA genes  were determined, whereas none of Palaemonetes has been reported.It had becoming increasingly popular to employ entire mitogenomes for phylogenetic relationship analysesPalaemonetes sinensis , is one of the important species of Palaemoninae, and widely distributed in China, Myanmar, Vietnam, Japan, southeastern Siberia and Sakhalin, with crucial ecological value and a certain degree of ornamental and economical value22. Except mitochondrial 16S rDNA and nuclear Histone (H3) gene sequences6, there was no report about mitogenome of P. sinensis. In this study, the complete mitogenome of P. sinensis was obtained through NGS. As the first mitogenome of Palaemonetes, it would contribute to a better understanding of phylogenetic relationship within Palaemoninae, particularly among Palaemonetes and other genera mentioned above. Additionally, it is of great importance for future study of genetic biodiversity of P. sinensis.Chinese grass shrimp, P. sinensis mitogenome was slightly smaller than that of Palaemon serenus , but it is in the range of the known Palaemoninae mitogenomes . Nucleotide BLAST (blastn) of the entire mitogenome between P. sinensis and closely related species presented high similarity .Approximate 5.2 million clean reads were obtained from raw sequences data and were clustered into 4,169,064 high quality reads. After further assembly, a circular mitogenome of 15,955 bp in length was finally generated  and higher than that of Palaemon and Exopalaemon , but lower than that of Macrobrachium (0.100 in M. lanchesteri to 0.157 in M. bullatum)  presented a strong A + T bias, which account for 66.7% of the bases, and showed a AT skew ([A − T]/[A + T] = 0.085) and negative GC skew ([G − C]/[G + C] = −0.275). The AT skew was similar with P. sinensis mitogenome, and was 11,166 bp in length totally. Among the 13 PCGs, nine genes  were coded on H strand, while the rest four genes  were on L strand. The 13 PCGs ranged in size from 159 to 1725bp . With the exception of P. sinensis, species of Macrobrachium showed slight smaller negative AT-skew (−0.138 to −0.150) and bigger negative GC-skew (−0.052 to −0.080)  were greater than 1, with higher frequency of usage. And this result was consistent with the result of E. carinicauda10.The average frequency of the protein-coding genes codon and was calculated and shown in Table P. sinensis mitogenome contained a set of 22 tRNAs genes . Furthermore, they showed a positive AT skew (0.062)  arm was replaced by a simple loop  and negative GC skew (−0.145) (Table The 5) Table .26, CR of P. sinensis mitogenome had different size. That was a common phenomenon, because it was generally believed that the length of control region has the largest variation of mitogenome16. P. sinensis had the highest composition of A nucleotides, and the lowest composition of G nucleotides, as well as the highest AT-skew value  and three species of Exopalaemon  with the gene order was 5′-nad4L- trnP- trnT -nad6-3′)26. However, a rearrangement of translocation between trnP and trnT (gene order: 5′-nad4L-trnT-trnP-nad6-3′) was identified in all four Macrobrachium , which was similar with the out group (Panulirus stimpsoni30 & Panulirus ornatus31) in this study.Among all known Palaemoninae sequences, gene order and orientation of the complete mitogenome of 34. Shen et al. identified nine different rearrangements in the comparison of 23 Pancrustacea mitogenome archived in GenBank, and found the same translocation between E. carinicauda and M. rosenbergii, which was identical to this study10. Wang et al. inferred that this invasion between trnP and trnT might be the unique mitochondrial character of genus of Exopalaemon23. However, from the results of present study, because the other three genera were all consistent with the same gene order pattern, Macrobrachium was supposed to be the unique genus due to its rearrangement  archived in GenBank so far. Phylogenetic analyses were based on the concatenated PCGs derived from 11 Palaemoninae mitogenomes belonging to four genera   Table . As a rem) Table . Apart f3. Ashelby et al. strongly supported that Palaemonetes, Exopalaemon, Coutierella, and certain Palaemon belonged to single monophyletic clade based on the analyses of mitochondrial 16S rDNA and nuclear Histone (H3) genes in Palaemoninae6. Therefore, Ashelby et al. suggested a further re-appraisal of morphological characters combined with further genetic work at generic-level were needed to establish a reliable classification in Palaemoninae6.The phylogenetic relationship within subfamily Palaemoninae Rafinesque, 1815, has been always debatable in their morphological cladistics study and molecular phylogeny. Pereira demonstrated the paraphyly in Palaemon, Palaemonetes, and Macrobrachium according to the analysis a matrix of 81 morphological characters in 172 specieset al.24 and Shen et al.10. And also genus Exopalaemon present monophyly with high support values. Interestingly, the only species which does not distribute in Asia-Australia in this study, P. capensis merged into Macrobrachium clade with comparatively low support value. Adult P. capensis inhabit in freshwater after a more saline planktonic larval phase35. Its life cycle reflects the evolutionary history of freshwater palaemonid shrimp. And in this study, P. sinensis, M. bullatum, P. capensis characterized by abbreviated larval development, which has been considered as a primitive trait took place early in the origin of the family Palaemonidae36. The Palaemon and Palaemonetes clade confirmed their morphological similarity demonstrated by merging of species of both genera37, while, Cuesta et al.5 and Botello & Alvarez38 suggested that Palaemon and Palaemonetes were more similar, and both different from Macrobrachium according to the analysis of mitochondrial 16S rDNA. However, taking into account the tiny proportion of archived mitogenome (11 species from 3 genera in 372 species from 26 genera), more mitogenome from more complete taxon are indispensable to reveal the phylogenetic relationship within Palaemoninae.In this study, apparent heterogeneity of Macrobrachium was proved by both topology and mitogenome gene order rearrange. This result supported previous study by Kim P. sinensis was determined in this study. This result can help us to understand the basic features and gene arrange of this species. As for PCGs, in the comparison with other known mitogenomes of Palaemoninae, P. sinensis characterized by highest composition of A and C nucleotides, as well as the lowest composition of T and G nucleotides. Additionally, P. sinensis has slight positive AT-skew value and the biggest negative GC-skew value, whereas the other species all have negative AT-skew values. As for control region, P. sinensis featured by highest composition of A nucleotides, and the lowest composition of G nucleotides, as well as the highest AT-skew value. Gene order comparison of P. sinensis and previously-sequenced Palaemoninae revealed a conservative order among genera of Palaemonetes, Palaemon and Exopalaemon, and a unique translocation between trnT and trnP in Macrobrachium. The phylogenetic analysis using Bayesian Inference (BI) and Maximum Likelihood (ML) based on concatenated set of nucleotide sequences of 13 PCGs indicated that Exopalaemon formed a monophyletic group and then clustered with two Palaemon species and P. sinensis successively whereas Macrobrachium formed a monophyletic group and then clustered with P. capensis in the other clade.The first complete mitogenome of genus of Palaemonates, P. sinensis were collected from Shenyang Longwei Lake, Liaoning, China . The whole body of one individual shrimp was immediately preserved in liquid nitrogen until DNA extraction. Total genomic DNA was extracted using the TIANamp Marine Animals DNA Kit , and the quality of extracted DNA was assessed by electrophoresis on a 1% agarose gel and Thermo Scientific NanoDrop 2000.The 39, SPAdesv3.9.040 and BLAST v2.2.31 (https://blast.ncbi.nlm.nih.gov/Blast.cgi), were performed by using software mummer v3.141 to determine the position relation of contig sequences. The complete mitogenome sequence was revised and confirmed by pilon v1.1842. All these procedures were performed by Shanghai Personal Biotechnology Co., Ltd., China.After random break by Covairs ultrasonic breaker, DNA was fragmented for constructed genomic DNA library using Whole Genome Shotgun (WGS) strategy, which was sequenced by Illumina Miseq instrument based on NGS technology. Colinear analysis for mitochondrial splicing sequences obtained by A5-miseq v2015052243 and MITOS29, and the precise location was identified by the mitogenome of the related species based on Palaemoninae sequences archived in GenBank. Identification of initiation and termination codons were carried out by using an alignment generated through ClustalX version 2.044, with other related species sequences as references, and verified by utilizing ORF finder and Blastn of NCBI. The location and secondary structure of tRNA genes were predicted and annotated using MITOS29 and tRNAscan-SE with default settings28. Nucleotide composition and the relative synonymous codon usage (RSCU) were determined using MEGA 745.The locations of putative protein-coding genes and rRNA genes were preliminarily predicted by software DOGMA46. Online mitochondrial visualization tool mtviz was utilized to drawn the graphical diagram of the complete mitogenome (http://pacosy.informatik.uni-leipzig.de/mtviz/mtviz). In the end, the complete mitochondrial DNA sequence was uploaded to GenBank database under the accession number MH880828.To describe base composition, AT skew = [A − T]/[A + T], GC skew = [G − C]/[G + C] were analyzed as described by Perna & Kocherhttps://www.ncbi.nlm.nih.gov/genbank/) for phylogenetic analysis within Palaemoninae. GenBank sequence information of eleven species was shown in Table Panulirus stimpsoni (GQ292768.1) and Panulirus ornatus (GQ223286.1) were employed as an out-group taxon from GenBank. Nucleotide sequences from 13 mitogenome PCGs were aligned using Clustal Omega . Moreover, Gblocks was utilized to remove poorly aligned region and divergent site47.Eleven others complete mitogenome sequences of subfamily Palaemoninae (ten species) were obtained from GenBank (49 through online server Phylemon 2 (http://phylemon.bioinfo.cipf.es/evolutionary.html) and MEGA 745 based on Akaike Information Criterion (AIC) value for Maximum Likelihood method (ML) and Bayesian Information Criterion (BIC) value for Bayesian inference (BI). Consequently, GTR + I + G was selected as the best-fit evolutionary model for ML analysis by both MEGA 745 and jModelTest49, whilst GTR + I + G and Tpm3uf + I + G were considered as the best model for BI analyses given by MEGA 745 and jModelTest49, respectively. Because Tpm3uf model was not implemented in Mrbayes v3.2.150, it was replaced by the closest over-parameterized model (GTR)52. As a result, GTR + I + G model was selected for further phylogenetic analysis.The optimal nucleotide substitution models were given by jModelTest (v2.0)45. The BI analysis was carried out as 4 simultaneous Markov chain Monte Carlo (MCMC) for 100,000 generations, sampled every 100 generations by using Mrbayes v3.2.150, the average standard deviation of split frequencies was less than 0.01. Both topology tree and the Bayesian posterior probilities (PP) was derived after the first 250 “burn-in” trees were excluded.Afterwards, ML analysis was performed on 1000 bootstrapped datasets by MEGA 7


The treatment lasted for 6 weeks. Sesame oil composition and plasma lipid profile were analyzed. Nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), phosphorylated eNOS, and inducible NOS (iNOS) protein expressions were determined in the left ventricle and aorta. Oxidative load, measured as conjugated diene (CD) and thiobarbituric acid reactive substance (TBARS) concentrations, was detected in the liver. Neither sesame oil nor cotreatment with simvastatin affected plasma lipid profile in Zucker fa/fa rats. Sesame oil and similarly cotreatment with simvastatin markedly increased NOS activity and phosphorylated eNOS protein expressions in the left ventricle and aorta of Zucker fa/fa rats. There were no changes in eNOS and iNOS protein expressions within the groups and tissues investigated. Hepatic CD concentration was higher in Zucker fa/fa comparing Zucker lean rats, and sesame oil treatment decreased it significantly. Interestingly, this decrease was not seen after cotreatment with simvastatin. In conclusion, phosphorylation of eNOS and decreased oxidative load may significantly contribute to increase in total NOS activity with potential beneficial properties. Interestingly, simvastatin did not affect NO generation already increased by sesame oil in obese Zucker rats.Current treatments for cardiovascular and obesity-associated diseases, such as statin therapy, may be associated with several side effects. Products from food sources with polyphenolic compounds may represent promising agents in the treatment of cardiovascular and metabolic diseases with minimal side effects. Thus, we aimed to study the effect of sesame oil and simvastatin treatment on plasma lipid profile, nitric oxide generation, and oxidative load in obese Zucker rats. 12-week-old male Zucker rats were divided into the control and sesame oil- (1.25 ml/kg/day) treated Zucker lean groups, the control and sesame oil (1.25 ml/kg/day), or simvastatin (15 mg/kg/day) together with sesame oil-treated Zucker fa/fa groups,  In the modern society, metabolic syndrome clustering obesity, hypertension, dyslipidemia, and hyperglycemia represent one of the major causes of atherosclerosis, heart failure, and stroke. The average prevalence of metabolic syndrome is 31% and is associated with a twofold increase in the risk of coronary heart disease and cerebrovascular disease and a 1.5-fold increase in the risk of all-cause mortality , 3. CurrThe cardioprotective effects of polyphenolic compounds have been linked mainly to its antioxidant properties; however, recent findings attribute its antiatherosclerotic potential to modulation of different signaling pathways . EmerginSesame, with several polyphenolic ligands and high oil content (50–60%) , becomesThus, the aim of our study was to evaluate the effect of the sesame oil and simvastatin treatment on plasma lipid profile and nitric oxide generation in the left ventricle and aorta, as well as on hepatic oxidative load in obese Zucker rats.Most of the chemicals and reagents were obtained from Sigma-Aldrich; when not, the company is indicated.All procedures and experimental protocols were performed in accordance with institutional guidelines and were approved by the State Veterinary and Food Administration of the Slovak Republic (Ro-1998/15-221) and by an ethical committee according to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Directive 2010/63/EU of the European Parliament. Male lean Zucker and obese Zucker fa−/fa− rats were obtained from Charles River, USA. All rats used in the study were born in an accredited breeding establishment. They were housed in groups of 3 animals, under a 12 h light-12 h dark cycle, at a constant humidity (45–65%) and temperature (20–22°C), with free access to standard laboratory rat chow and drinking water.12-week-old male Zucker rats were divided into the control Zucker lean group, the Zucker lean group treated with sesame oil in the dose of 1.25 ml/kg/day, the Zucker fa/fa control group, the Zucker fa/fa group treated with sesame oil in the dose of 1.25 ml/kg/day, and the Zucker fa/fa group treated with simvastatin (15 mg/kg/day) and sesame oil in the dose of 1.25 ml/kg/day. Each group consists of 6 animals. Treatment was administered via gavage from the 12th week of age for 6 weeks. Daily water consumption was estimated individually for every animal and adjusted, if necessary. All animals were housed at a temperature of 22–24°C and fed with a regular pellet diet ad libitum. Blood pressure was measured noninvasively, using tail-cuff plethysmography weekly. At the end of treatment, the animals were sacrificed, and body weight (BW), heart weight (HW), left kidney weight (LKW), and tibia length (TL) were determined. Relative heart and kidney weights were calculated as HW/tibia or LKW/tibia ratio. Samples of the left ventricle and aorta were used to determine NO synthase (NOS) activity and endothelial NOS (eNOS), phosphorylated eNOS, and inducible NOS (iNOS) protein expressions by Western blot analysis. Lipid profile was analyzed in the plasma and conjugated dienes and thiobarbituric acid reactive substances (TBARS) in the liver.The composition of sesame oil has been commercially determined, and plasma lipid profiles were detected by commercially available kits .Briefly, rat blood was collected from the aorta after anesthesia. The levels of triglyceride (TG), total cholesterol (TC) high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) were measured in the plasma using a biochemical enzyme kit  according to the protocol by 800TS Absorbance Microplate Reader .3H]-L-citrulline from [3H]-L-arginine  as previously described and slightly modified by Pechánová et al. [3H]-L-citrulline was measured with the Quanta Smart TriCarb Liquid Scintillation Analyzer .Total NOS activity was determined in crude homogenates of the left ventricle and aorta by measuring the formation of [á et al. . [3H]-L-Protein expressions of eNOS and iNOS were determined in the left ventricle and aorta by Western blot analysis. Protein expression of phosphorylated eNOS (peNOS) was determined in the left ventricle only. The samples were probed with polyclonal rabbit, anti-eNOS, anti-peNOS, anti-iNOS, and anti-GAPDH antibodies . The intensity of bands was visualized using the enhanced chemiluminescence system , quantified by using ChemiDoc™ Touch Imagine System  and normalized to GAPDH bands.2 atmosphere. After the addition of cyclohexane, the absorbance  was determined. The concentration of CD was calculated using the extinction coefficient ε = 29,000 l/mol/cm and expressed as μmol per g tissue.Samples of the liver were homogenized in 15 mmol/l EDTA containing 4% NaCl. Lipids were extracted using 1 : 1 chloroform-methanol mixture. The concentration of CD was estimated as described by Kogure et al. . Chlorofg for 10 min, 1 ml of the supernatant was added to 0.5 ml of 0.7% 2-thiobarbituric acid and incubated in a hot water bath for 10 min. After cooling, TBARS were measured at 532 nm . An extinction coefficient of 156,000 mol−1·l·cm−1 was used for the calculation of the results.To determine TBARS, 1 ml of 10% tissue homogenates of the liver  was added to 2 ml of 7.5% trichloroacetic acid and mixed. After centrifugation at 1000p < 0.05 (for both ANOVA and Duncan test).The results are expressed as mean ± SEM. One-way analysis of variance and Duncan test were used for statistical analysis. Values were considered significant with probability value Relative heart and kidney weights did not differ between Zucker lean and Zucker fa/fa-treated or Zucker fa/fa-untreated rats. There were no differences within the groups. Blood pressure did not differ between Zucker lean and Zucker fa/fa rats. Treatment with sesame oil (SO) or SO + simvastatin (SIM) did not affect blood pressure in Zucker rats (data not shown).The sesame oil contains mainly unsaturated fatty acids; 46.7% from which was linoleic acid and 37.6% oleic acid. Sesame oil had low amount of saturated fatty acid, specifically 1.8% of palmitic acid.The elements  were detected and their concentrations were determined. Calcium was present at the highest concentration 1.24%, potassium at the concentration of 0.49%, and phosphor 0.20%.From polyphenolic compounds, the sesame oil contains 4.73% of sesamin and 0.13% of sesamol.Plasma concentrations of total cholesterol, TG, HDL, and LDL were higher in Zucker fa/fa compared with Zucker lean rats. SO treatment increased TG concentration in Zucker lean rats. Neither SO nor SO + SIM affected plasma lipid profile in Zucker fa/fa rats .There were no changes in total NOS activity in Zucker lean and Zucker fa/fa rats. SO and SO + SIM cotreatment significantly increased NOS activity in the left ventricle  and aortThere were no changes in eNOS protein expressions within the groups and tissues investigated Figures . ExpressSimilarly as in eNOS, there were no changes in iNOS protein expressions within the groups and tissues investigated .Hepatic CD concentration was higher in Zucker fa/fa compared with Zucker lean rats, and SO treatment decreased it significantly. Interestingly, this decrease was not seen after SO + SIM treatment . There wOur study documented that neither sesame oil nor sesame oil and simvastatin cotreatment affected plasma lipid profile in Zucker fa/fa rats. Interestingly, sesame oil and similarly sesame oil and simvastatin cotreatment markedly increased NOS activity in the left ventricle and aorta of Zucker fa/fa rats. Phosphorylation of eNOS, decreased lipid peroxidation, and oxidative load may significantly contribute to increased NOS activity in these rats. To the best of our knowledge, this is the first study investigating the effects of sesame oil and simvastatin cotreatment on nitric oxide generation in obese Zucker rats.Zucker fa/fa rat represents a spontaneous genetic obesity model, which exhibits some components of metabolic syndrome including hyperlipidemia . HyperliIn Zucker fa/fa rats, total cholesterol was increased by 119% in comparison with the lean control. However, simvastatin was not able to decrease cholesterol level significantly. This fact might be explained by a relatively lower dose of simvastatin in our experiment, statin resistance, or insufficient absorption. However, in our parallel experiments (not published data), we were able to see increase of serum bile acids in the presence of simvastatin. Thus, beneficial simvastatin effect may be related to the accelerated transformation of cholesterol into bile acids, which plays important role in the improvement of physiological functions . Sesame 1179 or 1177 [Recent studies suggested that statins may increase NOS activity under different conditions , 26. Li  or 1177 –29. On t or 1177 . Althoug or 1177 .Unfortunately, there are only limited data concerning the effects of sesame oil on NOS activity. Hsu and Parthasarathy  found thHepatic CD concentration was higher in Zucker fa/fa compared with Zucker lean rats, and sesame oil treatment decreased it significantly. Interestingly, this decrease was not seen after sesame oil and simvastatin cotreatment. There were no significant changes in TBARS concentration within the groups; however, decreasing trend in TBARS concentration after SO treatment in Zucker fa/fa rats was seen. In agreement with our study, antioxidant effect of sesame oil and its polyphenolic compounds were documented in atherosclerotic and diabetic conditions , 34 or iIn conclusion, our study clearly documented that neither sesame oil nor an addition of simvastatin affected plasma lipid profile in Zucker fa/fa rats. Sesame oil and similarly sesame oil and simvastatin cotreatment increased NOS activity in the left ventricle and aorta with potential beneficial properties in the cardiovascular system. We suggest that phosphorylation of eNOS and decreased oxidative load may significantly contribute to increase in total NOS activity. Interestingly, simvastatin does not affect nitric oxide generation already increased by sesame oil in obese Zucker rats.Statins that lower LDL cholesterol and have additional pleiotropic effects are widely used in the treatment of cardiovascular and obesity-related diseases –37. Howe


A term male newborn born to a mother who had hereditary spherocytosis presented with neonatal jaundice at 20 hours of life. Complete blood count showed hemoglobin 17.1 g/dL, MCV 104.2 fL, MCH 32.9 pg, and MCHC 31.6 g/dL. The patient had indirect hyperbilirubinemia requiring phototherapy. The maximum total bilirubin level was 12.15 mg/dL at 20 hours of life. Peripheral blood smear revealed spherocytes, crenated red cells, and polychromasia. A flow cytometric test with eosin-5-maleimide- (EMA-) labeled RBC was performed in the patient and parents. The fluorescence histograms of EMA-labeled RBC from the patient and mother were shifted to the left, and the fluorescence ratio when compared with normal was 0.69 and 0.84, respectively. The flow cytometric test with EMA is useful in supporting the diagnosis of hereditary spherocytosis during newborn period. Hereditary spherocytosis (HS) is an inherited red blood cell (RBC) membrane disorder resulted from deficiencies of cytoskeletal membrane protein involving in the vertical connection between the phospholipid bilayer and structural proteins of the cell membrane . The cliHS is generally diagnosed by the significant presence of spherocytes in peripheral blood smear. The diagnosis can be confirmed by osmotic fragility test (OFT), autohemolysis test, acid glycerol lysis test, or pink test –5. Howevξ-NH2 group of Lys-430 on band 3 protein which is a predominant protein in the RBC membrane [Flow cytometric analysis with eosin-5-maleimide- (EMA-) labeled RBCs has shown a high sensitivity and specificity for diagnosing HS –14. The membrane . The defmembrane .A term male newborn with gestational age of 38 weeks was born by normal delivery. The birth weight was 2,350 g. His mother's blood type was O, Rh positive, and prenatal thalassemia screening test was negative. His mother and aunt has had mild hemolytic anemia and recently been diagnosed with HS. The patient was noted with jaundice at 20 hours of life. Physical examination showed jaundice without pallor and nonpalpable liver and spleen, while the rest of physical examination was unremarkable. He was investigated for the cause of neonatal hyperbilirubinemia. The peripheral blood smear showed spherocytes, crenated red cells, and polychromasia. The diagnosis of hereditary spherocytosis was suspected, and flow cytometry with EMA-labeled RBCs was performed to support the diagnosis.Complete blood count (CBC), peripheral blood smear, and flow cytometric analysis with EMA-labeled RBCs of the patient and his parents were performed. The results are shown in μL of whole blood in EDTA from each subject were washed twice with 1 mL of sterile phosphate buffered saline (PBS), pH 7.4, and centrifuged at 1,500 rounds per minute (rpm) for 5 minutes. Five μL of red blood cells were incubated with 25 μL EMA dye or PBS (as an unstained control) at room temperature in the dark for 1 hour. The cell suspension was centrifuged at 1,500 rpm for 1 minute. Then, the unbound dye was removed. The pellet with EMA-labeled RBCs was washed twice with 0.5 mL of sterile 0.5% bovine serum albumin (BSA) solution in PBS. Those EMA-labeled RBCs were suspended in 0.5 mL of 0.5% BSA in PBS. This cell suspension 100 μL was added to 1.4 mL of 0.5% BSA in PBS solution for flow cytometric analysis. Mean fluorescence intensity (MFI) as mean of geometric mean (X-GMean) was determined for 10,000 events in FL1 channel of Beckman Coulter CyAn™ ADP High-speed Analyzer .The flow cytometric analysis with EMA-labeled RBCs was performed as previously described . In brieRed blood cell parameters and flow cytometry results of the patient and parents are summarized in Indirect hyperbilirubinemia in newborns is caused by overproduction of bilirubin, impaired bilirubin uptake, or impaired bilirubin conjugation. Hemolysis is an important cause of increased bilirubin production resulting in neonatal indirect hyperbilirubinemia. The common causes of hemolysis in neonates include red blood cell membrane disorders, alpha-thalassemia disease, or G-6-PD deficiency. Impaired bilirubin conjugation could be found in G-6-PD deficiency or other inherited diseases, Gilbert syndrome, or Crigler–Najjar syndrome. HS was the most likely the cause of hyperbilirubinemia in our patient.The patient was treated phototherapy for indirect hyperbilirubinemia for 2 days. He also received folic acid supplementation. RBC transfusion was not required. After phototherapy, the total bilirubin was reduced from 12.15 mg/dL at hour 20 of life to 9.53 mg/dL at 32 hours of life. The CBC of the last visit at the age of 9 months showed Hb of 11.1 g/dL, MCV of 66.7 fL, and MCHC of 32.9 g/dL with 15% spherocytes. His growth parameters and developmental milestones were normal for his age.This report demonstrates the usefulness of flow cytometric analysis of EMA-labeled RBCs to diagnose HS in a newborn. The patient and his mother who had HS showed a decreased MFI and fluorescence ratio when compared with a normal control. The findings are consistent with the previous reports of HS in newborns , 16, 17.The 2011 guideline for diagnosis of HS recommends the flow cytometry with EMA-labeled RBCs for diagnosing HS . This teThe main limitation of the flow cytometric analysis with EMA-labeled RBCs is the need of fresh red cells. Alternatively, the red cells can be stored at 4°C for a maximum of 7 days –25. The The definite diagnosis of HS can be made by identification of the causative mutation. However, as the information of the involved genes and mutations causing HS remains limited, the current genetic identification technique is usually based on the whole-exome sequencing (WES) method which is costly and not widely available. Using flow cytometry with EMA-labeled RBCs provides a fast and practical diagnostic test which is helpful to identify HS as the cause of neonatal anemia and hyperbilirubinemia.The careful examination of RBC morphology of the newborn and parents is the most crucial step of investigations of RBC disorders in newborns. EMA is a fluorescent dye that binds to band 3 protein. The deficiencies of band 3 and other adjacent proteins, ankyrin and band 4.2 which cause hereditary spherocytosis, result in a decrease of fluorescence intensity of EMA binding. Thus, the flow cytometry with EMA-labeled red blood cells is a practical and useful test for diagnosis of HS in newborn. The test is easily operated, rapid, and requires small amount of blood which is ideal to use in newborns.


LEA gene (IpLEA) related to salt tolerance from Ipomoea pes-caprae L. (Convolvulaceae). Sequence analysis revealed that IpLEA belongs to the LEA_2 (PF03168) group. IpLEA was shown to have a cytoplasmic localization pattern. Quantitative reverse transcription PCR analysis showed that IpLEA was widely expressed in different organs of the I. pes-caprae plants, and the expression levels increased following salt, osmotic, oxidative, freezing, and abscisic acid treatments. Analysis of the 1,495 bp promoter of IpLEA identified distinct cis-acting regulatory elements involved in abiotic stress. Induction of IpLEA improved Escherichia coli growth performance compared with the control under abiotic stresses. To further assess the function of IpLEA in plants, transgenic Arabidopsis plants overexpressing IpLEA were generated. The IpLEA-overexpressing Arabidopsis seedlings and adult plants showed higher tolerance to salt and drought stress than the wild-type. The transgenic plants also showed higher oxidative stress tolerance than the wild-type Arabidopsis. Furthermore, the expression patterns of a series of stress-responsive genes were affected. The results indicate that IpLEA is involved in the plant response to salt and drought, probably by mediating water homeostasis or by acting as a reactive oxygen species scavenger, thereby influencing physiological processes under various abiotic stresses in microorganisms and plants.Late embryogenesis abundant (LEA) proteins belong to a large family that exists widely in plants and is mainly involved in desiccation processes during plant development or in the response to abiotic stresses. Here, we reported on an atypical  The exposure to these forms of stresses do negatively results into low crop yield and quality. Of these challenges, water deficit is a pervasive cellular issue confronting both aquatic and terrestrial plants2. There are several types of adversity that could result in cellular water deficit in plants, including freezing, drought, and high salinity2. In the evolutionary adaptation process of plants to these adversities, plant cells have developed a series of physiological and molecular mechanisms to reduce or relieve injures caused by water deficit, including structural changes and alterations to molecular synthesis and metabolism3. A variety of protective proteins, chaperones, reactive oxygen species (ROS)-detoxification proteins, and transcription factors (TFs) are supposed to mediate the protective responses against external stresses to maintain cellular metabolic and structural integrity, as well as to ensure that the plants can complete their growth and development cycle1. Late embryogenesis abundant proteins (LEA proteins), as a sort of protective protein, are ubiquitous hydrophilic proteins in plants and are mainly involved in protection to desiccation during seed dehydration, or vegetative tissues under stress conditions by acting as cellular dewatering protectants4.During their life cycle, plants are constantly exposed to various adversities, including abiotic and biotic stresses, which affect their development and growthLEA mRNA was identified from cotton (Gossypium hirsutum) seeds and accumulated during late embryogenesis5. Since this discovery, numerous LEA members have been extensively characterized over the past three decades and are not only limited to plants2. Many reports have indicated that the accumulation of LEA proteins or mRNA is closely associated with the desiccation tolerance of plant tissues, but their specific function remains unknown, particularly in extremophytes. In general, these types of proteins are composed of a high percentage of charged amino acid residues as well as glycine (Gly) or other small amino acids, such as alanine (Ala), serine (Ser), and threonine (Thr), but lack or only contain small amounts of tryptophan (Trp) and cysteine (Cys) residues6, lead to their classification as ‘hydrophilic’ due to their high hydrophilicity7. LEA proteins are predicted to be intrinsically disordered in the fully hydrated state and then may become folded in the dry state and acquire α-helical structures. The ordered LEA proteins under dehydration possibly bind to enzymes, membranes, DNA/RNA, water, or ions, and ROS, thereby stabilizing proteins and membranes under abiotic stress8, as well as providing aid in the formation and stability of an intracellular glassy state that is indispensable for the survival of plant propagules in the dry state9.The first plant LEAs have been documented in Arabidopsis thaliana10, 34 LEAs in rice11, 27 LEAs in tomato12, 72 LEAs in sweet orange13, 23 LEAs in pine14, 108 LEAs in canola15, 23 LEAs in bamboo16, 17 LEAs in Dendrobium officinale (Orchidaceae)17, 26 LEA members in cassava18, and over 100 LEA members in three gossypium ecotypes19. Plant LEA proteins can be grouped into eight families in the PFAM database according to the eight PFAM motifs 20. Different LEA members appear to have specific subcellular distributions, such as in the cytosol, mitochondria, chloroplasts, endoplasmic reticulum, and nucleus10. This diversity in LEA proteins in plants indicates that their physiological function and modes of action under abiotic stress may be universal. However, their individual roles have not been well-characterized, especially in the halophytes of Convolvulaceae.LEA genes are highly represented in plant genomes; for instance, 51 http://forge.info.univ-angers.fr/gh/Leadb/index.php)20, only a few LEAs have been functionally characterized, and thus the biological roles of most members of the LEA family still remain unclear. Many studies on LEA proteins have focused on the protein structure, subcellular localization, transcriptional regulation, and acting as molecular chaperone or protective protein8. Some reports demonstrated that the heterologous overexpression of LEAs could improve stress tolerance in transgenic plants, yeast, and bacteria. For example, Thellungiella salsuginea  is a valuable halophytic genetic model plant, and the ectopic expression of TsLEA1 from T. salsuginea confers salt-tolerance in yeast and Arabidopsis21. Salvia miltiorrhiza is a well-known traditional Chinese herbal plant with strong environmental adaptability, and the overexpression of SmLEA1 and SmLEA2 enhances salt and drought tolerance in Escherichia coli and S. miltiorrhiza22. Foxtail millet is a remarkably drought-resistant plant, and the expression of a novel atypical LEA gene SiLEA14 in foxtail millet and Arabidopsis demonstrably improved the salt and drought tolerance of transgenic plants23.Although over 1000 LEA sequences have been gathered in the dedicated LEA protein database , as a typical marginal-marine halophytic plant with a high level of nutrient utilization efficiency, is widely distributed on beaches or islands in tropical and sub-tropical regions and provides one of the best known examples of oceanic dispersal24. This plant has strong environmental adaptability and can be used as an ecological ‘green shelter’ in sand fixation, wind resistance, landscape greening, and ecological restoration in tropical and subtropical coral islands and coastal zones25. It is well-known that I. pes-caprae possesses remarkable salinity and drought resistance, and therefore can be considered as a good germplasm for the characterization of salt/drought-tolerance-related genetic resources. However, the molecular mechanisms underlying these tolerances are not well-defined.I. pes-caprae seedlings, including both the aerial parts and roots, using a pYES-DEST52 shuttle vector, and described a series of salt stress-related genes from this library26. Of these, a full-length cDNA encoding a LEA protein  was further characterized in the present study. The overexpression of IpLEA resulted in enhanced resistance to abiotic stresses in E. coli and Arabidopsis. The IpLEA promoter mediated the remarkable induction of β-glucuronidase (GUS) expression in transgenic Arabidopsis under various stresses. Cis-acting regulatory elements in the IpLEA promoter were also predicted, and promoter-driven GUS expression was detected in transgenic Arabidopsis. These data reveal the potential application of IpLEA in the genetic engineering of plants with elevated salt or drought tolerance.We previously generated a cDNA library from salt-treated I. pes-caprae seeds and plants were gathered from beaches in Zhuhai City , China. The seeds germination, seedling planting and stress or hormone treatment of I. pes-caprae was performed as previously described28. In brief, the seedlings with 4–6 true leaves were used for stress treatment assays to assess the expression patterns of IpLEA. Subsequently, salt (300 mM NaCl), simulated drought or dehydration (300 mM mannitol), oxidative , and cold (0 °C) stresses and abscisic acid (ABA) treatment (0.1 mM) were applied to the I. pes-caprae seedlings to detect the expression pattern of IpLEA.The Arabidopsis thaliana (ecotype Columbia) plants used for the ectopic overexpression experiments were grown on solid Murashige and Skoog (MS) plates for about 10 d before being transferred into nutrient solution-soaked vermiculite as potted medium. All of Arabidopsis plants, including germinating seeds and seedlings, were put in a controlled environment greenhouse at 22 °C, with a 16-h light/8-h darkness photoperiod, a light intensity of 120 mmol m−2 s−1, and 60–80% relative humidity. Three T3 homozygous transgenic lines were picked out and confirmed by PCR, and then were accessed for further phenotypic analysis as previously described28. To identify the expression patterns of the salt/drought stress response genes in Arabidopsis, transgenic lines and WT (3-week-old) seedlings planted in vermiculite were immersed in 200 mM NaCl or 300 mM mannitol solutions for 24 h, following which the leaves were harvested for RNA isolation.I. pes-caprae was constructed and screened with a FOX (Full‐length cDNA Over‐eXpressing) gene hunting system using a yeast salt sensitive mutant (AXT3) complementary assay approach26. Thereafter, a full-length cDNA (IpSR26) encoding a LEA protein that could rescue the phenotype of AXT3 was selected for further investigation.A full-length cDNA library from https://www.ncbi.nlm.nih.gov/orffinder/). Two LEA_2 motifs (PF03168) were identified through the online NCBI blastp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). MEGA 6 was used for the protein homology comparisons. The grand average of hydropathy (GRAVY) and other physical and chemical properties for IpLEA were predicted with the ExPaSy program (http://www.expasy.org/tools), and the 3D structural diagram of IpLEA was predicted by PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).The full-length LEA protein cDNA sequence (GenBank accession no.: MF680612) was translated by the online ORFfinder translate tool . The amino acid sequences used were as follows: InLEA from Ipomoea nil (NCBI accession no.: XP_019174801), SmLEA2 from S. miltiorrhiza (NCBI accession no.: ADX9850), and Arabidopsis LEA_2 member (At2g44060.1).The amino acid sequence of IpLEA was aligned with known plant LEAs by ClustalW software (IpLEA was obtained by PCR amplification with primer pair IpLEAF and IpLEAR (Table I. pes-caprae genomic DNA and PCR were performed according to our previous study27. The 5′ flanking region upstream of the translation start codon (promoter sequence) of IpLEA was amplified from I. pes-caprae genomic DNA through genome walking using a Genome Walking Kit  according to the manufacturer’s instructions. Three gene-specific primers  and sequenced. The putative cis-acting elements of this promoter region (IpLEA-PRO) were analyzed using the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)29.The genomic DNA sequence of AR Table . The isoIpLEA CDS was amplified with the primers IpLEAEPF and IpLEAEPR , yielding the recombinant plasmid IpLEA-pET 28a. The recombinant plasmid and pET 28a (as a negative control) were then transformed into E. coli Rossetta (DE3). A single colony was then inoculated in liquid Luria-Bertani (LB) medium (containing 50 mg mL−1 kanamycin) and allowed to grow overnight at 37 °C with constant shaking at 200 rpm. Inoculum (1%) from the overnight grown culture was added to fresh LB medium  and allowed to grow at 37 °C with constant shaking at 200 rpm. The isopropyl β-D-thiogalactopyranoside (IPTG)-induced expression of His-tag IpLEA was conducted and confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), as previously described27.The PR Table , and theE. coli, with three replicates for each sample, basically according to our previous reports28. To evaluate salt, osmotic, and H2O2 stresses, cell cultures of E. coli containing pET 28a/IpLEA-pET 28a were adjusted to OD600 = 1.0 and then serially diluted . Two microliters of each sample was spotted onto the LB plates containing 0.2 mM IPTG and the stress treatment . For the drought test, 10 μL OD-adjusted cell cultures in tubes were immediately placed in a 40 °C drying oven where they were maintained for 4 h. Then, the samples were added to 100 μL liquid LB medium and dissolved at 37 °C for 1 h to recover. The samples were then diluted and spotted onto LB plates with 0.2 mM IPTG. The plates were incubated at 37 °C for 12–16 h. The bacterial colonies were then counted , and the differences were analyzed.A spot assay was performed to test the stress tolerance of the recombinant 2O2 (0.7 mM or 0.9 mM), and incubated at 37 °C with shaking (200 rpm). The aliquots were removed from each treatment every 2 h for 12 h and the absorbance (OD600) was measured. Abiotic stress  tolerances were determined with respect to the control cultures .For the growth curve in liquid culture assay, 1 mL inoculum  was added to 10 mL LB medium (containing 0.2 mM IPTG) containing salt (3% or 4% NaCl), sorbitol (0.8 M or 1 M), or HIpLEA cDNA generated by PCR amplification  vector to generate the recombinant plasmid IpLEA-pUC/GFP. After sequencing confirmation, the fusion construct and control (empty vector) vectors were co-transfected with another NLS-mCherry vector separately into protoplasts. GFP fluorescence was visualized using a confocal laser scanning microscope .The CDS of the 28, with a purpose of accessing assessing the function of IpLEA. In brief, total RNA was isolated from the different tissues of I. pes-caprae with HiPure Plant RNA kits , and the cDNA was synthesized from the total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix  with Oligo(dT)15 primers according to the manufacturer’s instructions. The expression levels of IpLEA in various organs of the seedling and adult I. pes-caprae plants, including the seedling root, seedling leaf, bud, mature root, vine, mature leaf, flower bud, petal, and young seeds, at 7 d after pollination (DAP), were detected respectively. The I. pes-caprae seedling samples  treated with salt, simulated drought or dehydration, oxidative stress and frost treatment (0 °C), and ABA were also assessed to examine the expression changes of IpLEA. All of the gene expression data obtained via qRT-PCR were normalized to the expression of IpUBQ (GenBank accession number: MF502417). The primers used for qRT-PCR are listed in Table qRT-PCR was performed basically according to our previously reportsCAT1, CSD1, APX1, and ERD5) and abiotic stress-related genes  in Arabidopsis (WT or transgenic plants), the total RNA was isolated from rosette leaves at different time points (with or without treatments), and cDNA synthesis was performed using the above procedure. The reference gene for the qRT-PCR was ACT2 (At3g18780) in Arabidopsis. The primers used for qRT-PCR are listed in Table For detecting the expression of antioxidation system-related genes  and confirmed by genomic PCR with the primer pairs IpLEAOXF/IpLEAOXR and IpLEAProF/IpLEAProR. Finally, T3 homozygous transgenic and WT seeds were germinated and used in the plant tolerance assays, or the T3 homozygous transgenic seedlings were used in the GUS staining assay.After sequencing confirmation, the constructs were transferred into IpLEA promoter driven by GUS) to GUS staining according to Jefferson et al.30. T3 seedlings of the transgenic Arabidopsis plants were treated with salt (200 mM NaCl) or osmotic stress (300 mM mannitol) for 24 h, with the untreated transgenic Arabidopsis plants as controls. The GUS-positive plant tissues were examined using a light microscope at low magnification and photographed. To further identify the characteristics of the IpLEA promoter, GUS expression in the transgenic Arabidopsis plants driven by the upstream 1,495 bp of ATG was also evaluated using qRT-PCR. The primer pairs for GUS and AtAct2 are listed in Table GUS expression analysis represent the typical results of at least three independent transgenic lines.We subjected the T3 seedlings of the transgenic Arabidopsis plants  was detected under NaCl  and mannitol  stress to detect the effect of the overexpression of IpLEA on improving the salt/osmotic tolerance of transgenic Arabidopsis seeds during germination. Additionally, root length was also calculated to evaluate the influence of the overexpression of IpLEA on transgenic Arabidopsis seedlings under abiotic stress . WT Arabidopsis and MS medium were used as control. The seed germination and seedling growth experiments were both performed on MS plates with or without stress factors, in the same greenhouse environment as Arabidopsis plants growing.The seed germination rate of the 28. Both WT and transgenic seeds  were grown on MS medium. Ten-day-old seedlings were planted in sieve-like square pots filled with nutrient solution soaked vermiculite. Thirty plants of each genotype were cultured in greenhouse as described above without watering for another 20 d to make sure seedlings growing up, and the water content of vermiculite in planting pots reduced but has not caused plants drought stress. The plants were then subjected to the following assays. For the drought tolerance assays, WT and transgenic plants  were maintained under continuous drought conditions for 9 d and then re-watered for 7 d. For the salt tolerance assays, plants of each genotype  were planted in sieve-like pots and well-watered as described for the drought tolerance treatment. Water was withheld for 20 d prior to irrigation with NaCl solution (150 mM and 200 mM) from the bottom of the plants. When the vermiculite was completely saturated with salt water, the NaCl solution was removed from the bottom the planting tray and the plants were cultured without watering. The plants grew in the salt-saturated vermiculite for another 23 d and were took pictures.Salt and drought tolerance assays were also performed on transgenic Arabidopsis adult plants according to our previous studyIpLEA OXs  and WT Arabidopsis were also assessed under H2O2 or MV challenges in similar method with the salt/osmotic tolerance of Arabidopsis plants. In brief, for the seed germination rate analysis on MS plates, the concentrations of H2O2 were 0 (as a control), 3.5 mM, 4 mM, and 4.5 mM respectively; for the seedling root length on MS plates, the concentrations of H2O2 were 0 (as a control), 2.5 mM, 3 mM, 3.5 mM, and 4 mM respectively. In addition, the 30-day-old seedlings of IpLEA OXs  and WT Arabidopsis adult plants grown in vermiculite were sprayed evenly with 20 μM methyl viologen (MV) , after which the plants were cultured with necessary watering for 18 d. The phenotype was recorded.For the oxidative stress analyses of the transgenic overexpression lines and WT plants, the seed germinnation, seedling growth, and adult plant of #1, #9, #11 and WT) growing in the soil, following which the leafstalks were immersed in 200 mM NaCl or 300 mM mannitol solution for 24 h. In situ detection of H2O2 and O2− was determined using vacuum-infiltrating with 1 mg/mL nitro-blue tetrazolium (NBT) or 1 mg/mL 3.3′-diaminobenzidine (DAB) solution, respectively, for 12 h, followed by clearing in 75% ethanol28.Rosette leaves were collected from 3-week-old seedlings .All of the experiments were repeated three times in independent experiments, and the data shown are the mean ± standard deviation (SD). In this research, the data were subjected to Student’s IpLEA cDNA was determined from a cDNA library of I. pes-caprae by FOX-hunting technique26. The cDNA sequence of IpLEA encodes a polypeptide of 313 amino acid residues with a 61 bp 5′ untranslated region (UTR) and a 390 bp 3′ UTR (including a polyA tail). The physical and chemical properties of IpLEA protein were summarized in Table 6 and IDP8, the GRAVY value and the instability index of IpLEA is −0.373 and 29.09 respectively . It is rich in hydrophilic amino acids, such as aspartic acid (Asp) , glutamic acid (Glu) , histidine (His) , lysine (Lys) , Ser , and Thr , but contains low quantities of hydrophobic amino acids, such as methionine (Met) , phenylalanine (Phe) , Pro , and Trp , and lacks Cys (C) and glutamine (Gln) (Q). A blastp search of the IpLEA protein in NCBI revealed that it contains two “LEA_2” motifs (PF03186)  in its coding region. Based on the genomic region of the IpLEA sequence and the Genome Walking Kit manual, a 1,495 bp promoter region sequence was obtained for IpLEA. The possible cis-elements were predicted by the online program PlantCARE, which indicated that a number of potential cis-acting elements that might be involved in binding some specific TFs were present , a binding site for myeloblastosis (MYB) transcription factors , and a TC-rich repeats (−1428 bp) that were identified in the IpLEA promoter. The ABRE, MBS, and TC-rich repeats elements are supposed to bind specific TFs to regulate gene expression under some distinctive environmental factors or developmental signals. The ethylene-responsive element (ERE) and two endosperm activating expression cis-elements (Skn-1 motif or GCN4_motif) were also found at −1,110 bp, −689 bp, and −1,200 bp  than other stress- or hormone- regulated elements, which might further implied the mRNA of IpLEA accumulating during the late embryogenesis of I. pes-caprae seed development. And also, the light regulated cis-elements can be found in the promoter region with high frequency , at least, the presence of these cis-elements in the IpLEA promoter region indicated that the transcription of IpLEA might be regulated dynamically by binding the TFs, and that IpLEA was probably involved in some abiotic stress responses by transcriptional regulation.The ent Fig. , and theABRE, −84 bp, a biE. coli by IPTG to further confirm that the accumulation of IpLEA could improve abiotic stress tolerance in the bacterium. As shown in Fig. E. coli cells.The recombinant IpLEA protein was induced in E. coli cells in liquid LB medium were spotted on LB plates with stress factors . When assayed with the liquid culture assay, the growth of E. coli cells expressing His-tag IpLEA increased rapidly with time compared to the E. coli cells expressing the empty vector (pET 28a) control , which showed that IpLEA has a relatively higher likelihood for distribution in the cytoplasm . Furthermore, the Plant-PLoc program (http://www.csbio.sjtu.edu.cn/bioinf/plant/) also indicated that IpLEA was mainly located in the cytoplasm.The subcellular localization of IpLEA was determined by an GFP Fig. . Our resIpLEA in I. pes-caprae, qRT-PCR was performed with total RNA extracted from various I. pes-caprae plant tissues. Our results revealed that IpLEA was expressed widely in most I. pes-caprae tissues  stress also showed obviously induced  expression and was constitutively lowly expressed in transgenic Arabidopsis.To test the activity of the promoter region, GUS Fig.  was tranGUS Fig. . Blue prGUS Fig. . The staIpLEA promoter, we also evaluated the GUS gene expression pattern in transgenic Arabidopsis seedlings by qRT-PCR. As shown in Fig. GUS gene exhibited an induced expression pattern, which indicated that even in Arabidopsis, the cloned IpLEA promoter region appeared to be a water-deficit/ABA-induced promoter , and MS medium containing NaCl  or mannitol  to evaluate their stress tolerance. On the MS plates, the germination rate and seedling growth of the WT and transgenic lines showed no differences. However, in the presence of NaCl and mannitol, the seeds and seedlings (7 or 21 d after germination) from the transgenic line exhibited significantly higher germination rates  and mannitol , following which the root length was measured after cultivation under these treatment conditions for 7 d. When growing on control MS plates, no significant differences in root length appeared both in the WT and in transgenic lines plants , the WT and IpLEA OX line seedlings appeared to be growing well and in similar condition. Following this, the salt/drought tolerance assay began. We selected 150 and 200 mM NaCl treatments for the salinity stress experiment. Under normal conditions without salt challenges, no growth differences both in the transgenic lines  and in the WT were observed. After the induction of salinity stress, almost all of the WT plants showed severe reduction in growth, and even death after 23 days’ high salinity stress, while the three transgenic lines remained alive , the three IpLEA OXs always exhibited a better seed germination status than the WT , and our results indicated that the overexpression of IpLEA in Arabidopsis improved the root growth of the seedlings under the oxidative stress caused by exogenous H2O2  or osmotic stress (300 mM mannitol). Compared to the WT, these three IpLEAOX lines showed significantly lighter NBT staining and less O2− accumulation under salt and osmotic stress conditions  and a Pro biosynthesis gene (ERD5) were dramatically upregulated by salt/osmotic stress challenges  indicated that the expression of these genes in the transgenic plants was significantly higher than in the WT under oxidative stress  from I. pes-caprae and verified its function. LEA genes have been shown to play pivotal roles in the abiotic stress responses of many plant species8. Although IpLEA showed some degree of hydrophilicity and IDP  characteristics8, the GRAVY of IpLEA is relatively low (–0.373), which indicated that the hydrophilicity of IpLEA is not as high as other IpLEAs 28. Accordingly, the 3D prediction by PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) also indicated that IpLEA contains several α-helixes and β-sheets  contained several potential cis-acting elements that respond to environmental stress, such as ABRE, MBS, and TC-rich repeats , also showed high salt/osmotic and ABA inducible features, which provides further evidence that the detailed salt/drought tolerant function of SiLEA1423. Based on our combined results regarding the promoter region of IpLEA, we can presume that the ABRE cis-regulatory motif binds ABA signal transcription factors and participates in the transcriptional regulation of IpLEA in response to ABA and osmotic stress signal transduction, as it does in other plants45. The MBS sites can bind MYB TFs and are suggested responding to drought stress and ABA signaling46, while TC-rich repeats cis-acting elements are involved in defense and stress responsiveness47. Our results provide insight into the molecular mechanism of the function of IpLEA in the extreme halophyte I. pes-caprae.Numerous studies have focused on the regulatory mechanisms of the promoter for specific functional genes, rather than simply the protein sequenceE. coli is an excellent experimental system for protein functional verification and has long been used as a powerful tool for exploring genetic function in multicellular organisms, especially for plant genes associated with osmotic stress48 or cold tolerance49. In the present study, after the induction of His-tag IpLEA protein in E. coli, we assessed the influence of IpLEA enhancing the stress tolerance of the transgenic E.coli strain Rossetta (DE3). Our research demonstrated that the His-tag IpLEA protein was rapidly and greatly induced in E. coli , binding membranes to maintain structural integrity (II), and safeguarding active enzymes by molecular shielding, as well as refolding misfolded proteins by acting as a disordered chaperone (III)8. In the present report, we focused on the anti-oxidative capacity of IpLEA and related it to the elevated tolerance to H2O2 in yeast26 and E. coli cells (Fig. IpLEA were generated in order to test the contribution of IpLEA to salt and drought tolerance (Figs. IpLEA overexpression transgenic plants. In addition, the IpLEA transgenic lines showed stronger ability to resist oxidative stresses than WT in seeds germination, seedling growth, and adult plants (Fig. 2O2/O2− distribution (Fig. 54. Our results demonstrated that IpLEA could effectively prevent or minimize cellular oxidative damage, thus enhancing the salinity and drought tolerance of the plant. We also evaluated the gene expression of some anti-oxidative genes and ABA/stress-related genes, and the results indicated that the overexpression of IpLEA was responsible for the higher expression of these genes and subsequent stress tolerance (Fig. Due to the diverse roles of LEA proteins in the response to abiotic stress, the cellular functional mechanisms of plant LEAs have been generalized into three patterns, namely, sequestering by binding Hlls Fig. . Transgece Figs. . The prece Figs. , therebynts Fig. . Changesion Fig.  in the snce Fig. . Here, wIpLEA, from I. pes-caprae. Our current data, together with available over-expression assays in microorganisms and Arabidopsis, support a functional role for IpLEA in salt and drought stress tolerance. Our work shows that IpLEA exhibits inducible expression patterns in response to high salt, osmotic stress, ABA, cold, and oxidative stress, which indicates that the promoter of IpLEA is abiotic stress-responsive. IpLEA is localized in the cytosol, and the accumulation of IpLEA in cells elevated ROS scavenging capability and improved the transcriptional level of stress-related genes. Our findings suggest that this protein has a protective function in water-deficit and ROS-detoxification due to its water-holding capacity and ROS scavenging capacity, thereby affecting the expression of genes involved in salt/drought stress. Based on the results in this study, we speculated that the pleiotropic effects caused by the cellular accumulation of IpLEA could lead to enhanced cellular water balance and improved ROS homeostasis, resulting in improved cell viability and plant growth under drought and salinity stress. The results indicate that this IpLEA protein has a significant role in improving the tolerance and survival ability of plants or organisms under abiotic stress and could constitute a promising candidate for salt/drought tolerance transgenic plant research.In conclusion, we identified a novel atypical LEA_2 subfamily gene, Supplementary Information