Parasutterella and reduced abundance of genus Corynebacterium, genus Odoribacter, and genus Acinetobacter. Increased Parasutterella abundance was positively correlated with serum testosterone level. A PICRUSt analysis revealed that reproductive and metabolic-related genes were enriched in rats of L/D group. In conclusion, the present study demonstrates that continuous light exposure, an important environmental factor, contributes to the occurrence and developmental progress of PCOS and changes in microbial component and structure. Continuous light exposure is one of vital causes of PCOS, which is closely related to microbial structure and functions.The interplay between genetic and environmental risk factors contributes to the pathogenesis of metabolic disease. Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in women of reproductive age. Circadian rhythm disruption is an important risk factor for PCOS. In this study, we evaluated the effect of circadian disorder on reproduction as well as metabolism, and determined its influence on gut microbiota in a rat model. Female Sprague Dawley (SD) rats were kept under continuous light exposure  or a control cycle  for four consecutive weeks. Manifestations in endocrine hormones and metabolism were detected and gut microbiota were analyzed with the 16s rRNA gene sequencing technique. To our knowledge, this is the first study to report PCOS-like reproductive manifestation, such as anti-Müllerian hormone (AMH) elevation induced by continuous light exposure. Moreover, continuous light resulted in abnormal glucose metabolism and gut microbial community variations, including enrichment of the microbial genus of  Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, with an estimated prevalence of approximately 10 to 15% worldwide . AccordiAmong the environmental factors that may contribute to PCOS, circadian rhythm disruption has recently received significant interest . In caseBacteroides, Escherichia/Shigella and Streptococcus, were positively correlated with clinical reproductive parameters, such as testosterone and BMI  rats and examined their reproductive and metabolic manifestations. Additionally, to determine the role of gut microbiota, we collected the fecal samples of SD rats and employed high-throughput sequencing of the bacterial 16S rRNA genes. The changes in reproduction, metabolism, and gut microbial constitution under influence of continuous light were evaluated. The results of this study provide new insights into the pathologic mechanism of PCOS occurrence and offer basic research evidence for clinical diagnosis and treatment in reproductive women suffering from circadian rhythm disturbance.N = 11/group). The control group was under a periodic cycle  while the light group was exposed to continuous light  for four consecutive weeks. All rats were housed in a temperature-controlled room (22–24°C) under specific pathogen-free conditions. Rats were allowed to adapt to the new environment for 1 week before our experiments with access to standard chow and demineralized water ad libitum.Female SD rats (200–220 g) of 6 weeks were purchased from the China National Laboratory Animal Resource Center  and randomly divided into two groups: control group and light group (Body weight (BW) was monitored once a week throughout the experiment and estrous cycles were monitored daily by exfoliative cytoscopy during the last 6 days. An intraperitoneal glucose tolerance test (IPGTT) was performed at 9 a.m the day before the rats were sacrificed. At the termination of the experiment, all rats were deeply anesthetized to sacrificed, serum, bilateral ovaries, parametrial adipose tissue, muscle, and liver were collected. The L/D group and L/L group were sacrificed at the diestrus stage of the estrous cycle.All experimental protocols were performed in accordance with institutional guidelines. The study was approved by the research committee of the Shanghai Jiao Tong University School of Medicine (number: 2016-1220).D-glucose normal saline solution  at 2 g/kg body weight. Blood samples for glucose measurements were obtained from the tail vein at the indicated times with an Accu-Chek glucose monitor .All SD rats were fasted overnight for 16 h (5 p.m. to 9 a.m.) followed by i.p. injection of 50% Trunk circulation blood samples were obtained and allowed to incubate for 4 h at room temperature. All samples were centrifuged  and serum was collected into 1.5 ml EP tubes and kept at −80°C for subsequent experiments.Total serum testosterone and estradiol were measured using ELISA kits from Cayman Chemical . Serum corticosterone and leptin were measured with ELISA kits from R&D Systems, Inc. . The levels of anti-Müllerian hormone (AMH), follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), progesterone, and insulin were measured using ELISA kits from CUSABIO Technology LLC (United States), Aviva Systems Biology, Corp. (United States), Enzo Life Sciences (United States), Uscn Life Science Inc. (Belgium), TSZ biological Trade Co., Ltd. (United States), and MyBioSource, Inc. , respectively. The sensitivity of ELISA kits of insulin, LH, FSH, estradiol, testosterone, corticosterone, leptin, progesterone, TSH, and AMH were lower than 0.312 μIU/mL, 5.2 mIU/mL, 0.156 mIU/mL, 15 pg/mL, 6 pg/mL, 0.028 ng/mL, 22 pg/mL, 15 pmol/L, 0.3 μIU/mL, and 0.1 ng/mL, respectively. The coefficient of variation of the kits mentioned above were 6.7%, 3.9%, 5.9%, 11.9%, 6.0%, 4.5%, 3.3%, 10%, 15%, and 15%, respectively. All ELISAs were used to detect indexes in rats and performed according to the manufacturer’s instructions.The homeostasis model assessment of insulin resistance (HOMA-IR) and homeostasis assessment of β-cell function (HOMA-β) were calculated for each rat using the following equations :The stage of the estrus cycle was determined by the predominant cell type in vaginal smears that were obtained from 9 weeks of age until the end of the experiment .Rat ovarian tissues were fixed with 4% paraformaldehyde, and then embedded in paraffin. Serial sections of 5 μm-thick ovarian tissue sections were deparaffinized and rehydrated through a graded ethanol series. Then the sections were stained in hematoxylin and differentiated by hydrochloric acid. Finally, the sections were incubated in eosin before covering the slides or visualization using a microscope . Total numbers of small antral follicles , large antral follicles (containing a single large antrum), atretic cyst-like follicles  and corpora lutea (CL) were classified and quantified as previously reported . To avoiParametrial fat pads were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 μm. Sections were stained with hematoxylin and eosin before images were taken for histomorphometry at 20 × magnification under a light microscope. Five distinct images were taken from each of three sections of the fat pad, with at least 200 μm separating these sections.-actin was amplified in parallel as an internal loading control. The primer sequences of targeting genes used were described in -actin was obtained as the target mRNA levels.Liver, parametrial adipose tissue, and muscle samples were rapidly dissected, snap-frozen in liquid nitrogen, and stored at −80°C for RNA extraction. Total RNA was extracted from these specimens using a total RNA kit following the manufacturer’s instructions . RNA concentration and quality were determined by measuring optical density at 260 nm (OD260) and the ratio of OD260/OD280 with NanoDrop ND-2000, and electrophoresis on a denaturing agarose gel. The mRNA from the total cellular RNA was reverse-transcribed to cDNA using PrimeScript RT Master Mix Perfect Real Time kit . 500 ng of RNA were used in reverse transcription and qRT-PCR was measured using the following settings: 2 min pre-incubation at 94°C followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 61°C for 20 s, elongation at 72°C for 20 s. Fluorescence was measured at the end of the elongation phase using 470 nm excitation and 530 nm emission (channel 1). Correct PCR products were confirmed by melting curve analysis. The absolute mRNA in each sample was calculated according to a standard curve setup using serial dilutions of known amounts of specific templates against corresponding cycle threshold (Ct) values . The houFive female rats were selected randomly from L/D group and L/L group, respectively. Fresh fecal samples were taken from these rats once a week throughout the experiment, collected into 1.5 ml sterile EP tubes, rapidly snap-frozen in liquid nitrogen, and stored at −80°C until further analysis.2O.Microbial DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit , according to manufacturer’s protocols. The V3-V4 region of the bacteria 16S ribosomal RNA genes was amplified by PCR  using primers 341F 5′-CCTACGGGRSGCAGCAG)-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′. PCR reactions were performed in 30 μL mixture containing 15 μL 2 × KAPA Library Amplification ReadyMix, 1 μL of each primer (10 μM), 50 ng of template DNA, and ddH® 2.0 . After preparation of library, these tags were sequenced on the MiSeq platform  for paired end reads of 250 base pairs (bp), which were overlapped on their three ends for concatenation into original longer tags. DNA extraction, library construction, and sequencing were conducted at the Realbio Genomics Institute .Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit , according to the manufacturer’s instructions, and were quantified using Qubit1 and chimeric sequences were identified and removed using Usearch (version 7.0). Each representative tag was assigned to a taxa by RDP Classifer2 against the RDP database3 using a confidence threshold of 0.8 OTU profiling table. To evaluate the community diversity and community richness, the Chao1 value was calculated using Mothur . Principal components analysis (PCoA) of the OTUs in different groups was conducted using R package  to compare the constitution of gut microbiota. LEfSe analysis coupled with the Kruskal-Wallis rank sum test was performed to identify the microbial differences among all groups. The linear discriminant analysis (LDA) score for each bacterium was calculated and a taxonomic cladogram was constructed to visualize the differences in microbiol composition. Predictive function analysis was performed using the PICRUSt algorithm based on the Kyoto Encyclopedia of Genes and Genomes Orthology (KO) classification  and no more than three ambiguous N . The copfication . Sequenct-test or one-way analysis of variance test followed by the Newman–Keuls multiple comparison test, where appropriate, to assess significant differences. In LEfSe analysis, a significant value of <0.05 and LDA effect size of >2 were used as thresholds. Significance was shown as ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001.All data are represented as mean ± standard error of the man (SEM). After examination for normal distribution, data were analyzed using a paired Student’s P > 0.05). The L/L group of rats had prolonged estrous cycles (P < 0.05). Histologically, the number of CL present was notably diminished in L/L group, demonstrating oligo/anovulation (P < 0.001). The number of small antral follicles as well as large ones significantly decreased . Additionally, as a previous study reported (P < 0.001). Similarly, oligo/anovulation is a key reproductive characteristic of PCOS.After experimental treatment for 4 weeks, there was no difference in BW or ovarian weight between the L/L and L/D groups , while the difference in testosterone abundance between the two groups was not significant (P > 0.05). There were no differences in concentrations of LH, FSH, estradiol, progesterone, or TSH between the two groups (P > 0.05). Moreover, to investigate the effect of stress on endocrine system induced by continuous light exposure, we measured one of the factors, the corticosterone level and found no significant difference between the two groups (P > 0.05).To explore the influence of continuous light on endocrine system, we measured the concentrations of important hormones in the two groups. Firstly, we found serum AMH level was clearly increased (P < 0.05), and dropped much lower at 60 min and 120 min compared to L/D group (P < 0.01). Although the area under curve (AUC) was not different between two groups (P > 0.05), abnormal glucose level and increased postprandial glycemia level indicated impaired glucose metabolism in L/L group. In addition, we found basal insulin concentration was significantly lower in L/L group of rats (P < 0.05). Leptin, another hormone that plays key role in the regulation of metabolism, remained unchanged in L/L group (P > 0.05). The adipocyte size in L/L group was comparable to that of L/D group , the HOMA-β was obviously lower in L/L group of rats (P < 0.05), indicating poor β−cell function after continuous light exposure.To learn more about metabolic abnormality in light-induced PCOS rats, we conducted homeostasis model assessment including HOMA-IR and HOMA-β in the two groups. HOMA-IR is a valid measure to determine insulin-resistance in rat model , while HLeptin mRNA expression levels were significantly higher (P < 0.01), while the expression levels of Ir (insulin receptor) remained unaffected in liver tissue (P > 0.05). Additionally, continuous light treatment reduced the relative expression of Ir (P < 0.05), but elevated the expression level of Irs1 (insulin receptor substrate 1) in adipose tissue (P < 0.001). In muscle of L/L group, the mRNA abundance of Glut4 (glucose transporter type 4) reduced significantly (P < 0.001), while expression of Leptin remained unchanged (P > 0.05). These data suggested poor β−cell function and impaired insulin signaling peripherally might contribute to lower basal insulin level and abnormal glucose metabolism. Continuous light exposure for 4 weeks resulted in glucose metabolic abnormality in PCOS rats.Then we detected the expression level of the genes that were closely relevant with glucose metabolism. Insulin receptor, insulin receptor substrate 1, leptin, and glucose transporter type 4 were reportedly associated with glucose metabolism in PCOS . After fP < 0.05). In the L/L group, alpha diversity was increased in rats in week 4 compared with that of week 0 (P < 0.05). Besides, the bacterial communities of rats in different weeks were clearly separated from each other by PCoA based on unweighted Unifrac in L/D group  and L/L group .To investigate the changes in gut microbiota as time went by, we compared the gut microbiota among different weeks. Chao1 index showed the alpha diversities of rats were significantly higher in week 4 compared with weeks 0, 1, and 2 in L/D group . To examine beta diversity between L/D and L/L group of rats, unweighted Unifrac distance was calculated to estimate dissimilarities in the community membership. PCoA showed L/D and L/L group of rats harbored distinct microbial taxa  revealed significant differences in the community membership between L/L and L/D group of rats.Microbial community richness  was measured by Chaol index. No significant difference was observed between L/D group and L/L group after 4 weeks of treatment  and eight being abundant in L/D group .Additionally, the relative abundance analysis showed that some microbial taxa differed in significance between the L/D and L/L groups . Continu/L group . It showParasutterella spp. was in positive correlation with serum level of testosterone . To infer the putative role of gut bacteria in the pathophysiological process, we performed PICRUSt analysis to predict the functional composition of a metagenome using marker gene data and a database of reference genomes  in gut microbiota, which was rather similar to the transformation of metabolic disorder  signaling,” “steroid biosynthesis,” and “glycan metabolism,” which implied a possibly weaker and unbalanced state of microbiota in continuous light group. In this view, we assumed that there might be some intricate interactions between imbalanced gut microbiota, hormonal, and metabolic disorders. Gut microbiota probably served as a vital link between circadian rhythm disturbance and PCOS phenotype. Imbalanced gut flora shaped by continuous light could interfere with gut epithelial function and accelerate inflammation reaction, contributing to further insulin resistance and reproductive disturbance in PCOS. The specific role and mechanism of gut microbiota in this pathophysiologic process shall be investigated in the future.On the other hand, we found the microbiome was drastically differed between L/D group and L/L group of rats at the termination of experiment. The relative abundance of several bacterial taxa significantly altered after light exposure, with the enrichment of microbial genus isorders . Some ofIn conclusion, we found circadian rhythm disorder could induce reproductive and endocrine system disruption, and glucose intolerance in SD rats. This study is to establish the impact of continuous light exposure on PCOS-like reproductive manifestation, as well as the diversity and composition of gutmicrobiota. The altered gut microbiota demonstrated a possible link between circadian rhythm disorder and PCOS-related manifestations. Imbalanced gut microbiota caused by circadian disorder might contribute to the pathology progress of PCOS, while further experiments are required to explore the underlying mechanism. This study confirmed the role of circadian rhythm disorder in developing PCOS-like manifestations including reproductive hallmarks and glucose intolerance, and discovered the possible mechanism of PCOS pathogenesis that is enrolled with gut microbiota. It shed light on an investigation direction toward the pathologic mechanism of PCOS occurrence and provided basic research evidence for clinical diagnosis and treatment in reproductive women suffering from circadian rhythm disturbance.PRJNA508095.The datasets generated for this study can be found in the NCBI Sequence Read Archive under Accession All experimental protocols were performed in accordance with institutional guidelines. The study was approved by the research committee of the Shanghai Jiao Tong University School of Medicine.WC, JZ, JX, SL, and YD designed the study. WC and JZ contributed to conducting the experiments and analyzing the data. WC, JZ, SL, WL, Z-JC, and YD drafted and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bathymodiolus azoricus’ molecular biology. We induced an in situ depletion of symbionts and compared the proteo-transcriptome of the gills of mussels in three conditions: symbiotic mussels , symbiont-depleted mussels and aposymbiotic mussels.Colonization of deep-sea hydrothermal vents by most invertebrates was made efficient through their adaptation to a symbiotic lifestyle with chemosynthetic bacteria, the primary producers in these ecosystems. Anatomical adaptations such as the establishment of specialized cells or organs have been evidenced in numerous deep-sea invertebrates. However, very few studies detailed global inter-dependencies between host and symbionts in these ecosystems. In this study, we proposed to describe, using a proteo-transcriptomic approach, the effects of symbionts loss on the deep-sea mussel Global proteomic and transcriptomic results evidenced a global disruption of host machinery in aposymbiotic organisms. We observed that the total number of proteins identified decreased from 1118 in symbiotic mussels to 790 in partially depleted mussels and 761 in aposymbiotic mussels. Using microarrays we identified 4300 transcripts differentially expressed between symbiont-depleted and symbiotic mussels. Among these transcripts, 799 were found differentially expressed in aposymbiotic mussels and almost twice as many in symbiont-depleted mussels as compared to symbiotic mussels. Regarding apoptotic and immune system processes – known to be largely involved in symbiotic interactions – an overall up-regulation of associated proteins and transcripts was observed in symbiont-depleted mussels..Overall, our study showed a global impairment of host machinery and an activation of both the immune and apoptotic system following symbiont-depletion. One of the main assumptions is the involvement of symbiotic bacteria in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens Symbiosis is a universal process defined as a long-lasting association between two organisms from different species; this definition encompasses the entire interaction spectrum, from mutualism to parasitism . In mutuin hospite [Riftia pachyptila) [Maintaining a long-lasting mutualistic association requires the establishment of adaptive interactions from both symbionts and hosts, including fine-tuned and specific mechanisms for partners recognition, symbiosis establishment, and maintenance of symbiont densities  hospite , 8. In t hospite , 10 or ohyptila) , 12. Beshyptila) , 14. Thehyptila) –17. Furthyptila) .2S, CH4, H2) [Deep-sea hydrothermal vents are characterized by challenging physico-chemical conditions, such as total darkness and sharp gradients in temperature, pH and concentrations of reduced compounds  . AlthougCH4, H2) , 20, 21.In these symbiosis “hot spots”, a large gap has been filled in the understanding of the ecology of hydrothermal vents and physiology of associated organisms , 23. In Bathymodiolus azoricus [B. azoricus is also capable of trophic plasticity; as a mixotroph, it may either use energy resources from its autotrophic symbionts or from the surrounding organic matter through regular heterotrophic filter-feeding. Indeed, unlike most other symbioses associated with hydrothermal vents, B. azoricus maintains a functional, though reduced, gut [B. azoricus and its endosymbionts and pave the way for possible experimental work. For instance, Kadar et al. (2005) performed a total symbiont depletion in mussels raised at atmospheric pressure, which induced a decrease in their fitness [B. azoricus and its symbionts has been evidenced using a proteo-genomic approach comparing aposymbiotic and symbiotic tissues [Among the diverse organisms associated with hydrothermal vents, the mussel azoricus  constituazoricus –29. B. aced, gut –32. Both fitness . More re tissues . While n tissues , 36. AltB. azoricus’ response to symbiont-depletion. In order to detect host’s proteins and genes involved in symbiotic functions, a depuration of symbionts was conducted in situ and the gill proteome and transcriptome of mussels were compared between control and symbiont-depleted individuals. To gain an insight into the adaptive features established by B. azoricus to specifically recognize and manage its symbionts, we first hypothesized that both immune and apoptotic pathways may be relatively over-represented in symbiont-depleted mussels, as these processes have been shown to be primordial in the regulation of symbionts in model symbioses. Finally, we hypothesized that in situ symbiont depletion may impact mussels’ energetic metabolism, and hence, investigated the effects of symbiont depletion on mussels’ carbon metabolic process in order to appraise the nutritional dependency of B. azoricus to its symbionts.In this context, and to gain a better understanding on symbiotic interactions in deep-sea hydrothermal vents, the objective of this study was to generate a comprehensive proteomic and transcriptomic overview of the mussel LC-MS/MS analyses led to the identification of a total of 1409 proteins with at least two unique peptides, in our three conditions: symbiotic mussels , mussels partially depleted in symbionts (T1) and aposymbiotic mussels (T2). A diminution of the total number of proteins identified was observed throughout the symbiont-depletion experiment, with a total of 1118 in symbiotic mussels, 790 in partially depleted mussels and 761 in aposymbiotic mussels  and symbiotic mussels. Among these transcripts, 799 were found differentially expressed only in aposymbiotic mussels and almost twice as many (1405) were found differentially expressed only in partially-depleted mussels as compared to symbiotic mussels  – were found to be up-regulated while two proteins were down-regulated  Table . In aposSimilarly to the activation of immune process, activation of programmed cell death seemed to occur mainly in aposymbiotic mussels . This activation was first evidenced by the up-regulation at both protein and transcript level of several activators of apoptosis such as cell death proteins  but also ligand and proteins associated with TNF receptors    were also found differentially regulated after symbiont depletion Table . In T1, Irrespective of the pathway, a low degree of congruency was observed between protein concentration and mRNA level (from 3 to 27%) Table . In the B. azoricus and its endosymbionts provides an interesting case study to deepen our knowledge on the molecular mechanisms at work in symbiotic interactions. In the present study, the in situ depletion of symbionts led to a global dysregulation – at both transcript and protein levels – of host machinery, impacting crucial process such as translation, cell cycle and division or transport. Furthermore, the activation of both immune system and programmed cell death processes in symbiont-depleted mussels suggest a fundamental role of these processes in adaptation to symbiotic lifestyle. In addition, the important modulation of enzymes involved in carbon metabolism suggests a putative nutritional dependency of B. azoricus on its symbionts, in spite of its ability to rely on particulate organic matter by filter-feeding.The symbiosis between the deep-sea mussel B. azoricus’ symbiotic tissue display an extensive and complex immune collection [Although traditionally thought to be solely involved in defense against pathogens, host immune system has been shown to be one of the key features allowing the establishment of fine-tuned associations between host and symbionts in metazoans/microbes mutualistic association , 14. B. llection , 38 whicllection , 39. Howllection . Interesllection  the lossllection .Concurrently to the activation of host immune system, activation of programmed cell death occurred mainly in aposymbiotic mussels . Apoptosis or programmed cell death-I is responsible for the maintenance of organisms’ homeostasis via the elimination of infected or unnecessary cells, or simply via their constant renewal . In our B. azoricus gills induced, after 7 days, a down-regulation of several enzymes involved in the energy yielded phases of the glycolytic pathway. The glycolytic pathway allows the oxidation of glucose into pyruvate which can be transported to the mitochondria and converted into acetyl-CoA that will in turn feed the TCA cycle and lead to the production of cofactors and ATP to fuel oxidative phosphorylation [B. azoricus’ symbionts which showed that thiotrophic symbionts may not be able to produce several TCA cycle enzymes [Metabolically, the in situ depletion of symbionts in rylation , 44. Therylation . However enzymes . The aut enzymes , 35. AltPocillopora acuta or Seriatopora hystrix and their Symbiodinium [. Such differences between mRNA levels and protein concentration can be explained biologically by distinct turnover of proteins and transcripts and/or the numerous posttranslational modifications in proteins. Furthermore, the divergence between protein concentration and mRNA level may also be due to the lower sensitivity of the proteomic analysis.Finally, among the 73 genes and proteins studied, only 8 and 11 (in symbiont-depleted and aposymbitic mussels respectively) differentially concentrated proteins were associated with DEG (10 and 15% respectively). Low correlation or congruency between protein concentration and gene expression has already been documented in yeast , plants iodinium , 50. SucB. azoricus on its symbionts, which could be perceived as an adaptive feature to symbiotic lifestyle. Our results uphold the nutritional inter-dependency already observed ex situ but also suggest that symbiotic bacteria may be involved in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens. However, our data do not allow us to disentangle between this hypothesis and a simpler explanation in which the drastic diminution of symbiont population compelled B. azoricus to produce its own defense, and to wipe bacteriocytes out. Further in situ studies will be needed to scrutinize these possible mechanisms. As for now, our in situ study provides a solid proteo-transcriptomics basis of the response of the deep-sea mussel B. azoricus to symbiont variations.Overall, our results confirm a significant reliance of Bathymodiolus azoricus were collected within a mussel bed in the vicinity of the hydrothermal fluid (chimneys) using the Remote Operating Vehicle (ROV), Victor 6000, remotely controlled from the Research Vessel “Pourquoi pas?”. These individuals - further referred to as “natural population”, corresponding to the first time (T0) of the in situ experiment – were then dissected to sample their gills, which were frozen in nitrogen, and stored at − 80 °C until further analyses. Subsequently, an in situ depletion of symbionts has been performed through a translocation of 30–40 individuals, divided in two wire steel cages, a dozen meters away from the hydrothermal fluid, capitalizing on a natural gradient in concentration of reduced compounds  between the mussel bed and the translocated site. The recovery of these cages was performed after 7 and 27 days (respectively T1 and T2) after the start of the translocation. Immediately after collection, gills were dissected, frozen in liquid nitrogen and stored at − 80 °C until further analysis. The relative quantification of thiotrophic symbionts (SOX) hosted in B. azoricus’ gills – measured by qPCR – indicated reductions of 50 and 90% of symbiotic content at T1 and T2 respectively [Sampling and in situ experiments were conducted at one hydrothermal vent site , located on the Mid Atlantic Ridge, during the BioBaz  and MoMAectively . In the Gill tissues were ground in a precooled mortar in the presence of liquid nitrogen and protein extraction was performed using the TCA/acetone method according to Mechin et al. (2007) . For eac4HCO3, 30 min at 56 °C; 25 mM iodoacetamide in 50 mM NH4HCO3, 20 min in dark at room temperature), proteins in gel pieces were digested by incubation with 12.5 ng.μL− 1 Trypsin  in sodium carbonate, overnight at 37 °C with gentle shaking. The reaction was stopped with one reaction volume (50 μL) 5% formic acid. Subsamples were sonicated for 10 min in an ultrasonic bath at room temperature and desalted using C18 packed column  prior to nano LC-MS/MS analyses.In-gel tryptic digestion method was used on the purified samples as described by Shevchenko (2006) . Brieflyv/v/v); buffer B: water/ACN/FA 10:90:0.1 (v/v/v)). The MS analysis was performed on a FT ICR mass spectrometer  with the top 7 acquisition method: MS resolution 60,000, mass range m/z 500–2000, followed by 7 MS/MS (LTQ) on the 7 most intense peaks, with a dynamic exclusion of 90 s. The raw data were processed using Proteome Discover v.1.4 (ThermoFisher Scientific) and Mascot v.2.5 (Matrix Science). Each sub-sample was analysed in triplicate. The database search was done on merged data using Mascot search engine on all taxa using SwissProt database  and B. azoricus libraries [http://heatmapper.ca).For each analysis, 8 μL of the peptide solution was loaded on a precolumn  and eluted on a capillary reversed phase column , at a constant flow rate of 220 nL/min, with a gradient 2 to 50% buffer B during 170 min followed by 50 to 60% during 10 min (buffer A: water/ACN/FA 98:2:0.1 (ibraries . The folibraries . Proteinibraries  with theibraries . Technichttps://earray.chem.agilent.com/earray/) including 61,300 unique cDNA expressed sequence tag (EST) and 27,062 annotated probes corresponding to around 13,000 unique genes. All sequences used have been obtained from previous sequencing of B. azoricus transcriptome (see [http://trace.ddbj.nig.ac.jp/dra/) with accession number DRA 004082). Probes were synthetized with positive and negative control using 8X60K-feature Agilent slide format. Probe sequences are available at the gene expression omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE124699.A custom heterologous 8X105K 60-mer oligonucleotide array has been produced using the Agilente Array application . Following extraction, RNA concentration purity and integrity were checked. Two pools containing 40 ng of total RNA extracted samples from T1 and T2 conditions  and four pools  for T0 were prepared. Labeled Cy3 or Cy5 probes were synthetized from 200 ng of total RNA using Agilent’s Low Input Quick Amp labelling Kit following manufacturer’s protocol. Labelled probes were then purified using Kit Illustra Cy Scribe GFx purification  and both quantity and quality were determined using NanoDropND-1000 spectrophotometer . For microarray hybridization 800 ng of probes were firstly fragmented (60 °C/30 min) using fragmentation buffer  and then added to 25 μL of 2X hybridization buffer . Arrays were hybridized in chambers  (65 °C/17 h). Following hybridization, arrays were washed twice with buffer-1 containing , once with buffer-2 containing  at 37 °C and finally once with acetonitrile . Hybridized microarrays were scanned with an Agilent scanner using the Feature Extraction Software . This process was repeated for all the hybridized slides with a 5-μm resolution mode. Data from each probe was then extracted and analysed.Total RNA was extracted from individual gills of http://www.rstudio.com) and BioConductor [2 signal intensity of the gene for one given sample by the average log2signal intensity of the gene from all samples in order to standardize gene expression to a mean of zero. Hierarchical clustering was performed using TmeV [http://www.tm4.org/) with the Euclidian distance and the complete linkage clustering parameters. KMC support parameters were used to identify clusters of genes that behave similarly in all samples. Prior identification, the optimal number of clusters was determined using the FOM  function. Differential expression patterns between conditions were identified by SAM  program. Briefly, SAM computes a statistic for each gene measuring the strength of the relationship between gene expression and the response variable. We applied a permutational test (100 permutations) to better estimate false discovery rate (FDR), which was used to estimate the proportion of false positives. Only genes with an FDR lower or equal to 5% were considered for further analyses.The microarray data were analysed as described earlier  using boonductor . Limma lonductor  was useding TmeV  . Due to the absence of replication in our proteomics analyses, correlation between proteomics and transcriptomics could not be assessed. Congruency analyses were performed as described by Mayfied and collaborator (2016) . Briefly


Seed germination, a complex, physiological–morphogenetic process, is a critical stage in the life cycle of plants. Biological changes in germinating seeds have not been investigated in poplar, a model woody plant.K-means method was used to identify multiple transcription factors, including AP2/EREBP, DOF, and YABBY, involved in specific seed germination and post-germination stages. A weighted gene coexpression network analysis revealed that cell wall, amino acid metabolism, and transport-related pathways were significantly enriched during stages 3 and 5, with no significant enrichment observed in primary metabolic processes such as glycolysis and the tricarboxylic acid cycle. A metabolomics analysis detected significant changes in intermediate metabolites in these primary metabolic processes, while a targeted correlation network analysis identified the gene family members most relevant to these changing metabolites.In this study, we exploited next-generation sequencing and metabolomics analysis and uncovered a series of significantly different genes and metabolites at various stages of seed germination and post germination. The Taken together, our results provide important insights into the molecular networks underlying poplar seed germination and post-germination processes. The targeted correlation network analysis approach developed in this study can be applied to search for key candidate genes in specific biochemical reactions and represents a new strategy for joint multiomics analyses.The online version of this article (10.1186/s12870-019-1862-3) contains supplementary material, which is available to authorized users. Plant seed germination and its immediate aftermath is a very complicated biological process involving a series of morphological, physiological, and biochemical changes under environmental influence –5. ExtenPoplar is a model woody plant that has long been studied, including in terms of physiological and morphological changes during germination . PreviouK-means clustering and weighted gene coexpression network analysis (WGCNA) to identify stage-specific gene clusters and network modules. In addition, we found that genes involved in cells and stress response were enriched at certain stages, whereas primary metabolic processes such as glycolysis and the tricarboxylic acid cycle (TCA) did not change significantly. Network analysis of enriched modules at different stages revealed a series of highly connected hub genes involved in transport, cell walls, and signaling. Finally, targeted correlation network analysis (TCNA) allowed us to find the most relevant gene family members potentially related to metabolite changes. Taken together, our results lay a foundation for further biochemical and functional analysis of poplar seed germination and its aftermath.By definition, seed germination begins with the uptake of water by the mature dry seed and terminates with the protrusion of the radicle through the seed envelope. On the basis of changes in fresh weight during seed germination, a three-phase germination model has been widely adopted in previous studies. The three phases of this model are as follows: I, rapid imbibition of water, which occurs as long as all matrices and cell contents are fully hydrated; II, limited water uptake; and III, increasing water uptake accompanied by embryo axis elongation and breakthrough of covering layers to complete germination (visible germination) , 35, 36.The poplar seed germination process was divided into stages 2 to 4 of the seed germination stages defined by Bewley 1997) [997 [1]. p ≥ 0.8. The number of observed transcript changes was as follows: 116 between stages 1 and 2 , 3694 between stages 1 and 3 , 4432 between stages 1 and 4 , 5207 between stages 1 and 5 , and 6378 between stages 1 and 6 . Compared with other stages, fewer changes in the number of transcripts were observed between stages 1 and 2. The number of genes with significant expression changes between successive time points during poplar germination and post germination is shown in Fig. For samples from each stage, the average number of raw reads per library was approximately 27 million , Clusters with similar expression trends were further combined into nine superclusters Fig. a. To undCPP (Zn), AP2/EREBP, EIL, ARR, and DOF, were only expressed at one stage  and ARR were only expressed at the end of germination , whereas genes in the brown module displayed the highest correlation with stage 5 . Genes in the pink module were relatively highly correlated with stages 1, 2, and 3 , the first principal component of a gene module, represents the gene expression profile of each module except for the gray module (Miscellaneous). The eigengene expression profiles of eight groups of modules are shown in Fig. To understand changes in biological processes in different modules, Fisher’s exact test was applied to identify enriched pathway categories and their mobilization trends in each module. The results of this test are given in Additional file Among the 10 modules, the green module was most strongly associated with stage 3, while the brown module was most relevant to stage 5 Fig. . The parANL2 (ANTHOCYANINLESS 2), a homeodomain protein belonging to the HD-GLABRA2 group that is highly similar to Arabidopsis AT4G00730 sequences. In a recent study, this gene was thought to be involved in the regulation of the root development process in Arabidopsis [ANL2 may therefore play a development-related regulatory role in poplar seed germination. The potri.016 g060600 gene, named ZFP8 (ZINC FINGER PROTEIN 8), encodes a nuclear C2H2 zinc-finger protein and was found to have high connectivity within the brown module. In Arabidopsis, the protein encoded by ZFP8 is a negative regulator of gibberellin-inducible seed germination and plays a key role in germination and seedling development [WUSCHEL-related homeobox 11 transcription factor (potri.019 g040800) found in the green module, are thought to be related to the germination of rice seeds [YABBY family member (potri.009 g000100) was located in the brown module; this transcription factor may play a key role in the germination of soybean seeds [WGCNA can also be used to construct gene networks in which each node represents a gene and the connecting lines between nodes, called edges, represent coexpression-related genes. The node gene with the highest connectivity, the hub gene, may play an important role in different modules. Brown and green module networks are shown in Fig. bidopsis , 39, andbidopsis . ANL2 maelopment . Some ofce seeds . In addian seeds . All of Some highly connected functional genes were also found in different modules. The distribution statistics of high-connectivity-node functional genes in brown and green modules are given in Fig. To understand the types of highly connected genes in different modules, we analyzed the top 10 node genes with the highest connectivity distribution Fig. d. In theIn this study, an untargeted metabolomics assay was performed to detect metabolic changes at different stages of seed germination and post germination. Because glycolysis, TCA, and pentose phosphate pathways play crucial roles in plant primary metabolism, changes in these three pathways during poplar seed germination were studied using metabolomics. Given the importance of the urea cycle and central amino acid metabolism, metabolites of those two pathways were also considered. A total of 19 metabolites related to the above five pathways were identified. These metabolites experienced significant changes in content during at least one stage. All metabolites and their patterns of changes are shown in Fig. cis-aconitate, alpha-ketoglutarate, succinate, and oxaloacetate. Changes in the contents of citrate and cis-aconitate followed similar trends, i.e., significant increases at stages 2, 4, and 5, whereas levels of alpha-ketoglutarate increased during stages 1 to 5 but were only significantly increased at stage 4. Oxaloacetate underwent significant decreases at stages 2, 3, and 6, while succinate was significantly decreased at stages 3 and 4 and significantly increased at stage 6. In regard to metabolites related to the urea cycle, argininosuccinic acid exhibited a significant increase from stages 2 to 5, ornithine was significantly increased during stages 2 to 6, and citrulline was only increased significantly at stage 4. Two metabolites in the pentose phosphate pathway, ribulose 5-phosphate and ribulose 1,5-bisphosphate, exhibited a consistent pattern of change, with significant increases from stages 2 to 6. In the central amino acid pathway, glutamate significant increased at stage 5, and glutamine significantly increased from stages 4 to 6. Aspartate was significantly increased at stages 5 and 6, while asparagine was significantly increased at stage 6.Five metabolites were closely related to the TCA: citrate, To investigate the characteristics of primary metabolic processes during seed germination, transcriptome and metabolome data for the entire germination process were subjected to an integration analysis. Figure To reveal the interrelationships of genes and their associated metabolites, TCNA was applied to intrinsically link RNA-Seq and metabolomic data. Detailed results of this analysis are shown in Fig. A strong positive correlation was uncovered between the change patterns of three metabolites: alpha-ketoglutarate, glutamate, and glutamine. The two families of genes controlling the synthesis and catabolism of alpha-ketoglutarate were negatively correlated with all isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes except for potri.008 g051600 . In the glutamate synthesis pathway, genes encoding alanine aminotransferase and glutamate dehydrogenase generally were strongly positively correlated with alpha-ketoglutarate and glutamate. Genes encoding glutamine synthetase typically have features highly associated with glutamate and glutamine.Adjacent metabolites in metabolic pathways generally have strongly related characteristics, and this correlation is typically positively correlated. As shown in Fig. K-means and WGCNA, were used to analyze the RNA-Seq data. This analysis produced several novel insights, including the identification of a series of time-specific modules, hub genes and transcription factors, some of the latter of which were consistent with previous research results. Many stress, cell wall, and protein-related genes were found to have a close relationship with germination and post-germination stages, while changes in glycolysis, oxidative phosphorylation, and the TCA were not significant. Further analysis showed that some metabolites in these pathways undergo significant changes at different stages of germination and that metabolite change patterns have similar characteristics under the same metabolic pathway. Enzymes associated with changes in metabolite content are commonly encoded by multiple gene families, and some members of these families were highly correlated with metabolite changes. Our study identified important genes and metabolites associated with germination and post-germination processes of poplar seeds and has laid a foundation for future biochemical and functional studies.Poplar is a model woody plant widely used in basic research. Because asexual reproduction is prevalent in poplar, the molecular mechanism of seed germination and its aftermath in poplar has not been investigated. In this study, samples from dry seed to seedling stages were collected and used for RNA-Seq and metabolomic analyses. Two different bioinformatic methods, Arabidopsis thaliana, rice, and soybean has been extensively studied. For example, two transcription factor genes, ZFP8 and WUSCHEL-related homeobox 11, as well as a YABBY family member are considered to be closely related to seed germination [CPP (Zn) and DOF are specifically expressed at different stages of seed germination, a phenomenon not observed in previous studies. We speculate that this discrepancy is due to differences among studied species.The seed germination process of mination , 41, 42.mination . Studiesmination ; this obGenes in green and brown modules in the WGCNA were found to be most relevant to stages 3 (slow water absorption period) and 5 (cotyledon expansion period), respectively. High-connectivity-node functional genes in green modules were associated with protein synthesis and decomposition, whereas those in brown modules were associated with CHO, cell wall, lipid metabolism and PS. We speculate that changes first occur in the early protein metabolism pathway, with significant changes in saccharide metabolism taking place during the cotyledon expansion period. Because the WGCNA method does not allow discovery of gene modules most closely related to other periods, such as rapid water absorption or hypocotyl elongation, we were unable to detect events occurring at the transcriptional level at each stage of seed germination. We plan to explore this issue in future research.K-means and WGCNA, to analyze transcriptome data, a strategy similar to that applied in previous studies of strawberry and Brassica oleracea [In this study, we used two different bioinformatics algorithms, oleracea , 45. Altoleracea , 46–51 aCorrelation analysis of metabolites during the germination process revealed a significant positive correlation among the most relevant metabolites. A significant difference was found, however, between glucose 6-phosphate and 6-phosphogluconate, 6-phosphogluconate and ribulose 5-phosphate, phosphoenolpyruvate and citric acid, and alpha-ketoglutarate and succinic acid. These transformations are situated at junctions with other metabolic pathways, and a strong correlation exists between branch metabolites and upstream metabolites. We have therefore inferred that substances at these branch sites flow in multiple, major directions. These results can thus serve as a useful reference for further study of metabolite flow.At the transcriptional level, many genes undergo complex changes during germination, even though the expression patterns of genes encoding the same enzyme are dissimilar. At the metabolic level, however, only a few significant changes to metabolites were observed Fig. a, b. ThiK-means and WGCNA methods, we identified transcription factors and metabolic pathways playing an important role in specific stages of seed germination. The application of targeted network analysis methods allowed us to discover candidate genes participating in specific processes. Such a method also represents a possible strategy for future joint sequential multiomics analyses. Our study was the first to use an integrated approach in regard to seed germination in poplar. The results of our study are thus an important starting point for future analyses and provide new insights to explore alternative strategies and possibilities.In this study, we investigated poplar seed germination and post-germination biological processes using a transcriptional combinatorial metabolomic method. In contrast to time-sequential methods used previously, we relied on a strategy based on morphological differences, which can better address the combination of molecular level and morphology. We compared our results with those obtained in earlier investigations of the germination of Arabidopsis , 53, ricPoplar simonii × Poplar nigra) were collected from the same female poplar plants producing half-sib families at the Experimental Forest Farm of Northeast Forestry University . Germination assays were carried out on three replicates (50 seeds per replicate). Seeds were incubated in covered plastic boxes at 25 °C on two sheets of absorbent paper moistened with 4.5 mL of distilled water. After removing excess moisture with absorbent paper, 20 germinating seeds were weighed (to ±0.0001 g) every 15 min at an ambient temperature of 25 ± 1 °C. Transcriptomic and metabolic profiling was performed at specific time points based on the fresh weights of germinated seeds. After weighing, seeds were immediately blotted with absorbent paper, snap frozen in liquid nitrogen, and subjected to transcriptomic and metabolic analyses.Seeds of poplar , using the HISAT2 alignment algorithm with default parameters [p ≥ 0.8 thresholds [Total RNA was extracted from approximately 200 seeds using Trizol reagent  and then incubated with 10 U DNase I  for 30 min at 37 °C to remove genomic DNA. Three experimental replicates per sample were used for RNA library construction. RNA-Seq libraries were prepared according to the Illumina Library Prep Kit manufacturer’s protocol. After sequencing, raw data were filtered to remove adaptor contamination and low-quality reads. All clean reads were then mapped to the poplar genome, which was downloaded from the Phytozome website (rameters . Mapped rameters . DEGs weresholds . Gene exK-means clustering with Euclidean distances in MeV4.8 [r ≥ 0.75 and p ≤ 0.05 thresholds (Student’s t-test). Networks were visualized using Cytoscape v.3.5.1 [n MeV4.8  yielded n MeV4.8 . Eigenge v.3.5.1 .p ≤ 0.05 threshold).The MapMan tool was used to assign pathway categories and screen transcription factors . Fisher’4OAc and 25 mM aqueous NH4OH (pH = 9.75). Gradient elution was carried out at a delivery rate of 0.3 mL min− 1 using the mobile phase (A) and acetonitrile (B) as follows: 0 min, 85% B; 2 min, 75% B; 9 min, 0% B; 14 min, 0% B; 15 min, 85% B; and 20 min, 85% B. The injection volume was 2 μL. Six biological repeats were performed, with all other details as described in a previous study [t-test (p > 0.05), and variables were discarded between the two comparison groups [Analysis of metabolites was performed using 50 mg of poplar seed collected from different germination periods, LC-MS/MS analyses were carried out on an UHPLC system  equipped with a UPLC BEH Amide column  coupled to a TripleTOF 6600 system  operating in EI mode. The mobile phase (A) consisted of 25 mM NHn groups .p ≥ 0.05 (Student’s t-test) and r ≥ 0.25 thresholds.TCNA is used to evaluate the contribution of each multigene family member to the change in a target metabolite. In this method, gene family members encoding proteins directly involved in the “synthesis” and “decomposition” of a target metabolite are considered. Genes responsible for target metabolite synthesis and decomposition were identified on the basis of gene annotation and metabolite rankings obtained from annotation information generated with the MapMan tool. A Spearman correlation analysis was performed and visualization was carried out using Cytoscape software (3.5.1) based on Additional file 1:Table S1. Changes in fresh weight of poplar seeds during germination. (XLSX 15 kb)Additional file 2:Table S2. The average number of raw reads per library and matching results. (XLSX 17 kb)Additional file 3:Table S3. IDs of genes in the 24 clusters. (XLSX 162 kb)Additional file 4:Table S4. The relative abundance of transcription factors at different stages. (XLSX 17 kb)Additional file 5:Table S5. Enriched pathway categories and mobilization trends in each module. (XLSX 22 kb)Additional file 6:Table S6. High-connectivity transcription factors. (XLSX 16 kb)Additional file 7:Table S7. Brown module and green module functional gene IDs and connectivity. (XLSX 48 kb)Additional file 8:Table S8. Primary metabolite changes during seed germination and post germination. (XLSX 10 kb)Additional file 9:Table S9. Primary metabolism-related gene profiling. (XLSX 22 kb)Additional file 10:Table S10. Genes and correlations involved in a targeted correlation network analysis (TCNA). (XLSX 14 kb)


The human demographic history of Mainland Southeast Asia (MSEA) has not been well studied; in particular, there have been very few sequence-based studies of variation in the male-specific portions of the Y chromosome (MSY). Here, we report new MSY sequences of ∼2.3 mB from 914 males and combine these with previous data for a total of 928 MSY sequences belonging to 59 populations from Thailand and Laos who speak languages belonging to three major Mainland Southeast Asia families: Austroasiatic, Tai-Kadai, and Sino-Tibetan. Among the 92 MSY haplogroups, two main MSY lineages (O1b1a1a* [O-M95*] and O2a* [O-M324*]) contribute substantially to the paternal genetic makeup of Thailand and Laos. We also analyze complete mitochondrial DNA genome sequences published previously from the same groups and find contrasting pattern of male and female genetic variation and demographic expansions, especially for the hill tribes, Mon, and some major Thai groups. In particular, we detect an effect of postmarital residence pattern on genetic diversity in patrilocal versus matrilocal groups. Additionally, both male and female demographic expansions were observed during the early Mesolithic (∼10 ka), with two later major male-specific expansions during the Neolithic period (∼4–5 ka) and the Bronze/Iron Age (∼2.0–2.5 ka). These two later expansions are characteristic of the modern Austroasiatic and Tai-Kadai groups, respectively, consistent with recent ancient DNA studies. We simulate MSY data based on three demographic models  of major Thai groups and find different results from mitochondrial DNA simulations, supporting contrasting male and female genetic histories. Thailand and Laos occupy a key location in the center of Mainland Southeast Asia MSEA; , which iHigham 2014Higham 2014The census size for Thailand was ∼68.41 million in 2017 and for Laos was ∼6.76 million in 2016 . There aIt is generally thought that AA languages were brought to the Thai/Lao region by Neolithic farmers from southern China, whereas TK languages were brought by a later, Bronze Age migration, also from southern China . The NeoSchliesinger 2000Previous studies have reported an influence of postmarital residence pattern on genetic variation in northern Thai hill tribes, with lower within-population genetic diversity coupled with greater genetic heterogeneity among populations for patrilocal groups than for matrilocal groups for the male-specific portions of the Y chromosome (MSY), whereas the opposite pattern is observed for mtDNA . HoweverThe MSY are paternally inherited and exhibit lineages specific to populations/geographic regions, making the MSY an informative tool for reconstructing paternal genetic history and demographic change . HoweverWe have previously carried out comprehensive studies of the maternal genetic history of the Thai/Lao region, based on 1,823 complete mtDNA genome sequences . In ordeWe generated 914 sequences of ∼2.3 mB of the MSY, which combined with 14 published sequences brings the total to 928 MSY sequences belonging to 59 populations from Thailand and Laos  online. U tests between AA and TK for MSY: h: Z = 3.37, P < 0.01; mean number of pairwise difference [MPD]: Z = 2.40, P < 0.05; haplogroup diversity: Z = 3.74, P < 0.01 and for mtDNA: h: Z = 4.33, P < 0.01; MPD: Z = 1.47, P > 0.05; haplogroup diversity: Z = 4.37, P < 0.01). After the Maniq (MN), who have no MSY variation, and the Mlabri (MA), who have no mtDNA variation, the Htin (TN1), Lawa (LW3), and Bru (BU) show very low diversity values of MSY, whereas the Htin (TN1–TN3), Khmer (KH2), and Seak (SK) show low mtDNA diversity . LW3 showed very low MSY haplogroup diversity  , who speak a Mon language, show the highest MPD value for the MSY  D, sugges the MSY C, which  the MSY A. MO3 an the MSY A.P < 0.01 and 18.49%, P < 0.01 without MN, the hunter–gatherer group from southern Thailand) than among the TK  and ST-speaking Karen groups . For the AA group with more than one population sampled, the greatest within-group variation by far was among the three Lawa populations , whereas the seven Mon populations showed very low  within-group variation  (P > 0.01), Khon Mueang groups from northern Thailand , and Lao Isan groups from northeastern Thailand , indicating overall genetic homogeneity among these major TK-speaking groups. In agreement with the MSY, larger mtDNA variation is observed in the AA groups  than the ST  and TK groups , but interestingly the largest within-group variation is not among the Lawa  but rather among the Htin populations . In contrast to the MSY, each of the TK groups with more than one population sampled showed significant within-group differences for mtDNA, especially the Khon Mueang  (P < 0.01) than for mtDNA  for AA groups, but about the same for TK groups , and the ST groups have larger among-population variation for mtDNA  than for the MSY   indicates that the variation among populations (within group) accounts for 11.12% of the total MSY genetic variance . There i < 0.01)  online.  < 0.01)  online.  < 0.01)  online. P < 0.01) than by language family  , and the total variation among populations within group reduces to 10.54%. Thus, neither geography nor language family is a good predictor of the MSY genetic structure of Thai/Lao populations, indicating that these two factors are not important in the broad view  , there ioad view .r = 0.3381, P < 0.01), resistance distance  and least-cost path distance . However, the correlations are no longer significant when the MN group is removed from the analysis: great circle distance , resistance distance  and least-cost path distance . In contrary, no significance was detected (P > 0.05) between matrices of mtDNA genetic distance and geographic distances with and without MN (great circle distance: r = 0.0776 and r = −0.0323), resistance distance (r = 0.1433 and r = −0.1105), and least-cost path distance (r = 0.0997 and r = −0.0253).There are significant correlations between matrices of MSY genetic and geographic distance, estimated by Mantel tests, for all three types of geographic distances, that is, great circle distance  was carried out. This analysis attempts to maximize among-groups genetic differentiation and minimize within-group genetic variation; the results showed considerable overlap among groups defined by either language family or geographic location in both MSY and mtDNA  online. In sum, all results indicate lower genetic diversity of the AA groups than the TK and ST groups, except the Mon and Nyahkur, who exhibit high genetic diversity. The AA groups also show greater genetic heterogeneity than the TK and ST groups.h and MPD values are higher for matrilocal groups, but not significantly ; however, haplogroup diversity is significantly higher for the matrilocal groups   (Φst = 0.3109 for MSY and 0.0774 for mtDNA) , whereas the matrilocal groups (Htin and Karen) also show higher average Φst for MSY (0.1859) than for mtDNA (0.1553), but these are not significantly different . Contrasting genetic differences for the MSY versus mtDNA of Lawa, Htin, and Karen are clearly seen in the multidimensional scaling (MDS) and DAPC plots  and the Palaung, another minority group in the mountainous area of northern Thailand but not officially recognized as a hill tribe. The Khmu (KA), Lawa , and Palaung (PL) groups practice patrilocality, whereas the Htin  are matrilocal, as are the ST-speaking Karen . If residence pattern is influencing genetic variation, then lower within-population genetic diversity coupled with greater genetic heterogeneity among populations is expected for patrilocal groups than for matrilocal groups for the MSY, whereas the opposite pattern is expected for mtDNA . The MSY < 0.05)  online.  > 0.05)  online.  > 0.05)  online. PC plots  online. PC plots  online.However, in general, the AA-speaking groups, whether identified as hill tribes or as other minorities, are patrilocal groups. The AMOVA result indicates that the variation among AA populations is higher in MSY (20.01%) than mtDNA (14.03%), in accordance with expectations if residence pattern is influencing genetic variation. Conversely, the TK populations, where neither patrilocal nor matrilocal residence is preferred, exhibit similar among-population variances for the MSY (4.48%) and mtDNA (4.33%)  online. Φst values . After excluding these MA and MN as outliers, the MDS for the MSY showed that almost all AA-speaking groups are located along the edges of the plot, whereas most of the TK groups cluster in the center of the plot  over time in each group reveal overall five different trends  or remains stable (Lao Isan and Laotian); the Lue and Phuan show two increases in Ne, at about ∼5 ka and ∼10 ka; the Lawa show a stable population size since ∼30 ka and then a decline during the last 2 ka with a sudden increase ∼1 ka; and the Karen differ only slightly from the common trend with a population increase ∼1 ka.The Bayesian Skyline Plots (BSPs) of effective population size change fig. 7). Ne increases, at ∼9–11 ka, ∼5 ka, ∼2.0–2.5 ka, and ∼1.0 ka .The BSP by each major MSY haplogroup show four pulses of paternal  ∼1.0 ka , in agrePreviously, we used mtDNA genome sequences and demographic modeling to test different hypotheses about the origins of TK groups. Specifically, we tested whether different TK groups were primarily related to local AA groups , to a TK group from southern China , or were related to both . We found that the Khon Mueang (from northern Thailand), Lao Isan (from northeastern Thailand), and Laotian most likely originated via demic diffusion from southern China without substantial gene flow from AA groups . HoweverΦst and corrected pairwise difference among groups of populations used in ABC tests to estimate their genetic relationships  people (Test 1), the highest posterior probability (0.80) and rather highly selected classification trees (0.58) were found for the demic diffusion model  online. ionships  online. In order to gain more insights into MSEA genetic history, we here investigate the paternal genetic variation and structure by sequencing ∼2.3 mB of the MSY from representative ethnolinguistic groups from Thailand and Laos. In sum, most of the studied populations exhibit two major MSY haplogroups, O-M324* and O-M95* in different proportions, indicating two major paternal sources. O-M324* was widely spread in the TK groups, whereas O-M95* is predominant in the AA groups. However, some TK populations (BT2 and IS3) and some AA populations  exhibited the opposite pattern  online. The hill tribes, who occupy the mountainous northern region of Thailand, are notable for their variation in patrilocal versus matrilocal residence pattern , as wellHere, we analyzed the sequences of mtDNA genome and ∼2.3 mB of the MSY of the Khmu, Palaung, and Lawa groups, who practice patrilocality, whereas the Htin are matrilocal, similar to the ST-speaking Karen. The within-population genetic diversity values are in agreement with expectations, that is, greater diversity of matrilocal than patrilocal groups for MSY and the opposite trend in mtDNA  online. One factor in particular that could influence the within-population genetic diversity and between-population differentiation is geographic isolation, which enhances genetic drift and inbreeding, thereby lowering within-population genetic diversity and increasing between-population differentiation. This could explain the very low internal diversity and high differentiation from other groups of some groups of Htin (TN1) and Lawa  online tMoreover, these results are in keeping with previous observations that although the expected difference between patrilocal and matrilocal groups holds in some regions , in otheThe Mon groups showed genetic differences from other AA populations but closer relatedness to the TK populations, especially the central Thai, in both MSY and mtDNA . In addiPrevious genetic studies of G6PD mutations reported a high prevalence of the Mahidol type G6PD deficiency in the Mon, Burmese, and Karen, different from Thai, Laotian, and Khmer groups exhibiting the Vientiane-type G6PD mutation . Thus, bA connection between Indian groups and the Mon is suggested by South/Central Asian MSY lineages in the Mon, for example, R*, H*, J*, L*, and Q* , consistNe increase during the Holocene is primarily driven by the O2a2a* and O2a1c* lineages  among the groups used in the simulations does support a real contrast in the maternal versus paternal histories for the major TK groups in each region, and also finds genetic heterogeneity among these major groups. The northern Thai people showed closer genetic relationship with the Dai than AA groups in both mtDNA and MSY, supporting the demic diffusion model, whereas the ethnic Lao are closer to Dai for mtDNA but for MSY they are related to both Dai and AA rather equally, suggesting demic diffusion for the maternal history and admixture for the paternal history. The central Thai MSY sequences could be of AA origin because they are genetically more similar to the AA groups than the Dai, supporting cultural diffusion, but for mtDNA they are related to both Dai and AA rather equally, supporting admixture in central Thailand as found previously , AMOVAs, and Mantel tests of the correlation between genetic and geographic distances.We combined the 914 newly generated sequences together with 14 published sequences  belong tadegenet 1.3-1 , the MDS heat plot with five dimensions, showing per-dimension standardized values between 0 and 1, and heat plots of the Φst distance matrix and the matrix of shared haplotypes.A correspondence analysis based on MSY haplogroup counts was performed using STATISTICA 13.0 . Haplogroup assignment was performed by yHaplo . The R phttps://sharehost.hms.harvard.edu/genetics/reich_lab/sgdp/Y-bams/Y.tar; last accessed June 25, 2018)  using Heffalump v0.2 . We subset the variant file to sites that were overlapping the regions present on our capture bait and to samples that had a major haplogroup that was also present in our data set. These samples were combined with our samples; we then removed variant sites for which <25% of the samples had genotype information, and samples that had >25% of all sites with missing genotype information. The resulting data set provides 16,684 variable sites, which was imputed using BEAGLE v4.1  , the 1005, 2018)  and the 5, 2018) . The resGLE v4.1 . Additio−10 substitutions/bp/year  and compared three competing scenarios: demic diffusion ; cultural diffusion  and continuous migration  that were developed based on known historical hypotheses  online. /bp/year , and a p/bp/year  online. We simulated the derived site frequency spectrum (unfolded-SFS) for 2,364,048 loci using the fastsimcoal simulator  with theAll reads that aligned to the region of the MSY that was targeted by the capture-enrichment array were deposited in the European Nucleotide Archive (ENA) (study ID: PRJEB31636).Molecular Biology and Evolution online.Supplement_Material_msz083Click here for additional data file.


Compared to Q3, Q5 was also associated with decreased subscores on anterograde (β = −0.19 p = 0.020) and retrograde episodic memories (β = −0.18 p = 0.039). We found a non-linear U-shaped relationship between circulating leptin and cognition, with both lower and higher concentrations of leptin being associated with more severe cognitive impairment in community-dwelling older Asians.The role of leptin (a hormone related to fat mass) in cognition remains equivocal. Our objective was to investigate the relationship between circulating leptin concentration and cognition in older adults, accounting for potential confounders. We categorized 1061 community-dwelling older participants ≥60 years  from the Singapore Kidney Eye Study according to quintiles of leptin concentration . Cognition was assessed using the total and domain scores of the Abbreviated Mental Test (AMT). Age, gender, body mass index, mean arterial pressure, smoking, alcohol, education, memory complaint, anxiodepressive disorders, circulating concentrations of 25-hydroxyvitamin D, glycosylated hemoglobin, low-density lipoprotein cholesterol, and estimated glomerular filtration rate were used as potential confounders. Participants within the lowest (Q1) and highest (Q5) leptin quintiles exhibited lower  mean total AMT scores compared to those within the intermediate quintiles . Compared to Q3 as the reference, Q1 and Q5 were associated with decreased total AMT score (respectively, β = −0.53  Leptin is a hormone that is mainly secreted in white adipose tissue , and itsA few neuroepidemiological studies have suggested a possible association between serum leptin levels and cognitive performance in older adults ,15,16,17n = 2944) [Data for the current analysis were derived from a case–control study designed to examine the association of novel biomarkers including leptin with renal and retinal diseases  . The stu = 2944) ,19,20. ACirculating leptin concentration was measured from venous blood collected in the non-fasting state at baseline and stored at −80 °C. The collection and storage process was the same for all study participants. Serum samples were used in Chinese and Indian subjects, and plasma samples were used in Malays for measuring leptin. Circulating leptin levels were measured using a commercial ELISA kit  at the National University Hospital laboratory. The lower limit of detection of leptin in the current study was 0.78 ng/mL. The sensitivity of the assay was 0.135 ng/mL + 2SD. The coefficient of variation (CV) was below 10% (2.6%–6.2%) at concentrations ranging from 2.34 ng/mL to 28.9 ng/mL. First, the leptin concentration was categorized into ranges of 4 ng/mL to avoid saturated and unreadable scatter plot of the relationship between leptin concentration and cognitive performance. Data were grouped for circulating leptin concentration ≥20 ng/mL, as the number of participants was low. Second, leptin levels were categorized based on quintilization: quintile 1 less than or equal to 2.64 ng/mL, quintile 2 between 2.65 and 5.1 ng/mL, quintile 3 between 5.2 and 8.6 ng/mL, quintile 4 between 8.7 and 17.96 ng/mL, and quintile 5 greater than or equal to 18 ng/mL.Cognition was assessed using the Abbreviated Mental Test (AMT), a reliable standardized and validated screening test for cognitive impairment ,22. The The following potential confounders were assessed: age, gender, history of regular smoking and of regular alcohol consumption, education level, body mass index (BMI), mean arterial pressure (MAP), subjective memory complaint, anxiodepressive disorders, circulating concentrations of glycosylated hemoglobin (HbA1c), low-density lipoprotein (LDL) cholesterol and 25-hydroxyvitamin D (25OHD), and estimated glomerular filtration rate (eGFR).An interviewer-administered standardized questionnaire was used to obtain information on age, gender, subjective memory complaint, history of regular smoking, alcohol consumption, highest education level attained, and mood. High education level was defined as ≥6 education years. Regular alcohol consumption was defined as ≥5 days a week. Anxiodepressive disorders were defined as reporting moderate-to-extreme anxiety or depression. Clinical covariates were obtained from a standardized physical examination. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using a digital automatic blood pressure monitor after the participant was seated for at least 5 min and an average of two measurements were taken as the blood pressure value for that individual. MAP was calculated as: equation .2) test or ANOVA, as appropriate. Further post-hoc analyses were performed using Fisher’s least significant difference test. Secondly, univariate and multiple linear regressions were used to examine the association between cognitive performance (dependent variable) and each quintile of leptin concentration (independent variable), while adjusting for all studied potential confounders and using the intermediate quintile as the reference . Separate models were used to explain the total AMT score as well as all domain-specific AMT subscores . p-values < 0.05 were considered significant. All statistics were performed using SPSS .Participant characteristics were summarized using means and standard deviations (SDs) or frequencies and percentages, as appropriate. As the number of observations were higher than 40, comparisons were not affected by the shape of the error distribution, and no transform was applied . FirstlySubjects participating in the study were included after having given their written informed consent for research. The study was conducted in accordance with the ethical standards set forth in the Helsinki Declaration (1983). The SEED and Singapore Kidney and Eye Study were approved by the Institutional Review Boards  0796/2003, 1149/2008, STaR/0003/2008, and Biomedical Research Council (BMRC), Singapore 08/1/35/19/550; Singapore Kidney and Eye Study: NMRC/TA/0008/2012).A total of 1061 participants were recruited in this analysis . Overall, the mean ± SD measured leptin concentration was 12.1 ± 15.5 ng/mL .A first visual representation of the relationship between circulating leptin concentration and AMT score showed a U-curve, with a decrease in cognitive performance for low leptin concentrations, but also for high leptin concentrations .p for trend <0.001 using Jonckheere–Terpstra test). We also found that participants in Q1 and Q5 had significantly lower mean AMT scores compared to those within the intermediate quintiles . With regards to the domain-specific cognitive subscores, we found no difference between the lowest and intermediate quintiles. In contrast, the highest quintile was associated with worse score on anterograde (fully adjusted β = −0.19 with p = 0.020) and retrograde episodic memories (fully adjusted β = −0.18 with p = 0.039) compared to the third quintile. The other associations were not significant in multivariate models (e models .Our main finding was that, independent of all measured potential confounders, both lower and higher concentrations of leptin were associated with more severe cognitive impairment among community-dwelling older adults, specifically affecting episodic memory. This U-shaped relationship suggests that older adults with low or high circulating leptin concentrations are particularly prone to exhibiting cognitive impairments for which leptin may be a modifiable risk factor.Few studies with mixed results are available on the association between leptin concentration and cognitive function. Most of the previous ones reported a significant inverse association between leptin concentration and cognitive performance ,10,11,12First, it is possible that, up to a certain threshold, leptin is protective to the brain. The have been experimentation reports that leptin may prevent cortical atrophy ,13,30, bSecond, it is likely that such beneficial effects of leptin could be surpassed. For instance, higher serum leptin concentrations are known to accompany increased BMI and metabolic syndrome, which expose individuals to greater cerebrovascular risk. The consequence is that people with overweight and increased leptin status are more exposed to vascular dementia than the others. Moreover, a central resistance to leptin has also been described during obesity , which mThe finding of a U-shaped relationship between circulating leptin concentration and cognitive performance has interesting potential implications. To the best of our knowledge, there are no clear reference values for a “normal” leptin status in older adults. In general, there are two ways of establishing reference values for a biological variable. The first one is based on the use of “population-based reference values”, which comprises measuring a parameter in the reference population size and calculating the reference interval involving 95% of the population. The second method is to define abnormal status as the onset of adverse health effects, known as “health-based reference values”. In our study, using population-based reference values would define normality for circulating leptin concentration as a range of concentrations between 0.8 and 55.2 ng/mL. However, it appears clearly from Strengths of our study include the relatively large size of the sample of older participants representing two major ethnic groups in Asia—a geographic area in which the association between leptin and cognition has not yet been studied. Additionally, leptin concentration was measured using a standardized accurate and validated automated technique. Beside global cognitive performance, the use of AMT subscores allowed the assessment of specific aspects of cognition in relation to leptin. Finally, linear regression models were applied to measure adjusted associations.This study has a number of inherent limitations. First, the study cohort was restricted to community-dwelling Indian and Chinese adults who might not be representative of all older Indian and Chinese adults in Singapore. Second, no information was available on possible history of leptin repletion in studied participants. Third, a selection bias is possible since data were derived from a case–control study designed to examine the association of novel biomarkers including leptin with renal and retinal diseases . This alIn conclusion, we report a likely U-shaped association between circulating leptin concentrations and cognitive performance among community-dwelling older adults in Singapore. This suggests that older adults with both lower and higher circulating leptin concentrations are particularly prone to exhibit cognitive impairment for which leptin may be a modifiable risk factor. These novel findings need to be replicated in larger and preferably longitudinal cohorts with extensive evaluation of the diet, and ideally considering not only the cognitive outcomes but also some neurodegenerative biomarkers such as the TAU and amyloid beta peptides or brain atrophy.


In vitro, in vivo and ex vivo studies have demonstrated that lycopene-rich foods are inversely associated to diseases such as cancers, cardiovascular diseases, diabetes, and others. This paper also reviews the properties, absorption, transportation, and distribution of lycopene and its by-products in human body. The mechanism of action and interaction of lycopene with other bioactive compounds are also discussed, because these are the crucial features for beneficial role of lycopene. However, information on the effect of food processing on lycopene stability and availability was discussed for better understanding of its characteristics.By-products derived from food processing are attractive source for their valuable bioactive components and color pigments. These by-products are useful for development as functional foods, nutraceuticals, food ingredients, additives, and also as cosmetic products. Lycopene is a bioactive red colored pigment naturally occurring in plants. Industrial by-products obtained from the plants are the good sources of lycopene. Interest in lycopene is increasing due to increasing evidence proving its preventive properties toward numerous diseases.  Natural colored pigments from plant products have drawn great attention worldwide. These pigments display various colors and are made up of different phytochemicals commonly ound in the food matrix such as orange (β-carotene), yellowish-green (lutein), green (chlorophyll), and blue-purple (anthocyanin) . LycopenLycopene is a carotenoid hydrocarbon . The extended conjugated double bond system of these compounds is an important feature in the carotenoids responsible for their attractive colors because it forms the light absorbing chromophore . The exiin vitro and in vivo studies exhibited that lycopene has a beneficial role in chronic diseases such as cardiovascular disease, atherosclerosis, cancer and neurodegenerative disorders. However, some studies reported contrasting outcomes. This review offers an overview of the properties of ycopene with recent evidence on its contributions in human health and also provides broad information on lycopene in food processing by-products. Lycopene is one of the popular pigments highly accepted by food industry as a food additive and also for its health benefits ,6. As a The importance of natural food additives is given more attention due to an extensive use of the natural ingredients rather than synthetic compounds in food, cosmetics and pharmaceuticals. Meanwhile, the prices of raw materials are increasing and their availability is decreasing. Food processing by-products from orange , mango ,9,10, guLycopene is an unsaturated acyclic carotenoid with 11 linear conjugated and two non-conjugated double bonds. It is not the precursor for vitamin A, since it lacks the terminal β-ionic ring found in the basic structure of vitamin A. The red color of certain fruits and vegetables such as tomato, pink grapefruit, red grapes, watermelon and red guava is due to the presence of lycopene. Lycopene is reported as the most efficient singlet oxygen quencher in carotenoids group, whose quenching ability is mainly dependent on the number of conjugated double bonds, and to a lesser influenced by either the presence of cyclic or acyclic end groups . In additrans form and its chain containing seven double bonds that can be isomerized to mono-cis or poly-cis due to the exposure to high temperatures, light, oxygen, acids, catalyst and metal ions •  CCl3O2- + Lycopene+•(7) CCl2-•) through electron transfer can form the lycopene anion radical (reaction 8) [In addition, the reaction between lycopene and superoxide radical (Oction 8) .2-• Lycopene -• + O2Lycopene + OHowever, hydrogen abstraction is the reaction of carotenoids as hydrogen donor to reduce the radical. The reaction is presented in reaction 9 .•  Lycopene• + ROOH(9) Lycopene + ROOAgain, the modes of action for antioxidants were depended on their position in the cell . CarotenIn lipid bilayer of cellular membrane, lycopene is expected to be a poor antioxidant due to its lesser interaction with aqueous phase radicals. However, the role of lycopene as a lipid phase antioxidant should not be neglected. The combinations of lycopene and other antioxidants such as vitamin C, vitamin E and β-carotene has exhibited higher scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical than their individual antioxidant activity . Besides + TOH+˙  TOH + Lycopene+˙(10) Lycopene+˙ + ASCH2 Lycopene + ASCH˙ + H+(11) Lycopene+˙ + ASCH- Lycopene + ASCH˙-+ H+(12) Lycopeneet al. [Previously, lycopene was reported to react effectively with vitamin E radical in the lipophilic compartment . Inverseet al.  had sugget al. . Lycopenet al. ,80.+˙ + Lycopene  Carotenoid + Lycopene+˙(13) Carotenoid˙)(reaction 14) [Different interpretations of reactions between lycopene with vitamin E and vitamin C is also reported ,66,68. Ltion 14) .˙ + Lycopene  α-TOH + Lycopene+˙(14) α-TO+˙ to regenerate the intact lycopene (reaction 15) [On the other hand, some researchers suggestied that α-tocopherol (α-TOH) could reduce lycopenetion 15) .+˙  α-TO˙ + Lycopene(15) α -TOH + Lycopene+˙) and α-tocopherol (α-TOH) or δ-tocopheroxyl radical (δ-TO˙) was also reported [However, a different reaction of lycopene radical cation (lycopenereported  as the f+˙  α-TO˙ + Lycopene(16) α -TOH + Lycopene˙ + Lycopene  δ-TOH + Lycopene+˙(17) δ-TO+˙) rather than with α-tocopherol anion (α-TOH-) as given in the reaction 18 [In non-polar solvents, carotenoids will probably react with α-tocopherol radical cation (α-TOHction 18 :+˙ + Lycopene  α-TOH- + Lycopene+˙(18) α -TOH+˙ due to the following reaction (reaction 19) [However, the reaction between lycopene and ascorbic acid increase the decay rate of Lycopenetion 19) ,66,83:+˙+ AscH- Lycopene + Asc-˙+ H+(19) LycopeneLycopene in combination with other antioxidants such as vitamins E and C, polyphenols and other carotenoids have wide potential for human health ,84. ReceThe effects of lycopene towards various diseases have been previously reviewed by many researchers. The protective effects of lycopene have been shown on oxidative stress, cardiovascular disease, hypertension, atherosclerosis, cancers, diabetes and others. However, there are still no conclusive results reported due to the fact studies on the role of lycopene against these diseases is still ongoing. In vitro, ex vivo, and in vivo studies have been carried out to demonstrate the effects of lycopene against oxidative stress. In this context, lipid, protein and DNA oxidation are closely related to oxidative stress. Oxidative stress is one of the major risk factors of chronic diseases . Free raPrevious studies have reported lycopene-rich diet and lycopene supplementation provided protective effects against DNA damage in both normal and cancerous human cells ,88,89. Iet al. [Human plasma lycopene levels have shown an inverse association with oxidative DNA damage . Consumpet al.  reportedA decrease of lipid and protein oxidation was also obtained in human consumed lycopene in the form of ketchup or oleoresin capsules ,101. BesCardiovascular disease (CVD) affects the normal function of the cardiovascular system involving heart and blood vessels. The World Health Organization (WHO)  reportedStudy has shown that dietary lycopene supplementation  provided through tomato juice (50.4 mg lycopene), spaghetti sauce (39.2 mg lycopene) and tomato oleoresin (75.0 mg lycopene) will significantly increase serum lycopene . Their set al. [et al. [The circulating plasma lycopene has been thought to prevent the development of atherosclerosis, especially in smokers . Moreoveet al.  suggesteet al. . On the et al. . A study [et al.  exhibiteet al. [et al. [Nevertheless, there was a study reporting that a high serum level of total carotenes significantly decreased the risk of CVD mortality, but the inverse association between serum lycopene and risk of stroke mortality was not observed . The facet al.  that sig [et al.  suggesteCancer has emerged as a major public health problem around the world. This health issue has raised the awareness of people to go for natural products and their therapeutic or preventive value. The beneficial effect of lycopene is associated to decrease cancer incidence worldwide especially in prostate. Lycopene (1–4 µM) was also reported to reduce the risk of prostate, lung, leukemic and digestive tract cancers . Besideset al. [Study has reported higher plasma lycopene were inversely associated with prostate cancer risk . Lycopenet al. , frequenet al. [In contrast, some studies showed no beneficial effects of lycopene intake. Kirsh et al.  reportedet al. . Case-coet al. ,127. A set al. . Thus, iet al. [On the other hand, lycopene (1–10 µM) was able to inhibit human liver cancerous cells proliferation and preventing them from metastatic process ,130. Lycet al. ,46. Howeet al. .Study has shown that there was no association between some carotenoids and breast cancer among Chinese women, but increased intake of lycopene is associated to a reduced risk of breast cancer . A cohoret al. [et al. [Lycopene is closely related to various metabolic complications, especially diabetes. Serum lycopene is inversely associated with type-2 diabetes and impaired glucose metabolism. The fact is proven by Coyne et al.  that pla [et al.  found thet al. [et al. [In man, dietary lycopene was directly related to baseline serum concentrations of nonesterified fatty acids . Besideset al.  have eva [et al.  have fouLycopene has its ability to scavenge free radical. Thus lycopene may have health benefit effects and improvement of other disease conditions. Treatment of lycopene  in streptozotocin-induced diabetic rats has significantly attenuated cognitive deficit, increased acetylcholinesterase activity, oxidative-nitrosative stress and inflammation . et al. [The treatment of lycopene using 3-nitropropionic acid-induced rats has significantly improved the memory and restored glutathione system functioning . Akbaralet al.  also sugp < 0.001) restored glutathione and malondialdehyde levels, superoxide dismutase (p < 0.05), catalase and glutathione S-transferase (p < 0.01) [Lycopene supplementation in enucleated rat lenses culture has significantly ( < 0.01) . However < 0.01)  and subj < 0.01)  than theThe safety aspect of bioactive compounds in products has been received much attention from food scientists to avoid any side effects. Either synthetic lycopene or from natural sources have been reported to be safe  when used in as food additive . Toxicitet al. [Food processing by-products from the tomato puree and sauce industry are commonly used in the development of lycopene-rich products . Previouet al.  had repoet al. . Nowadays, there is an increasing trend towards utilization of food processing by-products as a source of functional components . Many stet al. [On the other hand, there are initiatives by food scientist to recycle the lycopene-rich by-products as food ingredients. Fortification with lycopene in dry fermented sausage was also done by adding dried tomato peel to the meat mixture during the sausage production . The devet al. . Besideset al. . The ideet al. . Anotheret al. .Thermal processing is used in the food industry to preserve food products and maintain the nutritional quality. Traditionally, sun drying is the easiest and cheapest technique, and it is commonly used in poor countries or small and medium industries for food preservation. However the disadvantages are food processing enhances lycopene destruction and increases the process duration . The altet al. [Chang et al.  reportedet al. . Besideset al. . In contet al. .et al. [cis isomers, lower total lycopene and trans isomers were obtained from tomato using air drying method at 95 °C for 6–10 h as compared to the vacuum drying and osmotic treatment methods. However, lycopene and other lipophilic antioxidant compounds in tomato pulps have high stability after air drying [Lycopene stability is always considered by researchers to ensure that lycopene is able to be preserved until utilization. A study done by Shi et al.  showed tr drying .cis isomers and decreased in trans isomers when the temperature and processing time increased [In air drying processing, total lycopenes was affected by isomerization and oxidation, while there was a significant increase in ncreased . Thus, tncreased  revealedet al. [Moisture content is closely related to lycopene degradation. When moisture is retained, the water soluble compounds will react as catalyst during lycopene degradation. Goula et al.  reportedLycopene is the red pigment that plays an important role in plant and animals. In human health, much evidence shows that consumption of lycopene rich foods can help in preventing degenerative diseases, but very limited studies have found a beneficial role of the consumption of lycopene alone. The interaction of lycopene with other active compounds is crucial for obtaining its optimal function in human health. On the other hand, some beneficial effects may due to the lycopene isomers or its metabolites but information about this is scarce. In addition, further studies on the biological activity of lycopene or with the combination of different compounds are also warranted. Besides, there are only limited studies on long term intake of lycopene that might provide information about the upper limit of lycopene intake. The development of clean technology for high quality nutraceutical products is also needed for the promotion of lycopene consumption. Nonetheless, further understanding of the clinical aspects of lycopene, its mechanism of action towards diseases, bioavailability, bioaccessibility, recommended intake, interaction with other compounds and its metabolites activities are needed due to the lack of conclusive results on the role of lycopene in human health.