ANR and LAR) involved in proanthocyanidin biosynthesis from catechins and derivatives. A total of 651 metabolites were identified, which include at least 257 phenolic compounds. Higher number of metabolites were differentially modulated in peel as compared to pulp. Overall, the relative quantities of amino acids, carbohydrates, organic acids, and other metabolites were increased in the pulp of TN cultivar. While the contents of phenolic compounds were relatively higher in HGF cultivar. Moreover, HPLC based quantification of catechin and derivatives exhibited cultivar specific variations. The ANR and LAR genes exhibited an opposite expression profile in both cultivars. Current study is the first report of numerous metabolites including catechin-based derivatives in mango fruit. These findings open novel possibilities for the use of mango as a source of bioactive compounds.Polyphenols based bioactive compounds from vegetables and fruits are known for impressive antioxidant activity. Ingestion of these antioxidants may promote human health against cardiovascular diseases and cancer. Mango is a popular tropical fruit with special taste, high nutritional value and health-enhancing metabolites. The aim was to investigate the diversity of phytochemicals between two mango cultivars of china at three stages of fruit maturity. We used ESI-QTRAP-MS/MS approach to characterize comprehensively the metabolome of two mango cultivars named Hongguifei (HGF) and Tainong (TN). HPLC was used to quantify selected catechin based phenolic compounds. Moreover, real-time qPCR was used to study the expression profiles of two key genes ( Mangifera indica (L.) Lam.) is an important tropical fruit and rank fifth in global production (55.6 million tonnes/year) after banana, apple, grapes, and oranges1. Numerous cultivars of mango are found worldwide, which show variations in fruit peel color, size, shape, and composition3. Apart from being consumed fresh, mangoes are also used to make desserts, juices, pickles, marmalades and jam4. During processing, a significant portion of the fruit is removed, which generates millions of tons of mango waste every year. However, the peel of mango fruit may be interesting for the presence of high levels of health-promoting compounds4. It seems in line with the efforts to explore the cost-effective potential of agri-waste for industrial use or to reduce its negative effects on the environment. The identification and reclamation of important metabolites from mango or its byproducts is a difficult task and its completion would promote the revaluation of mango as a natural source of antioxidants/bioactive compounds. In this regard, large scale metabolite profiling seems a promising way to explore metabolite potential of mango fruit.Fruits of tropical and subtropical regions are appreciated as energy suppliers, as well as for the presence of health-enhancing metabolites. Plant origin secondary metabolites are the focus of research for numerous health-beneficial properties and antioxidant activities. Mango -epicatechin and 2,3-trans-(+)-catechin6. Flavan-3-ols (as monomers or as PAs) promote plant resistance against various biotic and abiotic stresses7. These metabolites possess numerous pharmacological properties, hence involved in scavenging free radicals, antimicrobial, antioxidant, anti-nutritional, anti-cancer and cardiac protection activities9. Quantitative and qualitative differences in the phytochemical profiles of mango cultivars may contribute to distinguishing their health-promoting properties.Proanthocyanidins (PAs) are oligo/polymeric flavonoids that are naturally present in many vegetables, nuts, seeds and fruits12. However, very limited information is available about the identities of metabolites, which govern important properties of this fruit. In addition, there is a lack of studies for comprehensive identification, documentation, and quantification of flavonoids and other secondary metabolites. Instead of thoroughly evaluating all of the phytochemicals, researchers have attempted to study only particular metabolites of mango14. Recent advancements in widely-targeted metabolomics (supported by techniques like LC–MS/MS) have made possible a prompt and ultra-sensitive detection of a huge number of metabolites16. The liquid chromatography-tandem mass spectrometry is employed in the current study to identify and detect relative quantities of metabolites from two mango cultivars exhibiting contrasting features in terms of shape, size, taste and peel color2. This study aims at the revelation of the metabolic variations between two mango cultivars (from China) with distinct features17 and offers valuable data for appraising its nutritional importance in industrial utilization and breeding strategies.The biochemical composition of fruits  predominantly influences the consumer preference for visual features and taste. Several researchers have investigated the nutritional composition of mangoes24. In most of these studies, a standard metabolite was used to identify an exact compound or a relevant group of metabolites. However, based on these studies it was difficult to envisage comprehensive metabolic dynamics in mango. In current study, a total of 651 metabolites were annotated/ identified in three growth stages , we used multivariate statistics. Hierarchical cluster analysis (HCA), for relative differences in accumulation patterns at three growth stages, arranged metabolites from both cultivars into three groups Fig. . The metPCA is extensively applied in chemometric experiments to extract and rationalize important facts from biological systems with multivariate descriptions. By using this analysis, we can determine the core arrangement of variables in terms of principal components. According to PCA plots Fig. B, the QC2) and strong goodness of fit  were observed for these models. . As a result, higher predictability , 252 metabolites  at 2nd stage, and 261 metabolites  at 3rd stage ; indole and epigallocatechin (catechin derivative) were present in very high amounts (> 10 log_fold) in TN pulp (Table o-hexoside (Anthocyanins); protocatechuic aldehyde (catechin derivatives); syringic acid, 1-o-beta-d-glucopyranosyl sinapate (hydroxycinnamoyl derivatives); chrysin o-hexoside, chrysin 5-o-glucoside (toringin) (flavone); o-feruloyl quinic acid (quinate and its derivatives); eriodictyol c-hexoside (flavone c-glycosides); and aromadedrin (flavonol) were present in very high amounts (> 10 log_fold). Overall, the relative quantities of amino acids, carbohydrates, organic acids, and other metabolites were higher in the pulp of TN cultivars. While the contents of phenolic compounds were relatively higher in HGF cultivar. It explains better sweetness/taste of TN and better quality of HGF pulp17. Among others, syringetin (a flavonol) was detected only at the 2nd stage as ~ 10.71 log_fold higher contents in HGF as compared to TN. This compound has never been reported in mango. Besides, it was just reported in grape and wine26. This metabolite can induce human osteoblast differentiation through bone morphogenetic protein‐2/extracellular signal‐regulated kinase 1/2 pathway26. It can also enhance radio-sensitivity more effectively in cancer cells than in normal cells through enhancement of the caspase-3-mediated apoptosis pathway27. Moreover, growth stage or cultivar-specific variations in metabolite contents may provide important information in identifying respective molecular markers for mango fruit selection at different growth stages , 379 at 2nd (138 HGF vs 241 TN) and 375 at 3rd (136 HGF vs 239 TN) ; cucurbitacin D (terpenoids); gentisic acid (benzoic acid derivatives); Vanillic acid (hydroxycinnamoyl derivatives); N-sinapoyl hydroxycoumarin (coumarins) , following metabolites were detected in HGF peel (as compared to TN): DIMBOA glucoside (cyclic hydroxamic acid); spinacetin (flavone); sissotrin (isoflavone); s) Table . DIMBOA https://www.genome.jp/kegg/) was used to map the differential metabolites between both cultivars  database , B1 (29–88 µg /ml) and B2 (0–10 µg /ml) were reported in peel liqueurs of mangoes from Brazil37.Current study reports developmental stage dependent variation Fig. A in tota38. The TN mango fruit contains higher levels of total phenolics (TPs) and total flavonoids as compared to HGF2. However, total anthocyanin contents (TAs) were reported to be higher in HGF as compared to TN2. In phenylpropanoid biosynthesis pathway, both anthocyanins and proanthocyanins belong to the terminal steps39. Therefore, the increase in PAs  as discussed above . The expression patterns of these genes are highly correlated with PA accumulation in many plants. To determine whether the differential expression of catechin/derivatives correlated with the transcript abundance of MiANR and MiLAR, the expression levels of these genes were analyzed in fruits of both cultivars using relative qRT-PCR  for MiANR and decreased (with fruit maturity) for MiLAR  and ANR. In addition, there could be a potential competition between LAR and ANR enzymes. The activity of both of these reductases (ANR and LAR) is dependent on NAPDH. Therefore, if one of them is overexpressed, it will decrease the availability of NAPDH for the other enzyme. It is expected that such competition may govern the mutual inhibition of LAR and ANR expression observed in this study. Altogether, the PA biosynthesis is likely co-regulated by structural genes such as LAR and ANR, and the mutual inhibition between the ANR and LAR expression may affect PA accumulation16. A lack of clear association between the transcripts of LAR/ANR and the catechin/derivatives has already been reported in other plants as well46. Earlier studies indicate a potential importance of ANR and LAR genes in the biosynthesis of galloylated catechins47. Therefore, it could be explained in terms of variable amounts of catechin/derivatives and the fact that different forms of catechin are potentially interconvertible.The genes encoding anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) enzyme have been cloned and characterized in plants including poplar, buckwheat, lotus and fruits such as grapevine, strawberry, persimmon, apple, and mango . Mature fruit peel is purple-red and green  and Hongguifei (HGF), during three different developmental stages of fruit . Both varieties were maintained at a farm located at Basuo town, Dongfang County, Hainan province. The TN mango was developed by the Fengshan tropical horticulture branch institute of the Taiwan Agricultural Test Institute and introduced to southern provinces of China including Hainan and Guangdong in 1994. The TN fruit is oval-shaped and relatively small in size that weighs up to 300 g. Mature fruit peel is dark green to greenish-yellow. The peel is slightly thicker and ductile, which is good for storage and transportation The HGF mango, also known as Hongjinglong, is native to Taiwan. It is a hybrid of 'Irwin' and 'Kate' and was introduced to Hainan in the 1990sFruit samples from both varieties were collected at 40 , 65 (Enlargement or 2nd), and 90 (Ripening or 3rd) days after full bloom (DAFB) respectively. Samples for each stage consisted of 10 fruits from 10 mango trees. Fruits of each variety were peeled and cored, and the flesh was cut into small sections. Fruit samples at each stage were mixed and immediately frozen in liquid nitrogen, and then stored at − 80 °C until used.g for 10 min followed by filtration .A mixer mill with zirconia bead  was used to grind the freeze-dried samples for 90 s at 30 Hz. Then, an overnight extraction (at 4 °C using 70% aqueous methanol) was performed for 100 mg powder. Before LC–MS analysis, the samples were centrifuged at 10,000×www.shimadzu.com.cn/; MS, Applied Biosystems4500 Q TRAP, www.appliedbiosystems.com.cn/) was used to analyze the sample extracts. The analytical parameters were as follow: HPLC column, waters ACQUITY UPLC HSS T3 C18 ; solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 100:0 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 °C; injection volume: 5 μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS.An LC–ESI–MS/MS system  was set to 5 psi during QQQ scans based MRM analysis. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.Triple quadrupole-linear ion trap mass spectrometer  was used for Linear Ion Trap (LIT) and triple quadrupole (QQQ) scans. The equipment contained an ESI Turbo Ion-Spray interface, which was operated in a positive and negative ion-mode and the data was analyzed using analyst 1.6 software (AB SCIEX). The chromatographic method  is the same in both ESI acquisition modes. Following conditions were used for the source of ESI operation: turbo spray (ion-source); 550 °C (source temperature); 5500 V (ion spray voltage or IS); GSI (ion source gas I), GSII (gas II), CUR (curtain gas) were set at 55, 60, and 25.0 psi, respectively; CAD (the collision gas) was set at high. Polypropylene glycol (10 and 100 μmol/L) was used to tune the instrument and for calibration of mass in QQQ and LIT modes, respectively. The collision gas (Nhttps://kanaya.naist.jp/KNApSAcK/), Massbank (https://www.massbank.jp/), Metlin (https://metlin.scripps.edu/index.php), MoTo DB (https://www.ab.wur.nl/moto/), hmdb (https://www.hmdb.ca/), and other existing mass spectrometry public databases. The quantification of metabolites was accomplished using the multi-reaction monitoring model of the triple four-stage rod mass spectrometry . The detection standard of MRM is based on the parameters including Q1, Q3, RT, DP, CE from the database, which was built using the standards. The relative content of the compounds was determined by the signal intensity of Characteristic fragment ion Q3. The range of DP  was − 80 to 80 V and CE (collision energy) was − 50 to 50 V. In MRM mode, the four levers first filter the precursor ions of the target, the ions matching substances with different molecular weights are excluded for initial removal of disturbance, and the precursor ions are induced by the collision chamber to form many fragments of ions. The fragment ions are then filtered through the triple four-pole filter to select characteristic fragment ions, eliminate non-target ion interference, make the quantification more accurate, and improve repeatability. After obtaining the data of the different samples of the metabolite spectra, the peak area integral of all the material mass spectra was obtained, and the mass spectra of the same metabolites in different samples were corrected by integral correction.Based on the public database and the self-built database MWDB (metware) of metabolite information, the first-order spectrum and two-level spectral data of spectral detection were qualitatively analyzed. The structural analysis of metabolites is referenced by knapsack  was used for principal component analysis (PCA) and orthogonal projections to latent structures-discriminate analysis (OPLS-DA). PCA showed the distribution of the original data. In order to obtain a higher level of group separation and to get a better understanding of variables responsible for classification, supervised OPLS-DA were applied. Based on OPLS-DA, a loading plot was constructed, which showed the contribution of variables to differentiate between two groups. The first principal component of variable importance in the projection (VIP) was calculated to refine this analysis. The VIP values above one were designated as differential metabolites. In the second step, Student’s t-test was used to assess the remaining variables and variables with −1) was also mixed with the above mentioned reagents, After incubation for 30 min, the absorbance was measured to determine proanthocyanidins content. The concentration of proanthocyanidin of samples was calculated using the following equation based on a PAs standard curve: y = 1.038x + 0.046 Where X is the absorbance and y is the proanthocyanidin equivalent. For the precentage of PAs content in tested samples, the equation is as follow: D = (v Cn/1000 W) 100%. All tests were conducted in triplicate D: the percentage of PAs content of samples; V: The constant volumn of sample; C: the concentration of proanthocyanidin of samples (mg/mL); n: dilution times; W: weight of sample(dry weight). Then the percentage of PAs conent of samples was converted to mg/ 100 g FW based on the ratio of fresh and dry weight of mango.The proanthocaynidins content of the extracts were determined using the method described by DongRuixia[12]. Calibration curve was prepared by mixing methanol solution of standard proanthocyanidin  with 6 mL of 4% (g/v) vanillic aldehyde and 3 mL of concentrated HCl. After capping and shaking the tube, it was incubated in the dark for 15 m at 30℃ ± 1. The absorbance was measured at 500 nm  with methanol as blank control and the standard curve was plotted. 1 mL of each of the extract solution in methanol  was used for designing the gene-specific primers and NCBI Primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to verify primer specificity. The expression data were normalized using MiActin gene (GenBank accession number HQ830244) as an internal control. Following primers were used for RT-qPCR: MiANR , MiLAR , and MiActin . An equal amount of cDNA template was used for each sample including the internal control. The qPCR analysis was repeated in three independent experiments.Peel and pulp samples were used for RNA extraction with the help of RNAprep Pur Plant Kit for polysaccharides and polyphenolics-rich samples  following the instructions of the manufacturer. The concentration of RNA was estimated from each sample through NanoDrop spectrophotometer . All-in-one First-Strand Synthesis Mastermix, with DNaseI  was used to reverse transcribe 1.0 μg of total RNA. The qRT-PCR analysis was performed using an APPLIED BIOSYSTEMS StepOnePlus Real-Time PCR System and TB GREEN Premix Ex Taq II, Tli RNaseH plus kit (TAKARA). The Oligo Calculator  was significantly higher in both pulp and peel samples of HGF cultivar at all three stages of fruit development. Moreover, total proanthocyanidin contents and relative flavan-3-ols/procyanidins were also higher in HGF cultivar. It seems obvious that such variations are directly responsible for the detected differences in relative quantities of flavonoid. This study documented changes in absolute contents of important catechin/ derivatives and expression profile of key genes involved in their biosynthesis for three growth stages. Such knowledge of mango fruit will be helpful for producers in adding value to the fruit and increasing antioxidant components. Moreover, different derivatives show distinct contents towards maturity and higher concentrations in peels as compared to pulp. It advocates the need for further research to improve contents of these metabolites in edible portion of mango.The current study provides a global picture of metabolite dynamics between two mango cultivars by conducting an analysis of widely-targeted metabolomics based on LC–MS/MS data. The differential accumulation or absence of particular metabolites from either of cultivars indicates underlying differential metabolism. Important metabolites including catechin, its derivatives and procyanidins only up-regulated in HGF pulp and peel samples. The current study revealed that the expression of Supplementary Figure 1.Supplementary Figure 2A.Supplementary Figure 2B.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Legends.


ColNu mIHCF identified distinct patterns of nuclear receptor colocalization in breast cancers. Furthermore, in prostate cancer all cancer epithelium was positive for ERa at the plasma membrane; and in normal prostate a small ERa+/p63+/AR− basal population suggest stem cell commitment to differentiation. ColNu mIHCF could be used for improving diagnosis and treatment in cancer.At present, platforms for multiplex immunohistochemistry  identify markers in distinct cell populations within a tissue section using multispectral fluorescence and optic microscopy. However, the optic resolution is not enough to colocalize markers at the subcellular level in the main epithelial or cancer population. We use confocal microscopy in multiplex detection of nuclear hormone receptors since they are an important part of the diagnosis and treatment of breast and prostate cancer. Moreover, we increased the quantitative dynamic range and resolution through increasing the signal/noise ration through reducing autofluorescence and increased longer antibody incubations. ColNu) of nuclear hormone receptors  along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus ( Multiplex immunohistochemistry has been developed as a complementary diagnostic method in pathology also in its chromogenic or fluorescent-based (mIHCF) forms ,4,5,6,7.Moreover, if possible, the most difficult combination would be markers that not only stain the same cells, but also the same organelle within those cells. A previous publication had performed triple mIHCF using confocal microscopy, although only two of the markers were co-expressed in one cell and none in the same organelle .Nuclear markers are commonly used as tools in diagnostics. Sex hormone nuclear receptors, estrogen receptor α (ERa), progesterone receptor (PR), and androgen receptor (AR) are essential biomarkers in breast and prostate cancer pathology. These markers are indicators and/or targets of effective therapies in various clinical settings, making an accurate, essential assessment for patient management ,23,24. SHowever, we endeavored to detect three nuclear receptor or other nuclear markers in the same tissue section of breast and prostate using routine IVD reagents for pathology diagnoses, i.e., diagnostic primary antibodies with a common secondary antibody preparation for both mouse and rabbit sources. Nuclear receptors could be nuclear, cytoplasmic, or both, and confocal microscopy is required for subcellular colocalization. Moreover, using this methodological approach, there is a distinct possibility that the same cell population can be labelled by all three markers.The focus of our study was to design and standardize a technique to colocalize nuclear hormone receptors and other transcription factors in the same tissue section using validated IVD antibodies in multiplex staining. Once standardized, we wanted to explore its usefulness in the study of normal breast and prostate tissues and cancers.Autofluorescence of human FFPE sections is a technical problem when dealing with a highly sensitive system, such as immunofluorescence. Previous works in the literature address this problem through incubation in different solutions that quench or cover this background fluorescence, such as CuSO4 or Sudan Black B (SBB), respectively ,26,27,28We sought to test three cycles of staining/detach antibodies for nuclear receptors in a hospital setting, that would be practicable in routine clinical practice. Thus, the technique should follow protocols that did not exceed conventional working times and rely on validated commercial hormone receptor antibodies for diagnostic IHC with DAB. These commercial ready-to-use preparations rely in very tight dilutions, and short incubation times (<1 h) to detect nuclear hormone receptor abundance for selecting cancer cases known to respond to anti-hormonal therapy. These precise dichotomous (yes/no) conditions confer high specificity, impeding false positives in optical chromogenic microscopy. However, confocal microscopy detects an ample range of fluorescent intensities, thereby enabling quantitative detection. Therefore, we aimed to detect the entire receptor expression in both normal (low expression) and cancerous tissue (from negative to high expression). For both of these reasons , our IVD primary antibodies were incubated overnight in all three consecutive cycles.Several blocking solutions were assayed that could improve specificity by detecting low levels of ERa and PR in normal breast tissue in conventional IHC with overnight incubation of the primary antibody. We compared the obligatory commercial peroxidase/blocking solution alone or followed by 0.1% BSA or 0.1% I-block (purified casein) for two hours. All three yielded adequate results in regular IHC, albeit the I-block appear to yield a completely clean stroma without remnants of DAB. We therefore added this blocking step to our protocol.The next step to standardize mIHCF using fluorescent TSA, was to wash out the antibodies after the first cycle and test if the remaining signal was strong enough for confocal microscopy. We adapted the protocol of the group of Mezey combining high pH with microwave heating . The varColNu mIHCF in two tissue microarrays (TMAs), TMA-I and TMA-II keeping to a systematic order: first ERa  followed by PR , and, finally, AR  . per se does not yield an unspecific signal even after repetitive cycles of staining. We assigned a pseudocolor to each channel and overlaid them with DAPI or with a phase contrast plane  to reveal tissue structure  . Howevertructure B. ColocaProfiler C. No difProfiler . DetaileColNu mIHCF for ERa, PR, AR, p63, and TTF1 combined in groups of three markers were performed in TMA-I and TMA-II arrays  IHC, and negativity for HER2 FISH . ColNu mclear AR A,B. Therclear AR A. There clear AR A,B. The clear AR C. At theclear AR A–C.ColNu mIHCF could contribute to the sub-stratification of triple negative breast cancers in future studies.The striking ERa staining (and PR) at the plasma membrane in one (BC5) of the two triple negative cases , led to the interesting question as to whether it would be a frequent pattern in triple negative breast carcinomas. To explore this possibility, we studied a new array with four other triple negative cases (TMA-III cases BC6-BC9), three from breast and one lymph node metastasis . All fouColNu mIHCF was performed in NP from TMA-I and TMA-II arrays  enabled certain areas where nuclear AR was present in the absence of ERa to be distinguished. Normal prostate tissue appeared to be interspersed in cancer tissue. To confirm this, we performed ColNu mIHCF including p63 to reveal the basal cell layer still present in normal prostate epithelium but absent in prostate cancer  from the epithelium with a layer of p63 basal cells, some of which were positive for nuclear ERa  in all the prostate cancer samples from TMA-0, TMA-I and TMA-II arrays . All cancer samples exhibited apical ERa staining  against commercial peroxidase/blocking solution alone without any other additional blocking step. The result was identical, indicating that the key factor was overnight incubation of the ERa antibody. Thus, this staining could be incorporated immediately into routine clinical practice.We compared ERa IHC performed with the blocking step as Next, we performed ERa IHC (overnight incubation) in sections from needle-core biopsy . Of six ColNu mIHCF uses long primary antibody incubations, fluorescence detection and confocal microscopy, resulting in a highly quantitative technique.We have established a multiple IHC technique for nuclear hormone receptors and other nuclear markers that may be colocalized within the same cell. Most of our standardization has been done with three consecutive staining cycles; albeit we know that a fourth cycle is possible, as we have tested it with cytokeratin staining (AECK or AE1-AE3). We have combined the detection of three nuclear hormone receptors , a thyroid transcription factor , and p63 . Each positive or negative control yielded the expected result in all of the cycles tested. This implies that the colocalization results obtained for nuclear hormone receptors are reliable.The two breast cancer cases classified as ERa+ (BC1 and BC3) co-expressed abundant PR and AR in the same cell. This did not occur in normal breast tissue where the luminal epithelium frequently co-expressed ERa + AR or, less frequently, ERa + PR, but rarely all three receptors. Furthermore, we found a difference between both ERa positive cases in the sub-cellular localization of the PR and AR receptors. While in BC3 all three receptors were concentrated within the nucleus, in BC1 PR and AR were found throughout the whole cell (cytoplasm and nucleus). Intriguingly, BC1 has more than twice the Ki-67 index than BC3, which was also positive for HER2. While we do not know if any or both of these differences are related to the nuclear/cytoplasmic pattern of receptor colocalization in DCIS, it does deserve to be investigated in greater depth with large series of cases. At the molecular level, crosstalk among the three nuclear receptors has been reported and hormone receptors are capable of binding to common co-activators/corepressors . AdditioPR has a controversial role in breast tissue expressing ERa . In normAR also has a controversial role in ERa+ breast cancer. In a normal breast, AR counteracts the proliferative action of ERa . FurtherCase BC2 case expressed PR in the absence of any other nuclear receptor. This is a rare group of breast cancers that is not well studied ,46,47. TColNu mIHC in this study  five out of six were positive for AR. Only two presented strong plasma membrane (BC4) or weak nuclear (TMA-III-BC7) ERa expression. Again, this is merely a descriptive result of a small group of cases and more studies are needed with larger series of triple negative breast cancers.The last breast cancer case, BC5, expressed ERa, although it was located on the membrane. This case had been diagnosed as triple negative. ERa isoforms 3 (ERa-36) and 4 (ERa-46) are shorter isoforms lacking the N-terminal half (1-173 a.a.) and are able to be expressed and signal on the plasma membrane . Neverthlow/AR− population. In mice, injection of high doses of estradiol or testosterone induces prostate cancer; however, this does not happen in knockout mice for ERa, nor in aromatase knockouts in which hyperplasia and hypertrophy are observed, but not cancer [ColNu mIHCF staining identifies prostate stem cells on which it would now be possible to conduct numerous functional and quantitative studies. Case NP5 case exhibited areas of hyperplasia with abundant ERa+ cells at the stroma, also reported in a recent study [Our results for the prostate have been consistent. The cells of the normal  prostatic glands have an ERa+ nuclear subset among its basal population. These cells have previously been described as a small population (<10%) among basal cells ,58. We ht cancer . Interest cancer . These rt cancer . This sunt study . More stColNu mIHCF and IHC) as well as in other types of prostate samples such as needle-core biopsies and full-length sections of surgical specimens. We believe that this labeling is quite simple to implement in routine prostate cancer assessments, since the anti-ERa antibody is commonly found in clinical pathology laboratories. The only requirement is longer incubation times with ERa primary antibody. ERa staining accurately discriminates cancerous tissue from normal tissue and can be invaluable in needle-core biopsies, as well as in delimiting cancer margins in surgical specimens more easily.All prostate cancer samples displayed ERa expression on the apical pole of AR+ epithelium. We have replicated this result numerous times, with different samples from several TMAs, with two different techniques .This study includes normal human and tumor tissue samples from the TIROCHUS collection, registered with the National Biobank Register of the National Institute of Health Carlos III  and from the Biobank of the Clinical University Hospital of Santiago de Compostela (CHUS). The use of these samples is in strict compliance with legislated procedures of personal and pathology data protection and has been approved by the regional Territorial Ethical Committee (CAEI 2015/238 and 2016/239). Pathological and clinical data from tissue samples include age, gender, and tumor type  thyroid, normal (non-neoplastic) breast, and prostate cancer, TMA-0a—three per group of each, and TMA-0b—three breast, and prostate cancer cases including mixed normal tissue in the thick cores. In those two TMAs, techniques to reduce background noise were tested and parallel immunohistochemistry (IHC) with DAB and TSA as substrates were performed as single staining for each receptor to compare results.Next, two TMA were generated from FFPE tissue blocks with breast and prostate carcinoma including five cases of each group . All casA third TMA, TMA-III, was generated with another four triple negative breast cancer cases, two cores from each case, and one prostate cancer as control . Of thosColNu mIHCF in cases of seven normal breast, eight normal prostates and six normal thyroid tissues, and 15 breast and 12 prostate cancers cases.Globally, we have tested Needle-core biopsy sections obtained from transrectal biopsies  were staTM FLEX/HRP system (Dako-Agilent) for signal detection with DAB. When using ColNu mIHC conditions, all reagents were maintained but incubation with primary antibody was extended to overnight.3–4 µm sections of prostate needle-core biopsy were pretreated for antigen retrieval using PT Link with high pH . When using clinical conditions sections were stained using a standard automated staining  with EnVisionTM (Dako-Agilent). Gleason score was assigned according to the WHO criteria outlined in 2016 [Diagnosis evaluation of hormone receptors was performed considering only positive nuclear staining, following CAP and ASCO Guidelines ,24 and m in 2016 .ColNu mIHCF was performed manually, IHC for each antibody was also repeated manually since only the last step, the substrate for the HRP, varies: DAB for IHQ and TSA for IHQF.Since Antibodies and dilutions are shown in ColNu mIHCF protocol for a step-by-step description and .Paraffin sections were pretreated as above using similar antigen retrieval in the PT Link system. Cyclic immunohistochemistry stainings were performed in the same sections/slides by using IVD validated antibodies used for diagnostic IHC see . We usedAt the end of the IHCF cycles, slides were incubated in 0.1% Sudan Black-B  diluted in 70% ethanol for 20 min, washed three times with PBS-T  and mounted with Fluoro-Gel with Tris buffer  including 2 mg/mL DAPI (D9542 Sigma).Slides were photographed using a confocal microscope TC-SP5-AOBS with a white laser (470–670 nm) and a UV laser  equipped with the Leica Application Suite Advanced Fluorescence, LAS AF software, using serial sections (Z) every 1 µm for the 10× objective (PL APO 10×/0.40 CS) and 20× (PL APO 20×/N.A.0.70 CS) objective and 0.3 µm for the 63× objective (oil PL APO 63×/N.A.1.4-0.6 CS). Data were collected using Sequential Mode with the following order : first, Data were collected at resolution 1024 × 1024 pixels, with zoom 1×–3×, giving an XY field of 912.7 × 912.7 µm for objective 10×, of a range from 775 × 775 µm till 288.5 × 288.5 µm for objective 20× and of a range of 246 × 246 µm till 90.4 × 90.4 µm for objective 63×. Thus the final resolution was between 0.89 µm/pixel (10×), 0.75–0.28 µm/pixel (20×) and 0.24–0.08 µm/pixel (63×). Each section on the TMA was systematically registered in at least 5 ROI at 20×, 10 pictures per ROI at 63×.https://imagej.net/Fiji) [Adjust Brightness/Contrast to slightly adjust background for the automatic reconnaissance of nuclei; Merge Channel for identification of nuclei combining each channel, or more than one, with Ch00 (DAPI) allowing identification of single, double or triple positive nuclei; Cell Counter for manually counting each group of single, double or triple positive nuclei; FreeHand to select ROI when separating stroma from epithelium; Clear Outside to eliminate sections when counting a specific part of a picture.Fiji (Image J) v1.5.1 (et/Fiji)  was usedhttps://cellprofiler.org) [Cell Profiler v3.0.0   was usedResults are presented as Mean ± SEM using GraphPad Prism v.7 .ColNu mIHCF is a valid technique useful for cancer pathology. Our results suggest an immediate application in prostate cancer either to ameliorate diagnostic in needle-core biopsies or to propose additional therapies based on the expression of membrane ERa. With the advent of the new agonist and antagonist hormone therapies, we also foresee a future use of ColNu mIHC in the sub-classification of breast cancer types to adapt each case to a particular line of therapy, and in studies of tissue physiology of breast and prostate.In summary, our study is a proof of concept that


Tobacco use is a leading cause of preventable diseases and death for all individuals, even more so for people living with HIV (PLWH), due to their status of chronic inflammation. To date, in Italy no study was performed to compare smoking habits in PLWH and the general population. We aimed to investigate smoking habits in PLWH, as compared to the general population.Multi-center cross-sectional study. Smoking habits were compared between PLWH and the general population. PLWH were enrolled in the STOPSHIV Study. The comparison group from the general population was derived from a survey performed by the National Statistics Institute (ISTAT), with a stratified random sampling procedure matching 2:1 general population subjects with PLWH by age class, sex, and macro-area of residence.p < 0.001); quitting smoking rate was 27.1% vs. 50.1% (p < 0.001) and the mean number of cigarettes smoked per day was 15.8 vs. 11.9 (p < 0.001), respectively for PLWH and the general population. Smoking and heavy smoking rates amongst PLWH were significantly higher even in subjects who reported diabetes, hypertension and extreme obesity (p < 0.001). Logistic regressions showed that PLWH were more likely current smokers  =2.62–3.71; p < 0.001) and heavy smokers (> 20 cigarettes per day) . PLWH were less likely to have quitted smoking .The total sample consisted of 1087 PLWH  and 2218 comparable subjects from the general population. Prevalence of current smokers was 51.6% vs 25.9% (HIV-infected patients showed a higher rate of current smokers, a larger number of cigarettes smoked and a lower quitting rate than the general population. Our findings emphasize the need for smoking cessation strategies targeting HIV persons. Over the past two decades, HIV-associated morbidity and mortality rates have dramatically declined and the life expectancy of people living with HIV (PLWH) has considerably increased, due to effective antiretroviral therapy (ART). In Italy around 90% of PLWH starting HIV treatment achieve and maintain viral suppression , 2, so tEpidemiological studies performed in the USA and other industrialized countries have demonstrated that the prevalence of smokers among PLWH is two to three times higher than that of reference populations , 12 To dIn this study, we present the comparison between two cross-sectional surveys. We examined the characteristics of smoking habits in a sample of PLWH from Italy, enrolled in a study on smoking prevalence and cessation (STOPSHIV study) , 14, andwww.cisai.it), a nationwide network of Italian Infectious Diseases Clinics. PLWH were enrolled in the STOPSHIV study (Stop Smoking in HIV infection), a prospective cohort study on smoking habits. A detailed description of the STOPSHIV study was previously published [This study was conducted by the Coordinamento Italiano per lo Studio di Allergie e Infezione da HIV  [Specifically, data were drawn from the ISTAT annual sample survey “Aspetti della vita quotidiana” (“Aspects of daily life”), a cross-sectional nationally representative dataset covering the resident population in private households and involving more than 45,000 individuals (Pen-and-paper-interview technique). Information on gender, age group, ethnicity, region of residence, marital status, education, co-morbidities and smoking habits were collected by standardized questionnaire . Co-morbon (CDC) ).Since health-related variables were self-reported amongst the general population, it is likely that those subjects reporting hypertension or diabetes were being treated for these diagnoses. We harmonized data collection from STOPSHIV, classifying HIV patients as hypertensive or diabetics if they were treated for the condition when recruited.The overall comparison group sample was selected through a stratified random sampling procedure. Overall, this technique consisted in dividing the entire general population into different strata based on some specific characteristics and then randomly select the final subjects proportionally from the prevalence of the selected characteristics in the comparison population. More specifically, it consisted of subjects randomly chosen from a representative sample of the Italian population, to match the PLWH sample 2:1. Matching variables were age, sex and macro-area of residence .Though PLWH and people from the general population were interviewed for different projects, their smoking habits were examined using the same structured questionnaire. Subjects were questioned if they had smoked at least 100 cigarettes in their lifetime . If so, Smoking prevalence: percentage of current smokers in the overall sample. Specifically, current smokers were identified as those persons who reported smoking 100 cigarettes or more during their lifetime and were currently smoking every day or some days. On the other hand, never smokers were defined as persons who reported not smoking more than 100 cigarettes in their entire life .Quitting rate: prevalence of former smokers among all smokers (former + current). Specifically, former smokers were defined as those who reported smoking at least 100 cigarettes during their lifetime but were not currently smoking.Heavy smoking prevalence: percentage of those who currently smoke more than 20 cigarettes per day in the overall sample .Frequency of smoking: self-reported number of cigarettes smoked per day.All outcome variables were compared between PLWH and the general population (main comparison).Control variables were gender, age, citizenship , educational level , marital status , macro-area of residence, and alcohol abuse . Information on co-morbidities was also incorporated into the model.Categorical variables were described as frequency  and comparisons between PLWH and general population were performed using the Pearson’s or Mantel-Hanzsel chi-square test (as appropriate). Continuous variables were described as mean  and compared using the analysis of variance.Smoking, heavy smoking and quitting rates were calculated as percentage and 95% CI .Odds ratios (OR) and 95% CI for current smoking, heavy smoking, and quitting were calculated using the general population group as the reference. Potential confounders such as education, marital status, ethnicity, alcohol abuse and co-morbidities were included in the logistic regression equation to account for characteristics that were different between PLWH and the general population. We also included age class, gender and macro-area of residence.Since the presence of missing values, if any, was less than 6%, we presented estimates from the main variable complete cases. To check for robustness, we planned to replicate the analysis by using dummies for missing values. Data analysis was conducted using the statistical software STATA 14.Overall, 1087 HIV patients were enrolled in 10 participating clinical centers. In the PLWH group, mean age was 47.9 years (SD 10.8), and most subjects were on ART at study onset, with undetectable viral load..The comparison group consisted of a sample of 2218 subjects matched for gender, age class and area of residenceThe main characteristics of study groups are reported in Table p < 0.001).Prevalence of current smokers was 51.6% (95% CI = 48.6–54.6) for PLWH and 25.9% (95% CI = 24.0–27.7) for the general population (Table p < 0.001).Across gender and age groups smoking habits were consistently different between PLWH and the general population, with a remarkably higher proportion of ever smokers in the STOPSHIV group. Among current smokers, the mean number of cigarettes per day was 10.2 (SD 5.9) in general population women vs. 14.3 (SD 8.1) in PLWH women, and 12.4 (SD 6.1) in general population men vs. 16.3 (SD 8.9) in PLWH men (both p < 0.001) and in macro-area subgroups (data not shown). The quitting rate was lower in PLWH males aged 18–34 years  compared with the general population . The heavy smoking prevalence was significantly higher in PLWH  compared with the general population .Overall, the quitting rate was: 27.1% (95% CI = 24.0–30.3) in PLWH and 50.1 (95% CI = 47.1–52.9) in the general population. Across gender and age groups the quitting rate in PLWH was significantly lower in any age group compared with the general population (p < 0.001)  and more than four times as likely to smoke greater than 20 cigarettes per day . Moreover, our estimates show that PLWH were less likely to quit smoking  and that being a PLWH was associated with higher number of cigarettes smoked per day . All analyses were also run including dummies for missing values, without significant modifications in the results.In PLWH, as compared to general population, crude ORs was 3.04 95% CI 2.61–3.54) for current smoking, 4.30 (95% CI 3.44–5.38) for heavy smoking, and 0.37 (95% CI 0.30–0.45) for quitting  for smoking outcome measures of 1087 people living with HIV (PLWH) and 2218 subjects from the Italian general population. Table S3. Fully adjusted association between hypertension and smoking habits in strata of study (no hypertension is the reference category). Table S4. Fully adjusted association between diabetes and smoking habits in strata of study (no diabetes is the reference category). Table S5. Fully adjusted association between extreme obesity and smoking habits in strata of study (no extreme obesity is the reference category). Table S6. Fully adjusted association between study population and smoking habits in strata of hypertension . Table S7. Fully adjusted association between study population and smoking habits in strata of diabetes . Table S8. Fully adjusted association between study population and smoking habits in strata of extreme obesity .


Mitophagy is an autophagic response and plays essential roles in survival, development, and homeostasis of cells. It has been reported that mitophagic dysfunction is involved in several cardiovascular diseases. However, the effect of mitophagy on atrial fibrillation (AF) is still unknown. Therefore, we investigated the exact role of mitophagy in human chronic AF. Western blot was used to detect the protein abundance. The mitochondrial morphology and structure were observed by transmission electron microscopy. Immunofluorescent stainings were performed to analyze colocalization of mitochondria with autophagosomes or lysosomes. Totally, 43 patients with valvular heart disease undergoing cardiac surgery were selected, including 21 patients with chronic AF. Comparing with the sinus rhythm (SR) group, we found the size and number of mitochondria in atrial myocytes of patients with AF increased significantly. In addition, expression of LC3B II and LC3B II/LC3B I ratio was significantly decreased in the AF group. Moreover, the expression of p62 was markedly elevated in the AF group compared with that in the SR group. The results of immunofluorescence staining and western blot showed an enhanced expression of Cox IV in the AF group. Dual immunofluorescent stainings revealed that mitophagy defect in atrial myocytes of patients with AF resulted from dysfunction in the process of delivery of mitochondria into autophagosomes. For the first time, impaired mitophagy, during the phagocytosis of mitochondria, is associated with human chronic AF. Mitophagy could be a potential therapeutic target for AF. Atrial fibrillation (AF) is the most common arrhythmia, which increases the risk of stroke and heart failure and is associated with significantly increased mortality , 2. HoweAutophagy is a conserved cellular pathway that is characterized by the engulfment of cargo into double-membrane autophagosomes, which then fuse with lysosomes for degradation. Autophagy plays essential roles in survival, development, and homeostasis of cells. To ensure survival, the cell uses autophagy to selectively digest protein aggregates, oxidized lipids, and damaged organelles , 7. MitoA total of 43 patients with valvular heart disease undergoing cardiac surgery were selected and divided into a chronic AF group (21 cases with long-standing persistent AF) and a sinus rhythm (SR) group  tissues weighing about 100 mg were removed at the beginning of the surgical interventions under extracorporeal circulation. A portion of the tissues was fixed in 2.5% glutaraldehyde for ultrastructure analysis under transmission electron microscopy. The remainder of the tissues were snap-frozen in liquid nitrogen for protein isolation and frozen sections to perform an immunofluorescence staining.μm polyvinylidene difluoride membrane  using the Mini Trans-Blot electrophoretic transfer cell system . The membranes were blocked for 1 h at room temperature with 5% nonfat milk in TBST  and incubated with anti-COX II antibody  diluted at 1 : 5,000, anti-COX IV antibody  diluted at 1 : 1,000, anti-LC3B antibody  diluted at 1 : 2,000, or anti-p62 antibody  diluted at 1 : 1,000 overnight at 4°C. Subsequently, the membranes were incubated with IRDye 700CW goat anti-rabbit or anti-mouse IgG  diluted at 1 : 10,000 for 1 hour at room temperature. The signals were visualized and quantified with the Odyssey system . Protein band intensities were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was incubated with anti-GAPDH antibody  diluted at 1 : 10,000 overnight at 4°C.Total proteins were extracted from tissues by RIPA buffer  with PMSF . Protein concentration was determined with a BCA protein assay kit . The proteins were boiled with 5X SDS-PAGE sample loading buffer  and separated by 8%, 10%, or 12% SDS-PAGE, followed by transfer onto a 0.22 μm in thickness) were fixed with 4% polyformaldehyde for 15 minutes at room temperature. The sections were permeabilized with 0.1% TritonX-100 for 20 minutes followed by blocking with 10% goat serum for 1 hour at room temperature. Then, the sections were incubated with anti-Cox IV antibody  diluted at 1 : 200, anti-LAMP-1 antibody  diluted at 1 : 200, or anti-LC3B antibody (Abcam) diluted at 1 : 100 overnight at 4°C. Subsequently, the sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody  diluted at 1 : 500, or Alexa Fluor 594-conjugated goat anti-rabbit antibody  diluted at 1 : 500 for 1 hour at room temperature. DAPI  was used to stain the cell nuclei. Finally, the fluorescence images were captured by using a laser scanning microscope and further analyzed by ImagePro Plus 6.0 .The frozen RAA tissues were embedded in the optimum cutting temperature (OCT) compound and sectioned at −25°C in a cryostat. Sections  and postfixed by 1% osmium tetroxide solution for 2 hours. Subsequently, the samples were dehydrated in an ethanol gradient series  and 100% acetone, immersed in propylene oxide, and embedded in epoxy resin at 60°C. Ultrathin sections were obtained by using a Leica EM UC6rt ultramicrotome  and were doubly stained with 0.1% uranyl acetate and 3% lead citrate solution. The sections were observed and photographed by using a transmission electron microscope . A further statistical analysis was performed by Image J _v1.8.0 .Dual immunofluorescent stainings were performed to analyze colocalization of mitochondria with autophagosomes or lysosomes. As we know, Cox IV is an inner mitochondrial membrane protein. We used Cox IV to identify mitochondria. Cox IV was labeled with Alexa Fluor 488-conjugated goat anti-mouse antibody showing green fluorescence, which represented mitochondrial mass and localization. LAMP-1 that is an integral glycoprotein residing in lysosomes was used for identification of lysosomes . LAMP-1 t-test. The discrete variables between 2 groups were analyzed by the Chi-square test. p < 0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 16.0 .The continuous data are presented as mean ± standard deviation (SD). The discrete data are shown as percentages. The continuous variables between 2 groups were analyzed by the unpaired Student's p = 0.046; p = 0.013 and p = 0.03, respectively; p = 0.014; To explore the role of autophagy on AF, the autophagy markers LC3B and p62 were measured in the SR group and the AF group, respectively. The results of immunofluorescence staining suggested a decreased expression of LC3B in the AF group (p = 0.001 and p = 0.001, respectively; p = 0.023 and p = 0.047, respectively; The differences of mitochondrial morphology and number between the SR group and the AF group were observed by transmission electron microscopy. We found that the mitochondria were larger, and their numbers were obviously increased in atrial myocytes of patients in the AF group, compared with those in the SR group (p = 0.024; p = 0.001 and p = 0.032, respectively; Mitophagy is an autophagic response and plays a crucial role in the mitochondrial quality control process. To observe the change in mitophagy in patients with chronic AF, Cox II and Cox IV, which are localized to the inner mitochondrial membrane and are used as marks for mitophagy , 15, werp = 0.046; p = 0.485; To investigate the underlying mechanism of mitophagy defect in chronic AF patients, the mitophagy flux was detected by dual immunofluorescent stainings. The results showed that the yellow fluorescence intensity caused by merge of Cox IV and LC3 was obviously decreased in the AF group compared with that in the SR group (In the present study, we found impaired mitophagy in patients with chronic AF and further demonstrated that dysfunction in the process of engulfment of mitochondria into autophagic vesicles results in defective mitophagy. To the best of our knowledge, this is the first study to report that impaired mitophagy is associated with human chronic AF. Mitophagy could be a potential therapeutic target for AF.In this study, a decreased LC3BII level and LC3BII/LC3BI ratio were observed in RAA tissues of patients with chronic AF. Meanwhile, an elevated p62 level was found in chronic AF patients. These findings indicated impaired cardiac autophagy in chronic AF patients. Moreover, a reduction in LC3B expression in chronic AF patients, revealed by immunofluorescence staining, implied that the formation of autophagosomes was deceased. This finding further verified that there is impaired cardiac autophagy in chronic AF patients. Dual immunofluorescent staining confirmed the decreased engulfment of mitochondria into autophagosomes in atrial myocytes of patients with chronic AF.Intact autophagic responses are crucial to preserving cardiovascular homeostasis in physiological conditions . HoweverOn the other hand, the autophagy process includes three parts: autophagy vesicle formation, engulfment of cargo, and fusion with lysosomes. At the beginning of autophagy, an autophagy vesicle with a double membrane is formed to encapsulate protein molecules or/and organelles that need to be degraded. The autophagic vesicles form autophagic bodies after wrapping the material to be degraded. Finally, lysosomes and autophagic bodies fuse into autophagic lysosomes, which use the internal hydrolases to degrade the encapsulated cell components . ImpaireMitophagy is a special autophagy and plays a crucial role in the mitochondrial quality control and maintenance of mitochondrial function . Early sHowever, why did the mitophagy defect happen? The dual immunofluorescent stainings were used to investigate the underlying mechanism of mitophagy defect in chronic AF patients. Cox IV, LC3, and LAMP-1 were used for identification of mitochondria, autophagosomes, and lysosomes, respectively. The results showed that the fluorescence intensity caused by merge of Cox IV and LC3 was obviously decreased in the AF group, but the fluorescence intensity caused by merge of Cox IV and LAMP-1 was not significantly different between the SR group and the AF group. These findings suggested that mitophagy defects in atrial myocytes of patients with chronic AF may be due to dysfunction in the process of delivery of mitochondria into autophagosomes. However, our findings still need more in-depth studies to testify.Collectively, our finding provided convincing experimental evidence, for the first time, that impaired mitophagy, during the phagocytosis of mitochondrial, is associated with human chronic AF. Mitophagy could be not only a potential therapeutic target for AF but also a promising mechanism of AF, which is suitable for further exploration.


To examine the association between body mass index (BMI) and the outcome of in vitro fertilization or intracytoplasmic sperm injection embryo transfer- (IVF/ICSI-ET) assisted reproduction in women with polycystic ovary syndrome (PCOS) receiving the ultra-long agonist protocol.  We retrospectively identified all women receiving IVF/ICSI-ET for the first time using the ultra-long protocol between January 2013 and January 2018 at our hospital. Only women at ≤35 years of age receiving the first cycle were analyzed. P < 0.008). The number of oocytes retrieved was significantly lower in overweight and obese groups than in the normal weight group (P < 0.008). The number of transferable embryos was significantly higher in normal weight group than overweight and obese groups (P < 0.008). Embryo implantation rate, clinical pregnancy rate, full-term birth rate, and live birth rate did not differ among the 4 groups. The cycle cancellation rate was lower in the overweight and obese group than normal weight group (P < 0.008). The miscarriage rate was higher in the obese group than the normal weight group (P < 0.008). In multivariate logistic regression analysis, abnormal BMI was an independent risk for miscarriage .  A total of 1782 women were included in the analysis: 42 were underweight, 742 were overweight, 198 were obese, and 800 were normal weight. Gonadotropin dosage and duration were comparable between underweight and normal weight groups but were significantly higher/longer in overweight and obese groups ( Overweight and obesity are associated with poor outcomes in PCOS patients receiving ultra-long protocol. Measures to reduce body weight should be encouraged in overweight and obese PCOS women at ≤35 years of age prior to assisted reproductive technology (ART). Appr% 4.4%-5.% for sev0 kg/m2) , 40%-70%0 kg/m2) , and the% 1.3%-1.% for morg/m2) [2 . Additiog/m2) [2 .An increasing number of PCOS women are receiving ART. However, studies on the impact of overweight and obesity on the reproductive outcome of PCOS women who undergo in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) have yielded conflicting results . The disIn this retrospective analysis, we examined the potential association between BMI with reproductive outcomes in PCOS women receiving ultra-long protocol for IVF/ICSI-ET.We screened all PCOS women who received IVF/ICSI-ET for the first time between January 5, 2013, and January 29, 2018, at the Department of Reproductive Medicine, Second Clinical Hospital, Hebei Medical University . PCOS was diagnosed according to the 2003 Rotterdam consensus criteria . Women aThe study protocol was approved by the Ethics Committee (No. 2013L-25). Patient consent was not required due to the retrospective nature of the study. Patient data were anonymized.2), progesterone (P), and LH, and endometrial thickness on the day of human chorionic gonadotropin (HCG) administration, the number of oocytes retrieved, mode of fertilization, the number of transferrable embryos, embryo implantation rate, as well as clinical outcomes . Women were considered underweight if BMI was <18.5 kg/m2, normal weight if 18.5 kg/m2 ≤ BMI < 25 kg/m2, overweight if 25 kg/m2 ≤ BMI < 30 kg/m2, and obese if BMI was ≥30 kg/m2 according to the most recent BMI classification by the World Health Organization (WHO) [We retrieved the following information from electronic medical records: age, duration of infertility, BMI, basal follicle-stimulating hormone (FSH), serum basal luteinizing hormone (LH), and serum basal testosterone, the type of controlled ovary stimulating protocol, and the level of estradiol (Eon (WHO) .Clinical pregnancy was defined by the observation of gestational sacs, including ectopic gestational sacs, by transvaginal echographic screening at day 30 post embryo transfer. Biochemical pregnancy was defined as a positive urine or serum HCG test, defined by >5 IU/L of serum beta-HCG at day 14 post embryo transfer, in women who received luteal support until day 14 post oocyte retrieval.2 ≤ 50 pg/mL, and endometrial thickness < 5 mm. Serum hormone levels, including LH, E2, and P, were measured and women received a B ultrasound examination 30 days after GnRH-a treatment. Follicle growth was monitored 4-5 days after rFSH (150 IU to 300 IU/day) was given. The dose and duration of gonadotropin were adjusted based on serum LH and E2 levels until the day of HCG administration. When the number of dominant follicles (diameter ≥ 17 mm) was ≥3, and the diameter of less dominant follicles was ≥14 mm, 6000~10000 IU HCG was given to trigger ovulation. Thirty-seven hours after HCG triggering, oocyte retrieval was performed via transvaginal ultrasound-guided follicular aspiration. Embryos were cultured using a standard protocol. Two cleavage-embryos were transferred three days after oocyte retrieval under ultrasound guidance. Luteal support  was provided after oocyte retrieval and continued if pregnancy ensued and then tapered until discontinuation. Pregnant women received regular prenatal checkups until deliveries. Fresh cycle embryo transfer was cancelled and all embryos were cryopreserved by vitrification if any of the following occurred: (1) ovarian hyper-stimulation syndrome (OHSS) was present; (2) oocyte was obtained for ≥20 to prevent OHSS; (3) embryo developed slowly in ex vivo culture, quality of embryos was poor, or embryos failed to develop; (4) serum P level was >2 ng/mL on day of HCG injection; (5) endometrial lesions were observed on ultrasound.Subjects received 3.75 mg long-acting gonadotropin-releasing hormone agonist (GnRH-a) on day 2 of the menstrual cycle to achieve the goal of FSH < 5 mIU/mL, LH < 5 mIU/mL, EH test. Bonferroni correction was conducted for pairwise comparison for both ANOVA and Kruskal-Wallis H test, with P < 0.008 considered statistically significant. Categorical variables are expressed as number and percentage and analysed using the chi-square test or Fisher exact test (when the expected numbers are <5), with P < 0.05 considered statistically significant. Forward logistic regression analysis was performed to determine the risk factors for miscarriage.Statistical analysis was conducted using SPSS26.0 . Continuous variables with normal distribution are presented as mean and standard deviation and analysed using a one-way analysis of variance (ANOVA). Continuous variables not following normal distribution are presented as median and interquartile range and analysed using the Kruskal-Wallis 2 (range 16.53 to 42.78 kg/m2). 42 (2.36%) women were underweight, 800 (44.89%) women were normal weight, 742 (41.64%) women were overweight, and 198 (11.11%) were obese. The underweight group had significantly higher basal LH than the overweight and obese groups . The four groups were comparable in demographic and other baseline variables including duration of infertility, basal FSH, and basal testosterone.The study flowchart is shown in H test, Bonferroni correction; P < 0.008). The number of oocytes retrieved was significantly lower in the overweight and obese groups than in the normal weight group . The number of oocytes retrieved was significantly lower in the overweight group than the obese group . E2 on the day of HCG injection was significantly higher in the underweight and normal weight groups than in the overweight and obese groups , whereas progesterone on the day of HCG injection was significantly higher in the underweight and normal weight groups than in the overweight and obese groups . No significant difference was observed in LH and endometrial thickness on the day of HCG injection among the four groups. The number of transferable embryos was significantly higher in the normal weight group than in the overweight and obese groups .There was no statistical difference in the fertilization rate with either IVF or ICSI among the four groups . The totP < 0.008). In comparison to the normal weight group, the cycle cancellation rate was significantly lower in the overweight group . The miscarriage rate was significantly higher in the obese group than the normal weight group .No statistical difference was found among the 4 groups in embryo implantation rate, clinical pregnancy rate, live birth rate, twin rate, singleton rate, preterm birth rate, full-term rate, and ectopic birth rate . The cyc2 on HCG day, progesterone on HCG day, endometrial thickness and available embryos, and LH levels . BMI was independently associated with miscarriage   .PCOS is the most common endocrine disease in women of reproductive age. Major clinical features include hypertestosteronemia, high LH, and insulinemia and menstrual irregularities, obesity, and infertility. Follicles in PCOS women undergo premature luteinization and are prone to atresia due to prolonged hormonal aberration in the internal milieu. High LH levels and insulin resistance in PCOS women affected oocyte quality, predisposing these women to increased risk of abortion and reduced live birth rate . ObesityThe study showed that at baseline, women with lower BMI had higher LH, and underweight women had higher LH than women who were overweight or obese. LH and FSH are hormones essential for follicle development, maturation, and ovulation. The causes and mechanisms for low LH in obese women are still not clear, which could be related to higher insulin levels in women and abnormal pulsatile secretion of GnRH by the hypothalamus and LH by the pituitary gland. The total dose and duration of gonadotropin were significantly lower or shorter in the underweight group and the normal weight group than the overweight group and the obesity group, indicating that the dose and duration of gonadotropin increased with BMI. Obese women require higher doses of gonadotropin  for a lo2 were apparently lower than women with normal BMI. Meanwhile, compared with women with normal BMI, women whose BMI was >50 kg/m2 had a markedly higher early spontaneous miscarriage rate. In the current study, we also found that higher BMI was associated with a modest but significant increase in the risk of miscarriage . Normal weight PCOS women had the lowest miscarriage rate versus underweight, overweight, and obese PCOS women. The lowest miscarriage rate was approximately 30% in both underweight and obese PCOS women, suggesting that PCOS women are predisposed to abortion when BMI is too high or too low. Huang et al. [2) was markedly higher than that of overweight or obese PCOS women (BMI ≥ 24 kg/m2). Meanwhile, Plachot et al. [It remains inconclusive whether BMI affects the obstetric outcome of PCOS women receiving IVF/ICSI. Bellver et al.  reviewedg et al.  analyzedt et al.  retrospeThe current study is retrospective in design. As a result, the cause-effect relationship between BMI and obstetric outcomes cannot be drawn. Also, the sample size is not sufficient to allow us to recapitulate the previously reported differences. The cumulative live birth rate was not collected in the current study since a significant proportion (approximately 20%) of the patients who failed the initial cycle with fresh embryos were lost to further follow-up. As such, the outcome measures reflect only part of the entire clinical spectrum.In conclusion, overweight and obesity are associated with poor obstetric outcomes in PCOS patients at ≤35 years of age receiving ultra-long protocol. Weight reduction should be encouraged in such patients prior to ART.


Increasing evidence have indicated the relationship between intestinal dysbiosis and hypertension. We aimed to evaluate the effect of the electroacupuncture (EA) on intestinal microbiota in patients with stage 1 hypertension.93 hypertensive patients and 15 healthy subjects were enrolled in this study. Applying a highly accurate oscillometric device to evaluate the antihypertensive effect of EA. 16S rRNA sequencing was used to profile stool microbial communities from Healthy group, Before treatment (BT) group and After treatment (AT) group, and various multivariate analysis approaches were used to assess diversity, composition and abundance of intestinal microbiota.Firmicutes and lower Bacteroidetes abundance were observed in the BT group compared to the Healthy group. And EA treatment significantly decreased the Firmicutes/Bacteroidetes ratio compared to the BT group. Moreover, at the genus level, there was an increased abundance of Escherichia-Shigella in patients with hypertension, while Blautia were decreased, and EA reversed these changes.In this study, EA significantly decreased the blood pressure (BP) of hypertensive patients. Higher abundance of Our study indicates that EA can effectively lower BP and improve the structure of intestinal microbiota which are correlate with the alteration of blood pressure by electroacupuncture.Trial registration: Clinicaltrial.gov, NCT01701726. Registered 5 October 2012, https://clinicaltrials.gov/ct2/show/study/NCT01701726 Hypertension is China's leading cause of cardiovascular diseases (CVD), including stroke, heart failure, coronary heart disease . BecauseExcept for heredity and environment, intestinal microbiota has increasingly been recognized as a main risk factor for hypertension. Changes in composition and dysbiosis of intestinal microbiota were observed in multiple hypertensive patients and animals . ResearcA total of 108 subjects were enrolled:93 hypertensive patients and 15 healthy. All patients received EA treatment. Among them, 29 of whom had feces collected before and after treatment. 15 normal people were also collected feces after enrollment. The study protocol was approved by Sichuan Regional Ethical Review committee of the Teaching Hospital of Chengdu University of Traditional Chinese Medicine (2012KL-003) and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Patients of essential hypertension were included and clear diagnosis according to the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. Study participants were recruited from the third teaching hospital of Chengdu University of Traditional Chinese Medicine and its surrounding communities. Participants were enrolled if they fulfilled the following criteria: without aAfter inclusion, patients received 18 sessions of acupuncture treatment (3 times weekly for 6 weeks). Each session lasted 30 min. We chose bilateral acupoints of taichong (LR3), taixi (KI3), renying (ST9), neiguan (PC6) according to traditional Chinese medicine theory and based on review of the literature. Disposable stainless-steel needles were used in acupuncture treatments. Insertion was followed by stimulation performed by lifting and thrusting the needle combined with twirling and rotating the needle sheath to produce the sensation known as deqi .In addit24 h ambulatory blood pressure recordings were obtained by a highly accurate oscillometric device  within 24 h before acupuncture treatment and the second day of the final treatment respectively. The TM-2430 monitor measures blood pressure automatically, on the same principle as the conventional mercury sphygmomanometer, with a cuff and a microphone. The interval between measurements can be preselected, and BP readings are recorded on a data processor. Blood pressure measurements were taken automatically every 15 min from 8 AM to 10 PM and every 30 min from 10 PM to 8 AM. If a reading were considered faulty by the device, it was programmed to remeasure, which helped to avoid missing a measurement. The data from the device were then downloaded onto a computer for subsequent analysis. Before the study started, the investigators were trained for the appropriate use of the BP measurement device used in the study, and had to demonstrate proficiency according to the manufacturer’s instruction.A total 73 Stool samples were collected, of which 15 samples were from healthy, 29 samples were collected before acupuncture treatment and 29 samples were collected at the second day of the final treatment. Fecal genomic DNA was isolated from stool samples by using a QIAamp DNA Stool Mini Kit  according to the kit protocol with modifications. The quality of extracted genomic DNA was checked by using the 2% agarose gel, and the DNA concentrations were determined with QuantiFluor™-ST Handheld Fluorometer with Blue Channels . The extracted DNA was stored at − 20 °C.Bacteria within each sample were identified via 16S rRNA Sequencing. PCR amplification for the V4 hypervariable regions of the bacterial 16S rRNA gene was performed using the 515F forward (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R reverse (5′-GGACTACHVGGGTWTCTAAT-3′) primers. Nextera XT V2 index kit Sets A and B were used to barcode PCR products and the AMPure XP bead system  was used for purification. Barcoded DNA underwent quantification, normalization and pooling to develop sequencing libraries which were then sequenced on the Illumina MiSeq platform . The Quantitative insights Into Microbial Ecology (QIIME) v1.9.1 analysis tool was used to join and de-multiplex forward and reverse sequences. Using the Silva reference database, the open reference operational taxonomic unit (OTU) picking method was used for taxonomic assignments with a pairwise identity threshold of 97%. Prior to downstream analysis, the OTU table was rarefied to 3000 sequences/sample with no samples removed in this step. These OTUs were analyzed by global parameters (described below) including determination of diversity, evenness, richness, and relative abundance of the bacteria identified in each sample.www.majorbio.com). Chao1, Ace, Simpson and Shannon index were used for comparison of alpha diversity. Principle coordinates analysis (PCoA) (Bray–Curtis dissimilarity index) and partial least squares discriminant analysis (PLS-DA) were used to visualize the change of distribution patterns. The unpaired two-tailed t-test was used for comparisons between Healthy and Hypertension. The paired t-test was used for comparisons between BT group and AT group. Spearman’s correlation coefficients were used to assess bivariate relationships. All results with P < 0.05 between groups were considered statistically significant.Paired t-test were performed to compare the baseline and post-treatment blood pressure. 16S rRNA gene sequencing data was analyzed on the free online platform of Majorbio Cloud Platform  and diastolic blood pressure (DBP) were higher in the patients with hypertension, as shown in Table Among 397 patients screened, P < 0.001, P < 0.01, P < 0.05). While DBP and dDBP (daytime diastolic blood pressure) decreased significantly after EA treatment . nDBP (nighttime diastolic blood pressure) did not show any difference before and after treatment (P = 0.051).After the six-week EA treatment, the blood pressure of patients differed significantly Table . SBP . Each sample was normalized to an equal sequencing depth and clustering. After filtering the low abundant < 0.1%) OTUs, 870 OTUs were obtained at a 97% similarity level, from which 16 phyla, 30 classes, 55 orders, 98 families, 294 genera and 554 species were detected. Good's coverage was 99.82%, indicating the sequences identified in this study could represent the majority of bacterial sequences present in the samples. Although rarefaction curves of numbers of observed OTUs per sample suggested new bacteria would be expected with additional sequencing  and Chao1/Ace (species richness) index between the Healthy group, BT group and AT group Fig. : Fig. S2A Venn diagram was used to show the number of common and unique OTUs, thus intuitively describing sample similarity and overlap. The unique OTUs in the Healthy, BT, and AT groups were 10, 75 and 12 respectively. The common OTU number in three groups was 570, accounted for 92.08%, 68.75% and 74.51% of the total number of OTUs in the Healthy group (619), BT group (829) and AT group (765) Fig. a. 5 phylFirmicutes and Bacteroidetes were showed significant dissimilarities among three groups. Higher abundance of Firmicutes (P < 0.05) and lower Bacteroidetes abundance (P < 0.05) were observed in the BT group compared to Healthy group. And EA treatment significantly decreased the relative abundance of Firmicutes (P < 0.01) compared to BT group. Moreover, compared to Healthy group, the ratio of Firmicutes to Bacteroidetes was significantly increased in the BT group (P < 0.001) and reduced in acupuncture group (P < 0.05)  Fig. a.Fig. 4IBifidobacteriaceae and Lachnospiraceae. Bifidobacteriaceae in BT group decreased compared to the Healthy group(P < 0.05). In addition, lower Lachnospiraceae abundance were observed in the Healthy group compared to the BT group (P < 0.05).At the Family level Fig. c, differBlautia and Escherichia-Shigella. The relative abundance of Blautia in the BT group was significantly decreased compared to the Healthy group (P < 0.01), but increased after EA treatment (P < 0.05). In contrast, BT group showed greater abundance of Escherichia-Shigella than that in the Healthy group (P < 0.05) and EA treatment (P < 0.05) decreased the abundance of Escherichia-Shigella.At the genus level Fig. d, differf_Streptococcaceae  and g_Escherichia-Shigella  showed positive correlations with DBP. In addition, the negative correlations were observed for g_Lachnoclostridium  and g_Eubacterium hallii  with SBP. Moreover, g_Blautia were simultaneously found significant negative correlation with SBP  and DBP .We further assessed the correlation between the 10 most prevalent bacteria at family- and genus-level and blood pressure Fig. . The f_SFirmicutes and Bacteroidetes changed in the patients before treatment, while EA significantly reduced Firmicutes abundance and the ratio of Firmicutes to Bacteroidetes. Moreover, EA increased the relative abundances of Blautia and reduced that of Escherichia-Shigella compared with BT group. Therefore, we suggest that the mechanism of reducing blood pressure by EA might be closely related with structural changes of gut flora.There is convincing evidence suggesting that balance of intestinal microbiota is an important factor in hypertension although the mechanism is complex and in part unknown –23. In tP < 0.001) and DBP . This finding was similar with a randomized, controlled, assessor-blinded pilot trial to assess the effects of acupuncture on prehypertension and stage 1 hypertension, in which SBP was reduced 5.7 mmHg [This study showed that EA treatment at the aucpoints of taichong (LR3), taixi (KI3), renying (ST9) and neiguan (PC6) significantly decreased the SBP -salt hypertensive mice) [Firmicutes and a lower abundance of Bacteroidetes were exhibited in individuals with stage 1 hypertension compared to healthy group. These findings were in agreement with the findings of Nosheen et al., who have demonstrated higher Firmicutes/Bacteroidetes ratio in patients with grade 3 hypertension [In addition, our data showed that EA decreased the ratio of ysbiosis . The altrtension , 32, 33.ectively . Same reectively . In the ve mice) –37. In ortension . Thus, tBlautia and Escherichia-Shigella at genus level. Blautia is a group of bacteria that produces multiple SCFAs, including acetate [Blautia has been detected in hypertensive patients with preeclampsia compared to healthy, pregnant control subjects [Blautia was also negatively correlated with systolic BP in spontaneously hypertensive rats and healthy male volunteers [Blautia in EA group may be closely related to decreased blood pressure. Escherichia-Shigella is a group of gram-negative bacteria containing lipopolysaccharides (LPS) in their cell walls [Escherichia-Shigella after EA treatment may suggest an improvement inflammatory status of the host.Moreover, we observed the regulation of EA on  acetate , propion acetate , succina acetate . SCFAs a acetate , 41. Ace acetate . Another acetate . In addi acetate –44. Lowesubjects , which wlunteers , 47. In ll walls . It is kll walls , which ill walls –51. Bomfll walls , 52 descll walls . In thisF_Streptococcaceae,g_Eubacterium hallii and g_Lachnoclostridium were observed correlation with blood pressure. Gut Streptococcaceae is also associated with inflammatory bowel disease [Streptococcus from gut might involve in the formation of atherosclerotic plaque microbiota [Eubacterium hallii is a common member of the adult gut microbiota that can produce butyrate from lactate and acetate, and convert 1,2-propanediol to propionate [Eubacterium hallii increased energy expenditure and fecal butyrate concentrations, thus modifying bile acid metabolism of db/db mice. Lachnoclostridium, a species belonging to Lachnospiraceae, was also a butyrate-producing bacterium [We also identified a significant correlation between BP values and 5 microbial taxa in the family at genus level.  disease  and live disease . Moreovecrobiota . Eubacteopionate , 58. Udaopionate  demonstracterium , which cacterium .Ruminococcaceae, Clostridiales  were higher in non-treated hypertensive (SBP:153.1 ± 14.6) than normotensive (SBP:109.7 ± 7.1) [Clostridia in hypertensive rats in the colonic microbiota [Ruminococcaceae and Clostridiales should be enough attention in hypertensive patients.In addition, we noticed an interesting phenomenon that hypertensive patients seem to produce more types of bacteria, although the abundance of these bacteria is very low. Further analysis showed that most of these bacteria belong to rtension . Another7 ± 7.1) . An animcrobiota . TherefoAs to the underlying mechanism or the target of acupuncture, neuroendocrine immunity system may be considered as an important pathway. As we mentioned, the association between intestinal microbiota and the host are complex and bi-direction. On the one hand, intestinal bacteria can modulate the host by producing a wide range of metabolites  and by their own component. Some of these metabolites and the bacterial components are risk markers for hypertension, inflammation and metabolic syndrome. On the other hand, the metabolism of host can regulate the microbiota in gut. For instance, the colonocyte metabolism works as a control switch, mediating a shift between homeostatic and dysbiosis communities . Also, sThere were several limitations in this study. Firstly, we did not observe the long-term effect of EA on hypertension and intestinal microbiota. A study has reported that the antihypertensive effect of acupuncture could be decreased with the elapse of time . AnotherThe present study shows that electroacupuncture can effectively lower blood pressure and improve the structure of intestinal microbiota which are correlate with the alteration of blood pressure by EA. These findings could inspire future research for understanding the mechanism underlying the therapeutic effects of acupuncture on hypertension.Additional file 1: Fig. S1 Alpha diversity rarefaction curves of samples based on species-level OTUs.Additional file 2: Fig. S2 Analysis of principal coordinate analysis (PCoA) with the bray_curtis distance matrix on the OTU level.Additional file 3: Table S1. Unique OTUs in BT group.