Cochlodinium polykrikoides in the surface sediments of South Sea (Tongyeong coast), South Korea, were analysed using next-generation sequencing (NGS) and morphological approaches. Dinoflagellate cysts can be highly abundant  and have diverse species composition. A total of 35 taxa of dinoflagellate cysts representing 16 genera, 21 species (including four unconfirmed species), and 14 complex species were identified by NGS analysis. Cysts of Scrippsiella spp (mostly Scrippsiella trochoidea) were the most dominant and Polykrikos schwartzii, Pentapharsodinium dalei, Ensiculifera carinata, and Alexandrium catenella/tamarense were common. Thus, a combination of NGS and morphological analysis is effective for studying the cyst communities present in a given environment. Although C. polykrikoides developed massive blooms during 2013–2014, microscopy revealed low density of their cysts, whereas no cysts were detected by NGS. However, the vegetative C. polykrikoides not appeared during 2015–2017 in spite of the observation of C. polykrikoides cysts. This suggests that the C. polykrikoides blooms were not due to development of their cysts but to other factors such as currents transporting them to a marine environment suitable for their growth.The total dinoflagellate cyst community and the cysts of  Cysts are typically preserved in sediments, thereby providing an integrated record over time of the presence of cyst-producing dinoflagellates. Functions attributed to cysts include species dispersal, survival under unfavourable conditions, and bloom initialization4. Cyst surveys give information on history of harmful species in a given area, thereby providing an indication of the potential for future blooms, and it may reveal species rarely observed in the plankton, owing to rare, short-lived, or vegetative stages that are difficult to identify5.Dinoflagellates are one of the most diverse and abundant groups of marine plankton. Some dinoflagellates are known to cause outbreaks of harmful algal blooms in coastal waters around the world, and can be extremely toxic, leading to significant damage of local aquaculture and wildlife. The life stages of dinoflagellates form an important component of their ecology and biogeography. Around 200 of the approximately 2000 existing species of marine dinoflagellates are known to form resting cysts as part of their life historyCochlodinium polykrikoides has been one of the most frequently occurring harmful red tide species responsible for fish kills since 19956. In particular, the Tongyeong coastal area, located in the South Sea of South Korea, is one of the most severely bloomed areas of C. polykrikoides7. Although, C. polykrikoides blooms frequently developed in many coastal countries during the last decade, cysts of C. polykrikoides are still understudied10. Our previous study reported the cyst morphology of C. polykrikoides in the natural surface sediment samples9, but the mechanisms of C. polykrikoides bloom formation by germination of cysts are mostly unknown.In South Korea, 11. In other words, the challenge is not simply a matter of discovering ‘What is there’, but also ‘Why it is there?’ In this context, a major issue relates to how dinoflagellate cysts are temporally distributed and whether they follow any trends in their distribution.Advances in DNA sequencing technology and bioinformatics have significant potential to strengthen biological monitoring in the ocean. The challenge of modelling and predicting ecological changes requires the linking of taxonomic data to the functional roles of individual microbial groups in biogeochemical cyclesC. polykrikoides by focusing on 18S rDNA using next-generation sequencing (NGS) tool and morphological observation in samples harvested from the South Sea (Tongyeong coasts) during the red-tide and the non-red-tide season in South Korea.To this end, we investigated the diversity and distribution of cysts in dinoflagellate communities and The NGS results obtained for the eukaryotic organisms in surface sediments of Tongyeong coasts are summarized in Table Pentapharsodinium-Scrippsiella complex, which included Pentapharsodinium tyrrhenicum, P. dalei, Scrippsiella trochoidea, S. sweeneyae, and S. erinaceus, constituted the most predominant taxa with average proportions of 63.68% in 2013 and 62.20% in 2014. Gonyaulax spinifera (3.94% in 2013 and 11.99% in 2014), Alexandrium complex species I, including Alexandrium pacificum, A. catenella/tamarense, and A. fundyense (5.49% in 2013 and 6.50% in 2014), Woloszynskia complex species, including Woloszynskia cincta and W. halophila (6.09% in 2013 and 4.17% in 2014), Alexandrium complex species II, including A. minutum, A. insuetum, A. tamutum, and A. lusitanicum (3.23% in 2013 and 3.74% in 2014), Gymnodinium aureolum (3.47% in 2013 and 2.65% in 2014), Gyrodinium impudicum (2.29% in 2013 and 0.99% in 2014), and S. hangoei (2.31% in 2013 and 0.62% in 2014) were the next most abundant species and represent more than 1% of the total read counts of OTUs. These major taxa account for more than 90% of the total read counts of cysts of dinoflagellates. In particular, G. spinifera was present in higher proportion in 2014 compared to 2013.The composition of the dinoflagellate cyst assemblages is shown in Fig. −1 dry weight  in 2013 and 111–1,532 cysts g−1 dry weight (average = 716 cysts g−1 dry weight) in 2014  were the most predominant at average proportions of 40.72% (27.93% for S. trochoidea) in 2013 and 43.55% (21.05% for S. trochoidea) in 2014. Moreover, the predominant cysts  in 2013 were of Polykrikos schwartzii (9.65%), Protoperidinium sp. , Pentapharsodinium dalei (8.82%), Ensiculifera carinata (8.24%), Phaeopolykrikos hartmanii (5.23%), and Alexandrium catenella/tamarense (5.07%) and the predominant cysts in 2014 were of Ensiculifera carinata (13.77%), Protoperidinium sp , and Alexandrium catenella/tamarense (8.94%). Thus, the proportion of total common cysts (average cyst abundances > 5% in 2013 and 2014) were 77.92 and 77.63%, respectively , and five sampling sites had an abundance of 8–14 cyst g−1 dry weight in 2014 (average = 2 cysts g−1 dry weight)  Fig. .Figure 512, and it is also being used to monitor marine biodiversity14. However, we are unaware of any reports that have attempted to use eDNA to monitor dinoflagellate cysts in surface sediments. Approximately 200 dinoflagellate species produce resting cysts during their life cycle15 and these are deposited in the sediments. The cyst population can serve as inocula for the recurrence of blooms of harmful plankton species2. The dinoflagellate cysts in samples from surface sediments of the Tongyeong coast revealed a high abundance and diverse species composition  has begun to be employed in aquatic environmental researchS. trochoidea (the most predominant cyst) and Pentapharsodinium dalei in morphological results occurred at average proportions of 25.4 and 5.9%, respectively . In particular, they occurred frequently in spring and summer. In the NGS analysis, the assemblage composition is dominated by the Pentapharsodinium-Scrppsiella group, Gonyaulax spinifera, Woloszynskia group, Alexandrium group, and Gymnodinium aureolum. These dominant species detected by NGS were consistent with the morphological results. However, Pentapharsodinium spp. and some Scrppsiella spp. showed very high genetic similarity. Many researchers26 reported that Pentapharsodinium is a sister group or primitive lineage of Scrippsiella species based on molecular phylogeny of Calciodinellaceae. In this study, NGS analysis detected 35 taxa compared to only 27 cysts identified using microscopic observation. Thus, NGS analysis can allow researchers to detect higher number of species than microscopic analysis. This is because cysts have high morphological similarity and therefore are difficult to discriminate.Small and orthoperidinoid dinoflagellate species were the most common. Cysts of C. polykrikoides causes red tides in Tongyeong coast almost every year7. In particular, they developed massive blooms with density greater than 5,000 cells mL−1 in summer of 2013  and 44 sites during summer in September 2014 (i.e. during the blooming period of C. polykrikoides) using a gravity core sampler (diameter: 5 cm). The top 2 cm of the core samples were sliced and preserved immediately in the dark at 4 °C . This costal water is a eutrophic system that is subjected to strong mixing between the surface and bottom layers, in addition to supplementation of nutrients by food supply from fisheries. Currents in the studied area are not strong, because water depths are generally shallow (<20 m), and many islands are present. In order to analyse the distribution and composition of the dinoflagellate cysts morphologically and using NGS technology, we sampled the surface sediments from 50 sites during the winter, in December 2013 , and then photographed using an AxioCam MRm digital camera (Zeiss). Ten grams of sample was weighed in wet condition and dried in an oven to 110 °C for 1 day to measure the water content, and cyst concentrations in this study were estimated as the sum of living and empty cysts, which were presented as the number of cysts per gram of dry sediment.The core samples for morphological analysis were collected as described by Li The core samples were stored in dark and cool conditions (at 4 °C) for at least six months for decomposition of organic matter and organisms. Ten grams (wet weight) of the stored samples from each site were dried at 60 °C for 1 d in a drying oven. Thereafter, the dried sub-samples (2 g dry weight) from approximately 10–20 sites were merged and divided into the inner group (IG), middle group (MG), and outer group (OG) from the inner to outer sea, for the 44–50 sampling sites in the survey area , and 1 μL DNA template. The V4–V5 region of the 18S rDNA gene was targeted using the universal primers TAReuk454FWD1 (5′-CCA GCA SCY GCG GTA ATT GG-3′) and TAReukREV3 (5′-ACT TTC GTT CTT GAT YRA-3′)35. Three PCR reactions were performed in distinct tubes, and then mixed to obtain more accurate NGS results. The target region (V4–V5) was chosen because of its high coverage of almost all phyla in conventional and metagenomic studies36. Each primer was tagged using Illumina pre-adapter and locus-specific primers following the manufacturer’s instructions . The reaction profile consisted of an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 52 °C for 45 s, and extension at 72 °C for 1 min, and a final extension step of 72 °C for 5 min. Under these conditions, 800–1,000 ng PCR product was obtained using a BioRad T100 thermal cycler . Products from triplicate reactions were pooled and electrophoresed in 1.0% agarose gels, stained with ethidium bromide, and visualized under ultraviolet transillumination. Before NGS analysis, the amplified PCR products were individually purified using an UltraClean PCR Clean-up Kit (MoBio), and their DNA concentration was measured in a Bio-analyzer 2100 .To analyse dinoflagellate cysts, the 18S rDNA V4-V5 gene was targeted for the NGS analysis. The genomic DNA from cysts collected was extracted using beads in a Power Soil DNA Isolation kit  and diluted to a final concentration of 20 ng μL37 to check basic statistics, such as GC%, per base quality score distribution, and poor-quality sequences were flagged. Also, ambiguous and chimeric reads also were removed and noised sequences (denoising), which involved OTUs with read 1, 2, and 3, were removed at cut-off of 97% , low-quality sequences (score <33), singletons, non-target sequences. Using Basic Local Alignment Search Tool (BLAST)39, all of the sequence reads were compared with those at the National Center for Biotechnology Information (NCBI) database. Sequence reads with an E-value <0.01 were considered for further analysis. A pairwise global alignment was performed on selected candidate hits to find the most similar sequences. The taxonomy of the sequence with the highest similarity was assigned to the sequence read . To analyse operational taxonomic units (OTUs), the CD-HIT-OTU software40 was used for clustering, and metagenomic functional information to calculate alpha-diversity, including Shannon-Weaver diversity, Chao richness, and Simpson evenness based on the OTU table was generated using the closed-reference protocol in Mothur41 and QIIME42. All the six datasets were deposited in the Sequence Read Archive database at the NCBI under accessions SRS2412382 (PRJNA397358).Similar amounts of PCR product from each sample were analysed using a Mi-Seq platform. The data were pre-processed using MiSeq Control Software (MCS) v2.4.1. Raw sequences were first analysed using FastQC43 based on Bray-Curtis distance matrices obtained from the read count of OTUs.To test the significance of similarity between the six sequenced samples, the multidimensional scaling (MDS) analysis was performed using PRIMER 6Supplementary information


Dioscorea nipponica (DN), a medicinal plant used to treat myocardial ischemia (MI), metabolites generated by intestinal microflora from DN were identified, and the cardioprotective efficacy of these metabolites was evaluated. Our results demonstrate that diosgenin is the main metabolite produced by rat intestinal microflora from DN. Further, our results show that diosgenin protects the myocardium against ischemic insult through increasing enzymatic and nonenzymatic antioxidant levels in vivo and by decreasing oxidative stress damage. These mechanisms explain the clinical efficacy of DN as an anti-MI drug.Studying the biotransformation of natural products by intestinal microflora is an important approach to understanding how and why some medicines—particularly natural medicines—work. In many cases, the active components are generated by metabolic activation. This is critical for drug research and development. As a means to explore the therapeutic mechanism of  Cinnamomum cassia have been evaluated for their protective effect against isoprenaline- (ISO-) induced myocardial ischemia in rats [Dioscorea nipponica (DN) have been successfully developed as effective herbal medicines by the pharmaceutical industry for treating IHD. These herbal medicines developed from DN have been in use since the 1970s; they include “Polysponin” approved by the former Soviet Union's Ministry of Health, and Diosconin Tablet and Di'ao Xinxuekang Capsule produced in China and indicated for myocardial ischemia or angina pectoris.Ischemic heart disease (IHD) is a significant threat to human health, leading to high morbidity and mortality in the Western world, even in China. It is estimated by the World Health Organization that IHD will be the leading cause of death in the world in the coming decades . Recentl in rats , 3. In pDioscorea species in order to ensure the authentic origin of DN during herb collection [Dioscorea saponins but contained no free diosgenin [Dioscorea bulbifera extract, which is similar to the DN extract that both are rich in diosgenin, improves vascular function by superoxide dismutase (SOD) and catalase (CAT) activity. Thus, it is worthwhile further exploring the anti-MI activity of diosgenin [In the previous study, authors of the present report established a mixed microscopic method for differentiating DN from several llection  and lateiosgenin , 6 and tiosgenin . These fiosgenin . Thus, wiosgenin . It is riosgenin .Recently, identification of metabolites involved in the biotransformation of phytochemicals by intestinal microflora has been suggested as a potentially effective means to determine which compounds are active in living systems and to gain a better understanding of how herbs affect biological processes , 12. To General anaerobic medium broth (GAM broth), vitamin K1, and hematin chloride were purchased from Shanghai Kayon Biological Technology Co. Ltd. . Analytical grade ethanol purchased from the Merck  was used for the extraction of DN samples. Water was purified using a Milli-Q water system . Acetonitrile  and methanol  were used as the mobile phase for analysis. Formic acid  was added to the mobile phase for analysis. Isoprenaline was purchased from Sigma . Test kits for SOD, CAT, GPx, T-AOC, and MDA were all purchased from Nanjing Jiancheng Biotechnology Institute . Other reagents were of analytical purity.Standards (purity 98%) of protodioscin, dioscin, gracillin, diosgenin, protogracillin, and polyphyllin V were purchased from Phytomarker Ltd., Tianjin. Pseudoprotodioscin was provided by National Institute for Food and Drug Control . The chemical structures of standards are shown in The rhizomes of DN were collected from the cultivation base in the city of Lingbao in Henan Province, China. All crude drugs were of high quality and authenticated by Dr. Tao Yi, School of Chinese Medicine, Hong Kong Baptist University. Corresponding voucher specimens (number DN-01) were deposited in the Chinese Medicines Center, Hong Kong Baptist University.w/w) was thus obtained. The DN extract of 0.1 g was diluted in 10 mL sterile water, filtered through a 0.22 μm pore-sized filter . The filtrate was collected in a sterile tube. These tubes were used as in vitro biotransformation vessels. The dried extracts and diosgenin were suspended in 1% (w/v) aqueous carboxyl methylcellulose for administration to animals.The DN samples were dried at 60°C and then pulverized into powder. The powder of DN (200 g) was extracted in an ultrasonic bath with 1000 mL 80% ethanol at room temperature for 1 h. The operation was repeated twice. The combined extracts were evaporated to remove ethanol at reduced pressure in a rotary evaporator (50°C) and then were lyophilized with a freeze-drying system. DN extract  suspensions. The fecal suspensions were filtered through two layers of gauze. The filtered suspensions were then used to inoculate the in vitro biotransformation vessels.Fresh rat feces were immediately collected from ten healthy male Sprague Dawley rats (220–250 g). Samples were collected and mixed, on site, on the day of the experiment and were used immediately. Fecal slurries were prepared by mixing fresh feces samples with autoclaved PBS  to yield 10% (A 30 g GAM broth was dissolved in 1000 mL water (70°C), filtered, while hot, treated with antibacteria process with high pressure (0.15 MPa) and temperature (121°C) for 20 min, and cooled to 45°C. The GAM broth solution was then transferred to an anaerobic chamber , and 1 mg vitamin K1 and 6 mg hematin chloride were dissolved in the solution. Then biotransformation vessels were sterilized and filled with 30 mL of GAM broth solution.w/v), and then 1 mL of DN extract was added. In vitro biotransformation was run under anaerobic conditions for a period of 48 h. Two different control experiments were conducted: (i) incubations of the intestinal microflora in medium lacking the DN extract to monitor metabolites arising from basal metabolism and (ii) incubations of the DN extract in medium without intestinal microflora to monitor changes due to the purely chemical transformation of precursor compounds of the substrate.Vessels were inoculated with 3 mL of fecal suspension  was used for sample separation at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) using a gradient program of 0–2 min, 20% B; 2–12 min, 20–28% B; 12–20 min, 28–45% B; 20–35 min, 45–48% B; and 35–46 min, 48–75% B. The sample volume injected was 2 μL, and the solvent flow rate was set at 0.4 mL/min. For mass spectrometric determination, the UPLC system was hyphenated with an ultrahigh definition accurate mass quadrupole time-of-flight mass spectrometry (MS) system (Agilent Technologies G6540A) by a multimode ionization source (G1978-65339) interface. The conditions of MS analysis were optimized as follows: drying gas (N2) flow rate, 8.0 L/min; drying gas temperature, 300°C; nebulizer, 45 psi; capillary, 2500 V; and fragmentor voltage, 150 V. Mass spectra were recorded across the range m/z 100–1700 in both positive and negative modes. All operations and data analysis were controlled by Agilent MassHunter Workstation software version B.04.00.An Agilent 1290 series UPLC system  equipped with a binary pump, an autosampler, and a thermostatically controlled column compartment was used for the chromatographic analysis. A Waters ACQUITY™ BEH C18 column .w/v aqueous CMC-Na, i.g.); (2) model group (ISO injection only); (3) positive group ; (4–6) diosgenin treatment groups. For the diosgenin groups, diosgenin was administered at rates of 20, 40, or 80 mg/kg for 3 days after ISO injection. Dosage was determined from our previous study [A total of 42 rats were randomly divided into 7 groups: (1) normal control  to induce experimental MI twice at an interval of 8 hours on the first day. On the last day of experiment (4th day), the animals were sacrificed. After the rats were anesthetized with diethyl ether, then the blood samples were collected from the femoral arteries of rats. Serum was saved at −80°C following centrifugation at 4°C at 4000 rpm for 20 min.μm, Leica RM2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and examined by light microscopy  at 200x magnification.Immediately after the sacrifice of the rats, the hearts were removed, washed with iced normal saline, fixed in 10% formalin, and decalcified with formic acid (31.5% formic acid and 13% sodium citrate). The hearts were embedded in paraffin for sectioning by standard histological methods [P values less than 0.05 were considered statistically significant.Activities of SOD, GPx, CAT, T-AOC, and MDA were measured using kits according to the manufacturers' instructions. Values obtained from the experiments were expressed as the means ± standard deviation (SD). The statistical significance of the differences was assessed by ANOVA followed by post hoc test with LSD method , 15. P vDioscorea saponin aglycones), for structural and polar reasons, cannot produce enough strong ions with ESI; in these cases, APCI can be used to increase the ion yield. Therefore, in our previous study, ESI and APCI were used to detect saponin glycosides and saponin aglycones, respectively [Electrospray ionization (ESI) is a soft ionization technique. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry which utilizes gas-phase ion-molecule reactions at atmospheric pressure. ESI is today the most widely used ionization technique in chemical and biochemical analyses , 17. Howectively . Due to To solve this problem and save time, an ESI/APCI multimode ionization source was used for LC-MS analysis in the present study. The ESI/APCI multimode source is unique in that it incorporates both ESI and APCI into a single ion source, and it can simultaneously generate ions by ESI and APCI. The main advantages of the multimode source include eliminating the time required to switch ion sources on an instrument and eliminating the need to run samples twice to improve lab productivity . CompareThe effect of intestinal microflora on drug metabolism has received increasing attention in recent years. Anaerobic bacteria present in the small intestines are quite diverse in species, and different species produce enzymes which are different in functions; it is these enzymes that are also responsible for drug biotransformation in organisms. Incubation of test drugs, particularly phytochemicals, with fresh fecal specimen is a common means for investigating this kind of biotransformation , 12.m/z values and MS spectra with the data in the literature [In the present study, DN extract was tested by this method; the resulting metabolic profile is shown in terature . Compareterature .Light microscopy of heart tissue sections from normal control rat myocardium showed obvious integrity of myocardial membrane, a normal myofibrillar structure with striations, a branched appearance, and continuity with adjacent myofibrils . Tissue It is widely accepted that isoprenaline (ISO) injection can readily induce acute MI in rats; it is also widely accepted that antioxidant activity is one of the key mechanisms of anti-MI efficacy , 3, 19. 2O2), superoxide anion (O2−), and hydroxyl radicals (OH·), are generated during ischemia and particularly during reperfusion [2O2. Other endogenous antioxidant enzymes include catalase and glutathione peroxidase. Exogenous antioxidants are mainly derived from food and herbs. Numerous types of bioactive phytochemicals, such as polyphenolics, glycosides, and steroids, belonging to exogenous antioxidants, have gained attraction in clinical as well as research areas [Oxidative stress plays an essential role in the pathogenesis of MI injury. One cause is reactive oxygen species (ROS) resulting from mitochondrial dysfunction via the electron transport chain during MI. The major ROS, such as hydrogen peroxide , while MDA levels increased significantly (##P < 0.01) , except the SOD, GPx, T-AOC, and MDA serum levels in the low dosage diosgenin-treatment groups. These findings suggest that the anti-MI mechanism of diosgenin is related not only to more varieties of enzymatic antioxidant but also to nonenzymatic antioxidants.Groups administered with different dosages of diosgenin and DN extract exhibited varying degrees of antioxidant activity, as revealed by these five markers Figures . Patholoin vivo. Given their known benefits, these antioxidant properties may be responsible for the therapeutic effects of DN in the treatment of MI.Diosgenin administered orally at doses of 20, 40, and 80 mg/kg showed significant dose-dependent increment of the SOD, GPx, and T-AOC serum levels and dose-dependent reduction of the MDA serum levels. The peak treatment effects of diosgenin (78.9% and 77.3%) were recorded with the dose of 80 mg/kg in the SOD level and GPx level, which were higher than those of propranolol with the dose of 10 mg/kg (57.9% and 70.5%), compared with the ISO model group. These results demonstrate that diosgenin has distinct antioxidant properties β-carotene could significantly improve heart function of the isolated ischemic/reperfused (I/R) rat hearts through enhancing antioxidant capacity. However, increasing β-carotene dosage did not add any cardiovascular benefits. Moreover, the agent may mediate and, indeed, may exacerbate existing I/R pathological mechanisms [Reduction in oxidative stress caused by ischemia-reperfusion injury is clearly an appropriate countermeasure to the major challenges associated with ischemia. It is worth noting that the antioxidant is not a panacea. A recent study reported that administration of 30 mg/kg/day chanisms . Antioxiin vivo and to a decrease in lipid peroxidation. These phenomena help explain the clinical efficacy of DN as an anti-MI drug.The present study identified the metabolites from DN through analysis of organ-specific biotransformation and validated the cardioprotective effects of the screened metabolites using an isoprenaline-induced myocardial ischemia rat model. The findings of the present study provide evidence that, first, diosgenin is generated from DN by intestinal microflora; second, diosgenin can protect the myocardium against ischemic insult in a dose-dependent manner, almost comparable to the effect of DN extract; and, third, DN's protective effect can be attributed to the increase of enzymatic and nonenzymatic antioxidants


EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four  that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an  Patients with chronic kidney disease (CKD) have a high incidence of cardiovascular disease (CVD). In fact, the mortality from CVD is very high in CKD patients even from the early stages of the disease when the patient does not present with symptoms , increasing the expression of adhesion molecules  on the endothelial repair process developed by EPCs.Microvesicles (MV) can transfer proteins, cytokines, mRNAs, or miRNAs to target cells and influence their biological behavior  plus endothelial cell-growth medium supplements  and 10% fetal bovine serum . HUVECs were used for experiments between passages four and nine. HUVECs at 80% confluence were incubated with or without indoxyl sulfate (IS) at 256 μg/ml for 24 h. After the incubation period, cells were characterized for flow cytometry and culture supernatants were used for isolation of EMV. We first established the experimental model using a concentration- and time-response curve.Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Systems  and cultured at 37°C in a 5% COExpression of ICAM-1, PECAM-1, VE-cadherin, and VCAM-1 were measured in HUVECs after 24 h of treatment with IS (256 μg/ml). Next, VE-cadherin , PECAM-1 , ICAM-1 , and VCAM-1  antibodies were used to assess the expression in the experimental conditions. HUVECs were incubated with the antibodies for 20 min in darkness at room temperature. Then, cells were washed with PBS and fixed with BD CellFIX (BD). HUVECs without antibody were used as a reference (negative control). Mean fluorescence intensity (MFI) of different antibodies was used to analyze the cytometer data.EMV from the culture medium of IS-treated and untreated HUVECs were isolated by ultracentrifugation. The media was centrifuged (Heraeus Labofuge 400R) at 409 g for 5 min at 4°C to remove any intact cells, followed by centrifugation at 789 g for 10 min at 4°C to remove cell debris. The media was then transferred to ultracentrifuge 25 × 89 mm polypropylene tubes  and centrifuged at 18,000 g for 90 min at 10°C in an Optima XPN-100 ultracentrifuge with 70Ti rotor (Beckman Coulter). The EMV were sedimented owing to relative centrifugal forces. The supernatant containing EMV-free media was removed and the pellets containing EMV were resuspended in PBS and quantified by flow cytometry . Absolute values of MV were calculated using the following formula: (MV counted x standard beads/ L)/ standard beads counted . Results were expressed as the number of MV per microliter of culture medium. MV derived from IS-treated HUVECs were defined as indoxyl sulfate EMV (IsEMV).After 24 h incubation with or without IS (256 μg/ml), culture supernatants were collected. EMV were isolated as previously described. The pellet was resuspended in PBS and 10 μl aliquots were incubated with annexin V, ICAM-1, PECAM-1, VE-cadherin, or VCAM-1 for 20 min with gentle regular shaking at room temperature. EMV were quantified by flow cytometry (FC 500 Series). Prior to the sample acquisition, the samples were subjected to a separate and combined labeling reaction using all reactive  to compensate for the fluorescence using compensation tools on the flow cytometer. In a previous study, we established a MV gate on the FC 500 cytometer using a blend of size-calibrated beads with diameters of 0.3, 0.5, and 1.0 μm  according to the manufacturer's protocol. The NanoString nCounter® platform was used to screen the expression levels of 800 miRNAs using pools with miRNAs purified from controls and IsEMV. A total of 100 ng miRNAs (or 3 μl) was used per sample and conditions were set according to the manufacturer's recommended protocol . The nSolver 2.6 software was used to analyze and normalize the raw data using the top 100 most abundant miRNAs in all samples.Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study. EPCs were obtained from human peripheral blood mononuclear cells from healthy donors by density gradient centrifugation  and grown in EC basal medium-2 supplemented with growth factors  and 15% autologous plasma. Mononuclear cells were plated onto fibronectin  in coated, six-well plates at a density of 5 × 106 cells/well and then incubated at 37°C in a 5% CO2 atmosphere for 3 weeks. Four days later, cells in suspension were removed and fraction of attached cells was cultivated with EBM-2 supplemented with 15% autologous plasma. On 7th day of EPCs culture, 104 EMV or IsEMV (MV/ml) were added  (Invitrogen) is oxidized by ROS to become ethidium, which emits red color, and was used to measure superoxide anion. HUVECs and EPCs were exposed to HE (2 μM) for 15 min at 37°C. Quantitative analysis was performed on a flow cytometer .The percentage apoptosis was measured by annexin V staining. HUVECs and EPCs were obtained by mechanical disruption and washed once with annexin V binding buffer . Cells were then resuspended in annexin V binding buffer and annexin V was added following the manufacturer's protocol. Negative tube controls contained annexin V binding buffer. Quantitative analysis was performed on a flow cytometer .in vitro angiogenesis was evaluated using the Endothelial Tube Formation Assay Kit . After of treatment with EMV or IsEMV, EPCs were obtained by mechanical disruption and seeded on semi-natural Matrigel. Briefly, 50 μl of thawed gel solution was added to each well of a pre-chilled 96 well sterile plate. For the angiogenesis assay, a total of 2,500 cells/well were plated, of which 50% were EPCs obtained from MV treated cultures, and the remaining 50% were mature endothelial cells at low passage stages (3–4). Vascular endothelial growth factor (50 ng/ml) was administered in parallel as an internal positive control. After 4 h, photographs were taken with an optical inverted microscope , and an automated analysis was performed with the ImageJ software (http://rsb.info.nih.gov/ij/). Four parameters were taken for the quantification experiments . Results were expressed as previously described by Izuta et al. , characterized by a cluster of cells surrounded by elongated spindled-shaped cells, were counted manually by visual inspection using an optical inverted microscope  in a minimum of 10 random high-power fields.Cellular extracts from EPCs were prepared according to standard protocols .Data represent the mean ± SEM, and the analysis of variance (ANOVA) with a Bonferroni p = 0.004)   production by flow cytometry. After 24 h of treatment, we observed a significant increase in the MFI of HE in IS-treated HUVECs compared with controls  Figure . Further) Figure .p < 0.001), VCAM-1 (p < 0.001), and VE-cadherin (p = 0.001) in HUVECs at 24 h. However, at 24 h of treatment with IS at 256 μg/ml, no changes were observed in the MFI of PECAM-1 compared with controls.Moreover, we quantified the expression of adhesion molecules, indicative of endothelial activation, such as VCAM-1, ICAM-1, PECAM-1, and VE-cadherin. As shown in Figure p < 0.001) , PECAM-1 (p < 0.001), VE-cadherin (p = 0.002), and annexin V (p < 0.001). Although, VCAM-1 expression did not increase in IsEMV.EMV release was related to endothelial activation. We observed elevated EMV release in IS-treated HUVECs (EMV/μl) compared with controls . Likewise, IsEMV induced a significant increased in the percentage of annexin V+ EPCs  .We observed that IsEMV are able to modulate the differentiation of EPCs The ability of EMV to induce angiogenesis in EPCs was determined. As shown in Figure p = 0.033). Control and EMV-treated EPCs showed normal expression of this protein. We also examined whether IsEMV treatment activated the NFκB pathway. Activation of the NFκB pathway is usually marked by the phosphorylation and degradation of the NFκB inhibitory protein, IκBα, releasing NFκB and allowing its migration to the nucleus  and NFκB levels increased in nuclear fraction (p = 0.022) when compared with EMV-treated EPCs  were upregulated in IsEMV compared with control EMV. A recent study reported links between miR-181a-5p, miR-4454, and NFκB signaling in cartilage degeneration associated with osteoarthritis (Nakamura et al., Our study has some limitations related to the instrumentation and protocols used for identifying and obtaining the EMV. The size detection limits of standard flow cytometry are well known, causing smaller MV to be overlooked. The upper size limit of MV detection is likely >1 μm, as a 0.5 μm polystyrene bead reflects an MV around 1 μm (Chandler et al., In conclusion, our results show for the first time that IS, a difficult to remove uremic toxin in patients with CKD, is capable of inducing endothelial vesiculation with different characteristics that makes MV potential candidates for studying novel biomarkers being a very useful as diagnostic and prognostic tools for vascular diseases. IsEMV have membrane characteristics, miRNAs, and other molecules that reduce the ability of EPCs to regenerate and participate in the signaling pathways involved in apoptosis and oxidative stress. These specific mechanisms may constitute therapeutic targets in patients with CKD.AC, FG, PA, and JC participated in the conception and design of research; AC, FG, PB, and TO performed the experiments, acquired and analyzed data of work; AC, FG, PA, and JC performed interpretation of data; all authors drafted, edited and approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Sudden unexpected death in epilepsy (SUDEP) is a major cause of premature death related to epilepsy. The causes of SUDEP remain unknown, but cardiac arrhythmias and asphyxia have been suggested as a major mechanism of this event. Inflammation has been implicated in the pathogenesis of both epilepsy and ventricular arrhythmia, with interleukin-6 (IL-6) being recognized as a crucial orchestrator of inflammatory states. Our group previously reported that levels of IL-6 were increased in the hearts of epileptic rats. In this scenario, anti-inflammatory actions are among the beneficial effects of fish oil dietary supplementation. This investigation revealed that elevated levels of IL-6 in the heart were markedly reduced in epileptic rats that were treated in the long-term with fish oil, suggesting protective anti-inflammatory actions against dangerously high levels of IL-6. Based on these findings, our results suggest beneficial effects of long-term intake of fish oil in reducing the inflammation associated with chronic epilepsy. The mortality rate in people with epilepsy is substantially higher than that observed in the general population, which is a matter of concern among specialists . In thisStatus epilepticus (SE), in which postmortem examination does not reveal a toxicological or anatomic cause for death” .Adult male Wistar rats (220–280 g) were housed under standard controlled conditions  with food and water n = 9) were used in the experiments.Epilepsy was induced according to the procedure described previously . In brie®, 85 mg/kg). A single daily dose was given via oral gavage between 11:00 and 12:00 a.m. Volume was adjusted according to animal weight, which was verified three times a week during the 90-day treatment period. Each fish oil capsule contained eicosapentaenoic acid (EPA), 180 mg, and docosahexaenoic acid (DHA), 120 mg. Capsule contents were dissolved in 0.009% cremophor, yielding a final concentration of 21.25 mg/ml fish oil, corresponding to 3.82 mg/ml EPA and 2.55 mg/ml DHA, and the final composition of fish oil was administered as 1 ml/250 g of body weight. Animals were killed by decapitation. Hearts were removed, and ventricles were separated and split in half (for Western blot and ELISA procedures) and then stored at −80°C until use.Animals were randomly divided into the following groups: (1) animals treated daily with vehicle (0.009% cremophor) (control vehicle); (2) animals treated daily with 85 mg/kg fish oil (control fish oil); (3) animals with epilepsy treated daily with vehicle (epilepsy vehicle); and (4) animals with epilepsy treated with 85 mg/kg fish oil (epilepsy fish oil). During the 90-day treatment, animals received vehicle (cremophor 0.009%) or fish oil ] followed by Bonferroni posttest. F = 54.78; p < 0.0001]. Animals with epilepsy that were treated with fish oil for an extended time exhibited a marked reduction in IL-6 levels, since an interaction effect was observed between epilepsy and fish oil treatment , as shown in Figure The results of Western blot analysis showed that IL-6 levels in the heart of rats with epilepsy were substantially increased when compared with control rats . Furthermore, the ELISA results show the effectiveness of the fish oil treatment, which reduced IL-6 levels in both control and epileptic rats . There was no interaction effect between epilepsy and fish oil treatment .In Figure In this rodent pilocarpine model of seizures, fish oil supplementation reduced cardiac levels of IL-6, indicating an anti-inflammatory effect of fish oil.Consistent preclinical and clinical findings support the presence of a persistent inflammatory condition in the brain and plasma of subjects with epilepsy , 6, 11. Using the pilocarpine model of epilepsy, Ferrari and colleagues in our group demonstrated that chronic treatment with fish oil promoted neuroprotection and positive plastic changes in the brain of rats with epilepsy . The benOur findings report the beneficial anti-inflammatory effects of long-term oil fish intake in the hearts of rats with chronic inflammation associated with epilepsy. Our results suggest the potential therapeutic value of dietary supplementation with oil fish in patients with epilepsy.All animals were treated according to protocols for animal care, and this study was carried out in accordance with the recommendations established by the Federal University of Sao Paulo. All efforts were made to minimize animal suffering (Comissão de Ética no Uso de Animais—CEUA).MN: conducted the experiments, acquired data, analyzed data, critically discussed the manuscript, and wrote the manuscript. AAH: conducted the experiments, acquired data, analyzed data, and critically discussed the manuscript. AEH, LO, A-CA, and RC: analyzed data and critically discussed the manuscript. EC: conceived the work and wrote the manuscript. CS: critically discussed the manuscript and wrote the manuscript. FS: conceived the work, critically discussed the manuscript, and wrote 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.


Obese children had significantly higher sAA compared with overweight and normal weight children (p < 0.01). SIgA secretion rate was significantly lower in obese and overweight vs. normal weight children (p < 0.01). Multiple-linear regression analysis revealed that body mass index (BMI) (p < 0.05) and diastolic blood pressure (DBP) (p < 0.05) were independent predictors of sAA with CRF acting as a mitigator. Age and BMI predicted SIgA secretion rate (p < 0.05) with BMI (p < 0.001) found to be an independent predictor of SIgA secretion rate. Obesity, based on BMI, was associated with elevated SNS activity and lowered mucosal immunity. CRF-mitigated sympathetic activation was not associated with mucosal immunity.This study examined whether cardiorespiratory fitness (CRF) and body composition are associated with salivary secretory immunoglobulin A (SIgA), a mucosal immunity marker, and salivary alpha-amylase (sAA), a marker of stress-related sympathetic nervous system (SNS) activity, in South African children. Morning (7:30–8:00 a.m.) saliva samples were collected from 132 children . Body composition, resting blood pressure, and predicted maximal aerobic capacity (VO Obesity and associated diseases, such as type 2 diabetes, have reached worldwide epidemic proportions . In partThe combination of obesity and lack of physical activity (PA) have been shown to have negative effects on immune and neuro-endocrine function ,8. ReseaThere are a number of studies that have examined the role of the hypothalamic–pituitary adrenal axis in obesity, using salivary cortisol as a marker ,16,17. HAlthough the influence of obesity and PA on immunity has been well reported, research in the paediatric population is still limited, particularly in South Africa. The aim of this study was to investigate the effect that obesity has on both mucosal immunity and the SNS in black, South African children from a low socioeconomic area, and whether PA may mitigate these effects.From an urban school in a low socioeconomic area in Pietermaritzburg, KwaZulu-Natal, 132 black, South African children  participated in the study. The study was approved by the University of KwaZulu-Natal's Biomedical Ethics Research Committee and complied with the World Medical Association Declaration of Helsinki  regardinSamples were collected from each school grade on a separate morning over the course of a week, starting with grade 3 on Monday and ending with grade 7 on Friday. The children were requested to rest for 20 min upon arriving in the school hall at 7:00 a.m. and to complete with the researchers a short health questionnaire and interview (Wisconsin Upper Respiratory Symptom Survey (WURSS-44)  and the −1). Saliva flow rate was calculated by dividing the total amount of saliva obtained in each sample (mL) by the time taken to produce the sample (4 min) . This model was significant  and BMI (p < 0.05) and DBP (p < 0.05) were found to be independent predictors of sAA activity.The multiple linear regression results are presented in 2max]. This model was significant  and BMI (p < 0.05) was found to be an independent predictor of sAA secretion rate.The model that best predicted sAA secretion rate and accounted for 14.84%  of the variance was sAA (U/min) = −5.314 + 2.351 × [BMI] + 0.7135 × [DBP] − 0.2394 × [VOp < 0.0001) and BMI category (p < 0.05) was found to be an independent predictor of SIgA concentration.The model that best predicted SIgA concentration and accounted for 13.86%  of the variance was SIgA (µg/mL) = 303.71 − 1.681 × [Age] − 45.737 × [BMI category]. This model was highly significant  and BMI category (p < 0.001) was found to be an independent predictor of SIgA secretion rate.The model that best predicted SIgA secretion rate and accounted for 9.32%  of the variance was SIgA (µg/min) = 233.04 + 2.210 × [Age] − 34.886 × [BMI × Category]. This model was highly significant  and that age and BMI category predict SIgA (with BMI as an independent predictor). This indicates that obesity (based on the BMI categories), as well as elevated DBP and poor CRF, play major roles in elevating resting SNS activation and lowering mucosal immunity.Although the negative effects of obesity and lack of physical activity on immune and neuro-endocrine function have been identified in adults, information is limited for children ,8,38. Th2max) than both normal weight and overweight children. Similarly, Berndtsson, Mattsson, et al. [2max in Swedish obese children and adolescents when compared to the reference group.The results demonstrated that obese children had significantly greater weight, BMI, body fat percentage, DBP, and SBP, compared to overweight and normal weight children. A number of other studies have also demonstrated that obese children display elevated blood pressure, BMI, and body fat percentage ,40,41,42, et al.  found loA higher sAA activity and secretion rate were observed in obese children compared to normal weight or overweight children in our study. This finding is supported by Granger, Kivlighan, et al.  and GranOur study also demonstrated significantly lower SIgA concentration and secretion rate in obese compared with normal weight children, suggesting that mucosal immunity may be reduced in obese children. This finding is supported by Pallaroa, Tabernerb, et al.  who alsoIt is important to recognise the study’s limitations. Although salivary flow rate was controlled and clear instructions were provided to the parents and participants regarding brushing teeth and dietary and hydration practices prior to saliva collection, the health condition of the children’s gums and teeth was not determined using standardised methods. Furthermore, only one saliva sample was taken per child because of logistical and financial constraints. Additionally, an examination for Tanner stages for sexual maturation  was not This study provides support for an association between poor CRF and/or obesity, and compromised mucosal immunity and SNS activation in children. Results show that obese children have elevated sAA, lowered SIgA, and poor CRF. Replication of the study with a larger sample size is required together with longitudinal follow up of clinical outcomes. This may contribute to a better understanding of the pathways mediating the enhancement of mucosal immunity, control of SNS activation, and chronic disease in children and, subsequently, in adults.


The mean percentage of HRmax (%HRmax) and ratings of perceived exertion (RPE) were collected. Technical actions (TAs)  were calculated through notational analysis. Players’ genitalia development (GD) and pubic hair (PH) growth were assessed using Tanner scale. Results showed a higher %HRmax (p = 0.018), RPE (p = 0.042), dribbles (p = 0.007), shots (p = 0.003), and rebounds (p = 0.006) in two vs. two compared to four vs. four condition. Furthermore, a statistical difference was found for %HRmax (p = 0.005) and number of passes (p = 0.020) between bouts. In addition, no correlation between GD, PH, and training age with %HRmax, RPE, and TAs was found. These findings suggest that variations of the number of players involved affect ball-drills workload and that ball-drills training intensity varies across bouts. Finally, ball-drills elicit an adequate training stimulus, regardless of players’ maturation status and training age.This study aimed to assess the basketball ball-drills workload analyzing: (1) the effect of varying the number of players involved on physiological and technical demands; (2) the temporal changes in players’ responses across bouts; and (3) the relationship of players’ workload with their maturation status and training age. Twelve young male basketball players (mean ± SD; age 13.9 ± 0.7 years; height 1.76 ± 0.06 m; body mass 65.7 ± 12.5 kg; HR Such sport-specific conditioning has been found to develop players’ physical and physiological demands and both technical and decision making skills . Ball-dral games , underlial games ,13,14, cal games , and worThe number of players involved has been reported to be the main factor influencing the physiological and technical demands in basketball BDs ,13,14. AThe analysis of workload changes across bouts of the same BD could also provide additional interesting information to help coaches in designing sound training programs. Changes in physiological and technical demands across bouts have been extensively studied in soccer small-sided games (SSGs) ,17,18, wConsidering that studies in basketball BDs mainly investigated young players in a chronological age after the completion of their maturation process  ,13,14, aVariation in sexual maturity was also investigated in relation to basketball technical skills . This stIn many youth basketball teams, players with different levels of experience coexist. This difference could be responsible for different players’ physiological and technical demands. In fact, more experienced players might have a higher skill level and therefore be more involved in game situations than less experienced players. To the best of our knowledge, no previous investigations assessed the effect of different playing experience on BDs workload, calling for further studies in this area. Therefore, this study has multiple purposes, specifically to analyze: (1) the effect of varying the number of players involved on physiological and technical demands in basketball BDs in young players ; (2) the workload changes throughout bouts; (3) the relationship of BD physiological and technical demands with players’ self-assessed maturation status and training age.Based on previous investigations analyzing the physiological and technical demands in basketball BDs ,13, it hmax 202 ± 8 beat·min−1) volunteered to participate in this study. All players were members of an “Under 15” team participating at a regional-level of the Italian youth basketball championship. The study was conducted within two weeks during the 2013/2014 season. Players were familiarized with the procedures during the pre-season period, while the experimental protocol took place during the last macrocycle of the in-season period (March–May). Players completed a similar pre-season and in-season volume of training before the starting of the experimental procedures. Players performed three 90-min training sessions and one official game per week. Selection criteria for recruitment in the study included the absence of injury in the past 6 months. Ethics approval was obtained from the Institutional Review Board for Research involving Human Subjects of the University of Portland, approval number IRB00006544. Written informed consent was obtained from all the subjects and their parents after a detailed explanation of the aims of the study.Twelve (eight perimeter and four post players) young male basketball players , ratings of perceived exertion (RPE), and technical actions (TA) were the dependent ones. Moreover, the relationship between the dependent variables and players’ maturation status and playing experience was evaluated in each BDs setting.BDs were composed of three bouts of 4 min interspersed by 2 min of passive recovery, and were played during regular training sessions after 15 min of standardized warm-up. This set-up was selected because it represents one of the most used basketball practice drills with a duration 12 min) similar to that reported in previous studies ,12,13, a min simimax (beat·min−1). The test protocol involved 30-s shuttle runs interspersed by 15-s passive recovery periods executed on the full basketball court (28 m) at an increasing pace governed by a pre-recorded beep [max. Heart rate beats were recorded at 1-second intervals using HR monitors with internal memory  and exported and analyzed using Excel software . Ball-drill HR data were expressed as a percentage of HRmax (%HRmax). Moreover, players’ rating of perceived exertion (RPE) was collected at the end of each BD using the modified CR10-scale [Before participating in the study, players’ anthropometric measurements (height and body mass) were collected. Moreover, all players completed the 30–15 Intermittent Fitness Test (30-15IFT) specifically developed for basketball  to assesded beep . Playersll court  m at an www.Kinovea.org, accessed on 15 March 2015) This methodology has been previously used and described in previous investigations [Technical actions (TA) were assessed using notational video analysis through the Kinovea software  were calculated for the mean %HRmax and mean difference between the levels of each factor (number of players and bouts). Partial eta-squared (η2) was used as a measure of effect sizes, and values were interpreted as no effect (η2 < 0.04), minimum effect (0.04 < η2 < 0.25), moderate effect (0.25 < η2 < 0.64), and strong effect (η2 > 0.64) [Mean and standard deviation (SD) were calculated for all the dependent variables. Normality and homogeneity of variances were assessed using the Shapiro–Wilk test, showing a normal distribution for physiological parameters, while RPE and TAs data were not normally distributed. Therefore, a two × three (number of players × bouts) two-way ANOVA for repeated measures was applied to assess differences between two vs. two and four vs. four and the three bouts for %HR > 0.64) .p-values (adj-p) were calculated using a Bonferroni correction. In addition, 95% CIs for the mean differences for the RPE and TAs were calculated via bootstrapping with 10,000 replications. Effect sizes were calculated using the r-value for Wilcoxon signed-rank test [Differences between two vs. two and four vs. four conditions for RPE and TAs were assessed using Wilcoxon signed-rank tests, while differences between bouts for TAs were calculated using Friedman tests. For any significant differences of TAs between bouts, Wilcoxon signed-rank tests were used to perform pairwise comparison  and adjuank test , and valank test .max, RPE, and TAs with GD, PH, and playing experience. Statistical analyses were conducted using the software R (version 3.1.0 for Mac OS X) [Finally, Spearman’s rank test was used to correlate %HRac OS X) , and theac OS X) . The crimax registered during two vs. two and four vs. four drill conditions were 91.8 ± 3.0  and 89.7 ± 3.1 , respectively, with a greater value in two vs. two compared to four vs. four , mean difference ).The mean ± SD (95% CI) of %HRmax of 89.9 ± 2.7 , 91.1 ± 3.0 , and 91.2 ± 3.0  for the first, second, and third bout, respectively. A main effect was found for the bout factor ). Bonferroni post-hoc analysis showed a significant difference between first and second bout  = −1.145 ), a marginal statistical difference value between the first and third bout  = −1.282 ) ) between two vs. two (9.3 ± 0.9) and four vs. four condition (8.4 ± 0.9) with a mean difference (95% CI) of 0.9 .Moreover, results showed a mean and SD (95% CI) %HR 0.093)) , and no p = 0.007; r = 0.560 (large effect)), shots ), and rebounds ) were observed in two vs. two compared to four vs. four condition (p = 0.020). Post-hoc analyses highlighted a higher number of passes in the first bout compared to second ) and third bouts ), while no difference was reported between second and third bouts (adj-p = 0.909).Results related to technical actions are shown in ondition . Moreovemax, RPE, and TAs  the number of players involved influenced the physiological and technical loads during BDs in young  basketball players; (2) changes across consecutive bouts of the same drill were observed for the physiological demand, while no changes were found for most of the technical actions; (3) players’ self-assessed maturation status did not correlate with players’ physiological, perceptual, and technical demands in each considered BD, rejecting our hypothesis.max: 86%–87%; RPE: 8–9 AU) compared to four vs. four  [max and RPE in two vs. two compared to four vs. four condition. The increased physiological and perceptual demands are likely the result of an increased amount of work performed, due to an increase in the relative playing area per player [max  and an equal RPE  in both conditions. Overall, these results indicate a consistent psychophysiological response during basketball BDs played with a different number of players.Several variables, such as the use of different rules, court size, and number of players involved can influence the physiological demand of basketball BDs ,12,13,14While sport research has deeply focused on the physiological and perceptual demands in basketball BDs, less data are available on the effect of these training methodologies on the technical demand. Previous investigations analyzing BDs technical demand in young elite  and non-One of our results did not agree with that shown in previous investigations ,14, as nmax in the first bout compared to the following ones. Specifically, the results showed a significant difference between first and second bouts and a marginally significant difference between first and third bouts. This result is similar to that reported in soccer SSGs [A novel aspect of this study was the analysis of the variation of physiological and technical responses across bouts. Results showed a statistically lower %HRcer SSGs ,17,18, acer SSGs . These rcer SSGs , in whicThe analysis of technical actions documented no differences throughout bouts in overall TAs, a result in agreement with previous investigations in soccer ,16. In tThis is also the first study investigating the correlation between self-assessed maturation status and physiological and technical demands of basketball BDs. While maturation status influenced young basketball players’ strength outputs and individual time-based technical skills tests (dribbling and defensive movements) , no corrIn addition, no correlation was reported between the player’s playing experience and the physiological and technical demands in basketball BDs. It was expected that players with greater exposure to competitive basketball would show higher involvement during BDs showing greater physiological and technical demands. Interestingly, in our study, players with more years of training experience were involved to the same degree as novice athletes. This aspect could be explained by the fact that the BDs present a wide range of playing situations that could help players with fewer years of training to enhance their playing experience. This result could suggest to basketball coaches to use this training methodology independently from players’ training background.One of the limitations of this study is that only 12 players were analyzed and only a self-assessed Tanner stage questionnaire was used to determine players’ maturation status; thus, future research should use a larger sample and several more objective ways to determine players’ biological age. In addition, this study analyzed a specific sample composed by 14-year-old players, while future investigations should be provided on players with a different chronological age . Moreover, considering the contrasting results between this study and previous investigations on passing skills in basketball BDs, future investigations are warranted in this area. Finally, future studies should also correlate players’ skill ability levels with BDs physiological and technical demands.The high HR observed in this study suggests that basketball coaches can use BDs to train physical fitness simultaneously with the technical and tactical skills. In particular, coaches should use the two vs. two condition to impose greater internal load on their players when the periodization of training plans allows it. Furthermore, training sessions including BDs should be used to develop young players’ individual skills in a game-based condition. Specifically, the two vs. two should be preferred to the four vs. four in order to train dribble, shot, and rebound actions. Conversely, passing skills and the ability to intercept or steal the ball or to induce a turnover were not influenced by the different number of players involved, suggesting that coaches should use a different variable to modulate the learning of these abilities. Although the two vs. two condition elicited a higher number of technical actions, basketball coaches should also consider using four vs. four, because it likely stimulates better decision-making ability skills in young players due to more complex interactions with a greater number of players and opponents ,14. ConsThis study improves the knowledge about the use of BDs in young basketball players, suggesting that BDs are a viable training method that can be used by basketball coaches to train both players’ fitness and technical skills. The different number of players involved affected the physiological and technical demands of BDs in basketball, with a higher training load corresponding to a reduction of the number of players involved. Moreover, physiological demands change across bouts, while no variations were observed for the overall technical actions. Lastly, players’ maturation status and playing experience did not correlate with BDs physiological and technical demands in young basketball players.


Parathyroid failure is the most common symptom after thyroidectomy. To prevent it, a gland was preserved in situ or an ischemic one was autotransplanted. This study explored the relationship between in situ preservation of the parathyroid gland and gland failure.  Consecutive patients who underwent initial total thyroidectomy were enrolled retrospectively in a prospectively maintained database. Patients were divided into groups by parathyroid gland remaining in situ fraction (PGRIF) . Patients were graded by tertiles and followed at least one year after surgery.  559 patients were included. PGRIF is significantly inversely associated with transient hypoparathyroidism, protracted hypoparathyroidism, and postoperative hypocalcemia. PGRIF was identified as an independent risk factor for transient hypoparathyroidism, protracted hypoparathyroidism, and postoperative hypocalcemia . Autotransplantation of parathyroid gland would not affect the calcium level in the long term.  In situ preservation of parathyroid gland is crucial for parathyroid function. Less preserved is the independent risk factor for postoperative hypoparathyroidism and hypocalcemia, resulting in a worse function of parathyroid gland in the long term. Hypoparathyroidism is a well-recognized symptom after total thyroidectomy –3. EdafeParathyroid function failure is the main cause of postoperative hypoparathyroidism. Preserving all parathyroid glands, vasculature is still challenging, even for high-volume surgeons. Some authors think autotransplantation of discolored parathyroid gland would decrease the incidence of hypoparathyroidism , 5–8; hoIn the present designed study, we will explore the association between efficiency of in situ preserved parathyroid gland and parathyroid insufficiency.This retrospective study was conducted based on the database. All databases of patients and surgeries performed by Dr. Zhu were maintained prospectively. All thyroid surgeries were performed by one experienced surgeon (Dr. Zhu), with high volume of over 400 cases annually.Patients who underwent thyroidectomy in our department between 2013 and 2014 were identified. Inclusion criteria were (1) initial operation and (2) total thyroidectomy (TTx), and exclusion criteria were (1) reoperation, (2) incomplete data, (3) lobectomy and near TTx, (4) any disease that would affect the level of calcium and magnesium (like kidney disease), and (5) patients with preoperative hyper/hypocalcemia.3 fragments and autografted into the ipsilateral sternocleidomastoid muscle. To avoid incidental parathyroidectomy (IP), we systematically sought to remove the thyroid gland and tissue, in case intrathyroid cases happened. The same pathologist inspected and analyzed all the surgical specimens. After that, the parathyroid gland remaining in situ fraction  was graded by tertiles : IP = gland  in  specimen + intraoperative  IP. A complete central nodal dissection (CND) was performed, first, on the side of carcinoma.A conventional capsular dissection technique was adopted. Any parathyroid attached on the dorsal side of the thyroid was identified and freed carefully and retained in situ with intact vasculature. If the parathyroid gland was discolored at the end of surgery or was incidentally removed intraoperatively, gland autotransplantation will be performed. Then the parathyroid gland will be chopped into 1 mmThe demographic data about patients and blood  were collected upon admission. Postoperative calcium and iPTH were measured at 6 AM of the 1st morning. The iPTH level was determined by electrochemiluminescence immunoassay . Then patients with normocalcemia with no discomfort yet were discharged on the 1st or 2nd postoperative day. The other patients were instituted oral calcium and/or vitamin D supplementation on a case-by-case basis. Supply with calcium and/or vitamin D was decided by the hypocalcemic symptom or biochemical hypocalcemia. On average, each patient received 500 mL total intravenous fluid replacement after surgery until he (she) could drink.Patients were followed up rigorously in the outpatient department, from the end of the 1st month after discharge. Patients administrated with calcium and/or vitamin D were followed every month after discharge until 6 months, then 6 months thereafter, or they were followed 1, 3, and 6 month(s) after discharge until 6 months, then 6 months thereafter.Transient hypoparathyroidism is defined by a subnormal parathyroid hormone (<1.6 pmol/L), need of calcium replacement after surgery, and absence when followed in the 1st month. Protracted hypoparathyroidism is defined by a persistent subnormal parathyroid hormone (<1.6 pmol/L) and need of calcium replacement with or without calcitriol treatment 4 weeks after surgery. Permanent hypoparathyroidism is defined by a persistent subnormal parathyroid hormone (<1.6 pmol/L) and need of calcium replacement with or without calcitriol treatment 1 year after surgery. Parathyroid insufficiency and hypocalcemia are defined by subnormal parathyroid hormone and serum calcium concentration. The reference is calcium 2.1–2.7 mmol/L and iPTH 1.6–6.9 pmol/L. The Tumor Staging System adopted the standard of the Union for International Cancer Control (UICC) sixth edition.t-test; if not, variables were presented as median (interquartile range) and compared by U test. ANOVA test was used for mean comparison of multigroups. Pearson chi-square test or Fisher's exact test was used to compare frequency (percentage) for categorical variables. Logistic regression was used to determine the risk factor. P value < 0.05 indicated significant difference.Data analysis was performed by SPSS version 21 . If normally distributed, continuous variables were presented as the mean ± standard  deviation and compared by All experimental protocol in this study involving human participants was approved by the Ethics Committee of West China Hospital, Sichuan University . The informed consent forms were obtained from all individual participants in this study. All study participants provided written informed consent to indicate their agreement for the clinical data to be used in clinical research and publication. The methods were carried out in accordance with the Declaration of Helsinki and the guidelines of the Ethical Committee of the Cancer Hospital .Based on inclusion and exclusion criteria, 599 patients  were enrolled in the final analysis, with mean age of 43.25. A total of 238 patients (42.58%) developed transient hypoparathyroidism. 30 patients suffered from protracted hypoparathyroidism (5.37%), and only 3 developed permanent hypoparathyroidism (0.54%) .Autotransplantation was performed in 357 patients . An average of 3.55 parathyroid glands were identified . Parathyroid gland was found grossly in 17 specimens (3.04%). Most patients had thyroid carcinoma (539/559).P = 0.003). PGRIF showed a significant difference in transient hypoparathyroidism, protracted hypoparathyroidism, and hypocalcemia patients   .P < 0.001 and 0.033, resp.). Post hoc analysis showed that the mean of 1st day iPTH after surgery was 1.56 and 1.59 pmol/L in PGRIF 1 and 2, respectively, which was significantly lower than in PGRIF 3 . At 1 year of follow-up, the mean of iPTH in PGRIF 1 (2.73 pmol/L) was still significantly lower than that in PGRIF 2 (3.94 pmol/L) and 3 (3.79 pmol/L), P = 0.009 and 0.016, respectively  (P = 0.035), comparatively 49.26% (67/136) and 44.13% (173/392) of patients in PGRIF 2 and 3, respectively. This trend was not found at 1 month and 1 year of follow-up.In terms of serum calcium level, serum calcium of PGRIF 1 was significantly lower than that of PGRIF 2 and 3 on the 1st day after surgery ( pmol/L) . SpecifiP = 0.005). More patients developed hypocalcemia in the autotransplantation group, 180/357, than in the nontransplantation group, 82/202, P = 0.028. In contrast, a difference was no longer found at 1 month and 1 year follow-up  and PGRIF  were identified as independent risk factors for transient hypoparathyroidism, while only PGRIF  was identified as a risk factor for protracted hypoparathyroidism. Regarding postoperative hypocalcemia, sex   and PGRIF  were identified as risk factors   and calcium (P = 0.003) on the 1st day after surgery significantly. Song et al. [P = 0.028), consistent with Hallgrimsson et al.'s [In our study, less in situ preserved parathyroid gland led to lower serum iPTH , which is higher than previous results at 15%–50% , 25. OurOur research was limited by its retrospective nature, though the database was maintained prospectively. First, patient collection bias was unavoidable because over 95% of patients have malignancy, which resulted from the referring system. Second, as mentioned before, a high proportion of malignancy in the present study led to a large quantity of CND, so autotransplantation of parathyroid glands was performed in 357 patients. Therefore, we plan to conduct a multicenter retrospective study of recruiting more benign thyroid goiter patients to confirm this result. In addition, there lacked complete data about preoperative 25-hydroxy vitamin D (25OH-VD) level, so we did not take it into the final analysis. Yet, current meta-analysis evidence showed that lower preoperative 25OH-VD would not increase risk of transient hypocalcemia , 26.In conclusion, in situ preservation of parathyroid glands seems crucial for gland function. Though autotransplantation would not affect serum calcium in the long term, more gland preserved in situ was beneficial for gland function in the follow-up. Female patients with less parathyroid gland preserved in situ were vulnerable in transient hypoparathyroidism and postoperative hypocalcemia, and PGRIF was identified as a sole risk factor for transient hypoparathyroidism, protracted hypoparathyroidism, and postoperative hypocalcemia. At least preserving 3 parathyroid glands is a better way to avoid postoperative complications. Further prospective research with long-term follow-up is needed to confirm this result.


Identification and use of non-invasive biomarkers for kidney transplantation monitoring is an unmet need. A total of 121 biobanked sera collected from 111 unique kidney transplant (KT) patients (children and adolescent) and 10 age-matched healthy normal controls were used to profile serum proteins using semi-quantitative proteomics. The proteomics data were analyzed to identify panels of serum proteins that were specific to various transplant injuries, which included acute rejection (AR), BK virus nephropathy (BKVN), and chronic allograft nephropathy (CAN). Gene expression data from matching peripheral blood mononuclear cells were interrogated to investigate the association between soluble serum proteins and altered gene expression of corresponding genes in different injury phenotypes. Analysis of the proteomics data identified from different patient phenotypes, with criteria of false discovery rate <0.05 and at least twofold changes in either direction, resulted in a list of 10 proteins that distinguished KT injury from no injury. Similar analyses to identify proteins specific to chronic injury, acute injury, and AR after kidney transplantation identified 22, 6, and 10 proteins, respectively. Elastic-Net logistic regression method was applied on the 137 serum proteins to classify different transplant injuries. This algorithm has identified panels of 10 serum proteins specific for AR, BKVN, and CAN with classification rates 93, 93, and 95%, respectively. The identified proteins could prove to be potential surrogate biomarkers for routine monitoring of the injury status of pediatric KT patients. Organ transplantation is the treatment of choice for patients with organ failure . The numRecent developments in high-throughput assays such as gene microarrays and mass spectrometry-based proteomics have proven to be useful in profiling gene transcripts (mRNA) and proteins, and this global-scale molecular profiling has helped in a better understanding of homeostasis of organ failure –9. By anThe objective of this study was to identify signatures of serum proteins that are associated with KT injury in pediatric patient population. To meet the objective, we used a unique set of 121 serum samples from pediatric patients with kidney transplantation and performed mass spectrometry-based proteomics, followed by statistical and bioinformatic analyses to identify proteins that are associated with different kinds of transplant injuries in kidney. In addition, we also interrogated gene expression data from matching peripheral blood mononuclear cells (PBMCs) to investigate if there existed any correlation between the level of serum proteins and the gene expression of corresponding proteins in matching PBMCs.Serum samples were collected from 111 unique pediatric kidney transplant (KT) patients from Lucile Packard Children’s Hospital, Stanford University. Sera from 10 age-matched healthy normal controls were also included in the study as a non-transplant control. Each sample was matched with a biopsy collected at the time of serum collection. The study included 27 AR, 20 calcineurin inhibitor toxicity (CNIT), 25 chronic allograft nephropathy (CAN), 14 BK virus nephropathy (BKVN), and 25 normal graft function without significant injury [stable graft function (STA)] samples. All the study samples were collected from pediatric and young adult recipients transplanted between years 2000 and 2011 at Lucile Packard Children’s Hospital of Stanford University. The study was approved by the ethics committees of Stanford University Medical School and UCSF Medical Center. All adult patients and parents/guardians of non-adult patients provided written informed consent to participate in the research, in full adherence to the Declaration of Helsinki. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism. A summary of demographic information of KT patients and a summary of study samples and analysis scheme are provided in Table g for 5 min using a swinging bucket rotor. The upper layer of serum was then transferred to another cryotube and was stored at −80°C until use.Blood samples (4.5 mL) were collected into a 5 mL red top tube and incubated at room temperature for 30 min until the clot was formed. The sample was then centrifuged at 2,000 × Serum samples were depleted of the 20 most abundant serum proteins by ProteoPrep20 Plasma Immunodepletion Kit . The eluate from each sample was subsequently subjected to trypsin digestion with a standard trypsin digestion protocol. Tryptic peptides were reconstituted in a buffer containing 0.2% formic acid, 2% acetonitrile, and 97.8% water prior to mass spectrometry. The high-performance liquid chromatography utilized was an Eksigent nano2D (Eksigent) with a self-packed 150 μM ID C18, 15 cm column. The electrospray source was a Michrom Advance operated at 600 nL/min on an LTQ Orbitrap Velos (Thermo Fisher). Data acquisition was performed in a data-dependent manner in which the top 12 (Velos) most intense-charged peptide ions were selected for MS/MS fragmentation of charge state 2+ and 3+. Data were subsequently extracted with msconvert script into an mzXML format prior to Sorcerer (SAGE-N) analysis with the Sequest algorithm. The IPI human database was searched using a 50 ppm mass window on the precursor ion. We allowed for the static modification of propionamide on cysteine and variable modifications of methionine oxidation and lysine acetylation. All searches were compiled and displayed in a Scaffold  interface, which listed the identified proteins with cumulative spectral counts for each protein.t test, p value <0.05, and fold change 1.5.Gene expression analysis was performed for gene expression aberrations on a subset of whole blood collected at the same time from the study subjects for the proteomics study. This analysis included blood samples collected from 11 AR, 11 STA, 9 CAN, 9 BKVN, and 9 CNIT patients. Blood was collected in 2.5 mL PAXgene™ Blood RNA Tubes . Total RNA was extracted, and cDNA synthesis was done using a previously published protocol . Synthes® and the FDR at the unique peptide level 0.2%. The spectral counts for the identical IPI numbers were summed. After filtering for proteins that were not consistently identified in different samples, the identified proteins were used for further analysis. In the first step, the data were quantile-normalized and used for ANOVA analysis. Phenotype-specific proteins were identified with ANOVA with the criteria FDR <0.05 and at least twofold change in either direction. Principal component analysis (PCA) plots and heatmaps were generated using Partek Genomics Suite .Data analysis was done in three steps. First, we performed ANOVA to identify injury-specific serum proteins in KT injury. In this step, mass spectrometric data were analyzed with settings that included the false discovery rate (FDR) for protein identifications 0.1% based on target decoy analysis using Sequestvia Coordinate Descent for the estimations (x) = P(x) (case) and x1, …, Kx are the expressions of K proteins for observations x. The Elastic-Net fits this model by adding a mixed penalty term to the likelihoodIn the second step, we used penalized logistic regression to identify proteins that could serve as potential surrogate biomarkers for different KT injuries. This approach provides not only accurate estimation for the regression coefficients but also probability estimation for each patient , 30. We We fitted 100 Elastic-Net logistic regression models to the 137 proteins using bootstrapped samples maintaining the use of ~75% samples in the training set and ~25% samples in the test set to classify between transplant samples and non-transplant samples with different transplant injury status. For each bootstrap, a nested cross-validation loop estimated the best value according to the deviance. The parameter of the Elastic-Net was fixed at 0.95, the value recommended by Friedman et al. . The mea® (p = 4.79E−38) and peptidase inhibitor activity (p = 1.02E−13) as the top associations of the proteins identified.The objective of this study was to identify KT injury-specific proteins. By the proteomics analysis of serum proteins, we identified 137 proteins across all the samples with the criteria of (i) minimum two peptides per protein for a positive identification and (ii) 0.1% FDR for protein and 0.2% FDR for peptide identification Sequest® . An enriInjury vs no injury: for this, the samples were classified in to injury  and no injury (STA and HC). Ten differentially expressed unique proteins were identified specific to kidney injury after transplantation (Table Chronic injury-specific serum protein markers: the contrast between CAN/CNIT (Chronic) and AR/BK/STA (non-chronic) identified 22 differentially expressed proteins listed in Table Acute injury-specific proteins: we identified 6 differentially expressed proteins between patients with acute injury (AR and BKVN) and those with chronic injury (CAN and CNIT) or STA   on Table . These 1KT-specific panel: analysis of healthy normal control (HC) vs transplant samples identified 10 proteins , CP, ALB, apolipoprotein A-II (APOA2), IGHG3, ORM2, and SAA4) on the whole dataset as proteins specific to transplantation. The box-and-whisker plot in Figure Injury-specific protein panel: next, we sought to identify a panel of proteins that separate serum samples with kidney injury from those with well-functioning kidneys. A panel of 10 proteins was identified, which classified injury  (n = 86) vs no injury (STA and HC) (n = 35). The mean classification rate, sensitivity, specificity, PPV, and NPV were 0.92. 0.96, 0.84, 0.93, and 0.91, respectively. The interception and regression coefficients are shown in Table Acute injury protein panel: next, we identified 10 proteins that were specific to acute transplant injury (AR and BKVN) compared with no acute injury . The mean classification rate for the 10 protein panel was 98% [CI 95% = ], sensitivity = 0.95, specificity = 0.98, PPV = 0.95, NPV = 0.98. The interception and regression coefficients are shown in Table Biomarker protein panel for AR: the analysis that compared protein signal from AR samples against all non-AR samples identified 10 proteins to classify AR (n = 27) vs others  (n = 94). The mean classification rate was 0.91. The sensitivity, specificity, PPV, and NPV were 0.74, 0.96, 0.83, and 0.93, respectively. The interception and regression coefficients are shown in Table Biomarker proteins for BKVN: a panel of 10 proteins was identified that was able to classify BKVN (n = 14) vs others (n = 107) with a mean classification rate 0.93. The sensitivity, specificity, PPV, and NPV were 0.65, 0.96, 0.69, and 0.95, respectively. The interception and regression coefficients are shown in Table Serum protein biomarkers to distinguish CAN vs CNIT: a panel of 10 proteins was identified that classified CAN (n = 25) vs CNIT (n = 20). The mean classification rate was 0.95. The sensitivity, specificity, PPV, and NPV were 0.96, 0.94, 0.96, and 0.95, respectively. The interception and regression coefficients are shown in Table To identify transplant injury-specific panels, we fitted 100 Elastic-Net logistic regression models to the 137 proteins using bootstrapped samples maintaining the use of ~75% samples in the training set and ~25% samples in the test set to classify transplant samples and non-transplant samples with different transplant injury status. (i) p < 0.05). Among these, only 39 proteins (gene products) were identified in our serum proteomics data. With the significance criteria of an empirical Bayes t test p value <0.05 and a fold change of two vs stable graft (STA), a total of 626 upregulated genes were identified, which enriched for the biological system process (FDR = 0.0004). In addition, there were 7,316 downregulated genes enriched in AR, which are associated with cellular macromolecule metabolic process (FDR = 5.1E−14) and primary metabolic process (FDR = 2.1E−13). There were 12 upregulated and 713 downregulated genes in CAN. CAN-associated genes were highly enriched in immune system process (FDR = 7.4E−9) and response to stress (FDR = 2.4E−6). Blood from BKVN patients demonstrated 141 upregulated and 1,321 downregulated genes in BKVN, which were enriched in anatomical structure morphogenesis (FDR = 4.2E−05) and transcription regulatory region DNA binding (FDR = 0.005). There were seven upregulated and 290 downregulated genes associated with CNIT. These CNIT-associated genes were enriched in viral transcription process (FDR = 0.003). Even though we were not expecting to see correlation between serum protein level and gene expression in the whole blood, we noticed that some serum protein levels had aligned with corresponding gene expression levels in the PBMCs. Among those genes, complement factor properdin (CFP), attractin (ATRN), serum IgA (IGHG2 – gene), and clusterin (CLU) levels were downregulated in acute injury, including AR (both gene expression and protein level).A subset of PBMCs with matching serum samples from the same collection date for each patient was analyzed. A total of 9,665 genes were significantly different in whole blood by ANOVA  to gene expression data  was minimal, this is our effort to understand the molecular perturbations in different tissue types. This approach is important as analyzing different tissue types will help build a complete picture of biological events that occur during an immune or non-immune insult to the transplanted kidney.The third important finding of this study is that profiling of serum proteins and profiling of transcriptome from whole blood provided complementary information about the perturbations in the blood in case of other injuries and allowed us to compare complementary information about molecular pathways that were obtained from serum proteomics and gene expression of the blood cells. Even though the overlap in between the serum proteins  of University of California San Francisco.TS and MS conceptualized the study. TS carried out the experiments and data generation. Both TS and MS analyzed the data and prepared 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.