Rotavirus is the leading viral agent for pediatric gastroenteritis. However, the case-based surveillance for rotavirus is limited in China, and its circulation in the environment is not well investigated.From 2013 to 2014, rotavirus was detected in raw sewage samples of Jinan and Linyi by quantitative PCR (qPCR) and conventional reverse transcription PCR (RT-PCR). After sequenced and genotyped, sequences analysis was conducted.3 to 1.3 × 106 genome copies (GC)/L in Jinan, and from 1.5 × 103 to 3.0 × 105 GC/L in Linyi. A total of 318 sequences of 5 G-genotypes and 318 sequences of 5 P-genotypes were obtained. G9  and P[8]  were the most common G- and P-genotype, respectively. Multiple transmission lineages were recognized in these genotypes. Interestingly, an intragenic recombination event between two G9 lineages was observed.A total of 46 sewage samples were collected monthly for the detection of rotavirus, and rotavirus was positive in 43 samples . By quantitative assessment, the concentrations of rotavirus in raw sewage ranged from 4.1 × 10This study provided the first report of comprehensive environmental surveillance for rotavirus in China. The results suggest that the concentration of rotavirus in raw sewage was high, and multiple rotavirus transmission lineages continuously co-circulated in Shandong. Rotavirus, family Reoviridae. Its genome contains 11 double-stranded RNA (dsRNA) segments, encoding six structural proteins (VPs) and five (or six) nonstructural proteins (NSPs). Rotavirus is divided into seven groups, and group A, B and C can infect humans. Of these, rotavirus group A (RVA) is predominant, which contains at least 27 G- and 37 P- genotypes based on the VP7 and VP4 sequences  and P[4] were two most common P-genotypes, accounting for 56.0 % (178/318) and 35.8 % (114/318). P[9] and P[3] were only found in Jinan, with the detection rate of 3.5 % (7/200) and 0.5 % (1/200). Figure Homologous comparison was performed in all obtained sequences from Jinan and Linyi. To reduce workload, Shandong G9, P[8] and P[4] sequences displaying more than 99.5 % nucleotide identities within these genotypes have been deleted. Phylogenetic analysis was conducted between the remaining sequences and reference strains selected randomly from GenBank database.Phylogenetic relationships of G-genotypes were illustrated in Fig. Phylogenetic relationships of P-genotypes were shown in Fig. After RDP analysis and the alignment of 884-nt G9 sequences, an intragenic recombination event was observed in one G9 sequence (JN344-9) Fig. . The croEnvironmental sewage surveillance has been successfully conducted to obtain molecular characterizations of human enteric viruses in some regions –21. In tAt present, various detection rates of rotavirus in urban sewage have been reported , 23. In Currently, G1-G4, G9, P[4], P[6] and P[8] are the prevalent genotypes worldwide . In thisG9 infections are globally increasing at the exponential growth since 1993, and G9 have recently been the significant genotype in some regions. In this study, G9 was also observed as the predominant G-genotype and two main phylogenetic clusters were identified. The minor cluster was only recognized in Jinan in 2013, showing its local circulation in Jinan. The major cluster was continuously observed in Jinan and Linyi from 2013 to 2014, revealing that it was the main G9 transmission lineage in the two cities and the inter-city spread may exist. Interestingly, an intragenic recombination event between two G9 lineages was observed in this study. Intragenic recombination in rotavirus is rare until increasing reports recently , 32. To G6 and G8 strains were reported to be of animal origin , 35. In The genetic differences within P[4], P[8], P[6] and P[9] sequences were high, and more than one cluster was recognized, suggesting the co-circulation of multiple transmission lineages in these genotypes. P[3] sequence in this study showed the highest identity to the rhesus rotavirus P[3] strain . RRV is the recipient virus in human-rhesus reassortant vaccine . FurtherIn conclusion, this study shows that the concentration of rotavirus in raw sewage was high, and multiple rotavirus transmission lineages continuously co-circulated in Jinan and Linyi, eastern China. Additionally, this study proves that environmental surveillance is an ideal measure to obtain epidemiological data on circulating viruses in a given population.


There are inconsistences regarding the correlation between diabetes or fasting blood glucose concentrations and the risk and survival of amyotrophic lateral sclerosis (ALS) in the previous studies. Moreover, the association between hemoglobin A1c (HbA1c) levels, which reflect long-term glycemic status, and ALS survival was not examined.A prospective cohort study including 450 Chinese sporadic ALS patients . We identified 223 deaths during average 1.6 years of follow-up. We assessed levels of fasting HbA1c (primary exposure) and glucose (secondary exposure) via ion exchange high-performance liquid chromatography and hexokinase/glucose-6-pgosphate dehydrogenase methods, respectively. Multivariate Cox proportional hazards regression model was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of ALS mortality across the exposures.Our results indicated that, higher levels of HbA1c, but not fasting blood glucose concentrations, were significantly associated with higher risks of mortality. The adjusted HR was 1.40 : 1.02–1.99) for HbA1c of 5.7–6.4%, and 2.06 (95% CI: 1.07–3.96) for HbA1c ≥6.5%, relative to HbA1c <5.7% (P trend =0.01), after adjustment for age, smoking, obesity, disease severity, site of onset, lifestyle, and other potential confounders. The adjusted HR was 1.38  for fasting glucose concentrations ≥7.0 mmol/L vs <5.6 mmol/L. We did not observe any significant interactions between HbA1c levels and age, sex, smoking, body mass index, rate of disease progression of ALS, and site of onset .In this prospective study, we observed that individuals with higher HbA1c levels at the baseline had higher risk of mortality, which is independent of other known risk factors. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder characterized by degeneration of both upper and lower motor neurons, with a median survival of 3–5 years after symptoms onset . SeveralIn contrast, results regarding the association between diabetes mellitus (DM) and the risk, progression and survival of ALS were inconsistent or even contradictory –12. DM wTherefore, a large prospective study was conducted to examine the association between HbA1c and the mortality among 450 Chinese adults with ALS, adjusting for BMI, lipid profiles, disease status and lifestyle factors. We also explored potential interactions between HbA1c levels and these factors, in relation to ALS mortality. As a secondary exposure, the association between FBG concentrations and the mortality of ALS were also studied.The study was conducted in the Department of Neurology, West China Hospital of Sichuan University in the southwest of China. Patients who were diagnosed with definite or probable ALS according to the El Escorial revised criteria  were recBlood samples after an overnight fasting (> 8 h) were collected at the baseline. HbA1c levels were assessed by ion exchange high-pressure liquid chromatography on a Tosoh G7 standard mode  using reagents according to the manufacturer’s instructions in the clinical laboratory, West China Hospital. Intralaboratory analytical coefficients of variation were <2%. Participants were categorized into three groups based on their HbA1c levels (<5.7% (38 mmol/mol), 5.7–6.4% (38–46 mmol/mol), and ≥6.5% (48 mmol/mol)) and the lowest group was used as the reference . ParticiFBG concentrations were measured with the hexokinase/glucose-6-pgosphate dehydrogenase method. The coefficient of variation using blind quality control specimens was <2.0%. Participants were identified as having DM if they had a FBG ≥7.0 mmol/L, or were treated with insulin or oral hypoglycemic agents . And IGTBaseline concentrations of fasting serum total cholesterol (TC), and triglyceride (TG) were measured by an enzymatic colorimetric method using an automatic analyzer . Automatic hematology analyzer  completed blood counts. It performs hematology analysis according to the sheath flow DC detection method for erythrocytes and platelets and flow cytometry method using a semiconductor laser and fluorescent measurement for leukocytes and differential. Body height was measured, without wearing shoes, with an accuracy of 0.5 cm, using a stadiometer at the baseline. Body weight was measured to the nearest 0.1 kg with a steelyard scale, with underwear and no shoes. BMI was calculated as weight in kilograms divided by height in meters squared, and it was categorized according to the World Health Organization . Blood pAll the clinical data were collected at the baseline and during the follow-up evaluations, including age of onset, the region of symptom onset , disease duration at baseline, diagnostic delay, the ALS Functional Rating Scale-Revised (ALSFRS-R) score, and the use of riluzole, gastrostomy percutaneous endoscopy (PEG) and noninvasive positive pressure ventilation (NIPPV). The rate of disease progression was assessed by the changes of ALSFRS-R per month (Formula: (48 – ALSFRS-R score at the baseline visit)/ month intervals between first symptom onset and the baseline visit).P-value less than 0.05 were considered as statistical significance.Person-years for each participant were calculated from the date of recruitment to the date of death or tracheotomy which was taken as equivalent to death, or September 1st, 2015, whichever came first. Continuous variables were expressed as the mean ± standard deviation (SD). The Student’s t tests were performed for between-group comparisons of continuous variables. Chi-Square tests were used to determine the differences of categorical variables between groups. The multivariate Cox proportional hazards regression model was performed to calculate hazard ratios (HRs) and their 95% confidence intervals (CIs) of ALS mortality across the exposure  categories as the proportional hazards assumption was satisfied. We adjusted for potential confounders, including age, sex, site of onset, rate of disease progression, smoking, alcohol drinking, BMI, TC concentrations, anemia, hypertension, and use of riluzole, NIPPV and PEG  men and 196 (43.6%) women. The mean age of onset was 54.5 ± 12.2 years. The mean diagnostic delay was 15.7 ± 15.3 months. The ALSFRS-R total score was 39.1 ± 6.0 at the baseline (ranging from 12 to 48). For the site of onset, 244 (54.2%) patients had upper limb onset, 108 (24.0%) patients had lower limb onset, and 98 (21.8%) patients had bulbar onset. The average rate of disease progression was 0.77.Demographic and clinical characteristics of ALS patients among different HbA1c status are shown in Table SOD1 mutations, 3 patients  carried a C9ORF72 GGGGCC repeat expansion, 1 patient carried a FUS mutation , 1 patient  carried a CHCHD10 mutation, and no patient had TARDBP mutation (0/165). The frequencies of these ALS causative genes mutations were very low.Some patients also underwent an extensive genetic assessment using standard procedures. Four patients  had P = 0.20, HR =1.23), between patients with and without cognitive deficits , and between patients with and without frontal lobe dysfunction . As expected, higher BMI was associated with lower risks of mortality  after adjustment for potential confounders. In contrast, higher baseline HbA1c levels were associated with a higher risk of mortality during the follow-up .Sensitivity analyses generated similar results. After we excluded 21 participants who were using hypoglycemic agents, higher baseline HbA1c levels, but not FBG concentrations were associated with a higher risk of mortality. . Excluding participants who carried In this prospective study, we found that the risk of mortality was doubled for individuals with HbA1c ≥6.5% at the baseline, compared to those with normal HbA1c level (<5.7%). Pre-diabetes status, as defined by HbA1c of 5.7–6.4%, was also significantly associated with higher risk of mortality. The significant association between HbA1c and ALS mortality was independent of known risk factors, including BMI, blood cholesterol concentrations, and disease severity. Interestingly, no significant association was found between FBG and ALS mortality.A previous systematic review including 5 case-control and 2 cross-sectional studies  and a foConsistent with previous study , FBG wasSeveral potential mechanistic pathways including mitochondrial dysfunction, oxidative stress, and insulin resistance could underlie the observed relation between HbA1c and the mortality of ALS. On the one hand, mitochondria, which plays a crucial role in cell apoptosis, has shown to be an early target in ALS pathogenesis and contribute to disease progression  and its Systematic review study have demonstrated that the presence of iron deficiency with or without anemia (IDA) can lead to an increase in HbA1c values compared with controls, with no concomitant rise in glucose indices , which m2 or <23 kg/m2, respectively) exhibited steady increases in fasting blood glucose over time.Our current study was included in a Chinese population to find out that higher baseline HbA1c levels but not FBG concentrations were associated with a higher risk of mortality. Interestingly, there were some previous studies exploring DM and the risk of ALS in different ethnicity generated conflicting results: in Europe-based studies, presence of DM was associated with a lower risk of developing ALS , 35, butSeveral limitations of the present study should be noted. First, all the participants were solely recruited through a tertiary referral center in China; thus our results may not be generalized to other populations. However, the mean ALSFRS-R scores were similar 39.0 ± 6.0) between the current study and previous studies on this topic (39.0 ± 5.7) .0 ± 6.0 . FurtherIn this Chinese ALS cohort, we observed a strong dose-response relation between higher baseline HbA1c levels and higher future risks of mortality. Further studies performed in ALS patients with different ethnic backgrounds are needed to verify, whether higher HbA1c levels are also associated with increased ALS risks.


Besides, the sample pre-oxidized at 275 °C shows a specific capacity of 755 mA·g−1 at 100 mA·g−1 after 95 cycles. The improved electrochemical performance has been ascribed to the well dispersion of nanospheres, the improved electronic conductivity, and the structural integrity contribution from the carbon and cobalt coexisting nanocomposite. The strategy for preparing well-dispersed nanospheres by adjusting pre-oxidized and annealing processes could have insight for other oxide nanosphere synthesis.Well-dispersed Co/CoO/C nanospheres have been designed and constructed through a facile electrospinning method with a strategy controlling the morphology of nanocomposites via adjusting the pre-oxidized and heat treatments. Scanning electron microscopy results reveal that the as-synthesized sample pre-oxidized at 275 °C shows better spherical morphology with a diameter of around 300 nm without conspicuous agglomeration. X-ray diffraction analysis confirms the coexistence of cobalt and cobalt monoxide in the sample. Furthermore, the electrochemical tests reveal that the sample pre-oxidized at 275 °C displays excellent cycling stability with only 0.016% loss per cycle even after 400 cycles at 1000 mA·g Among various emerging novel anodes reported for lithium ion batteries, transition metal oxides (TMOs) have been regarded as the most appealing and competitive for their high theoretical capacity, easy manufacture, abundant raw materials, etc. SnO2, NiO, Co3O4, Fe2O3, and other TMOs have been studied widely both in their working mechanism and improvement methods [Nowadays, with the worsening energy crisis, lithium ion batteries (LIBs) have aroused widespread attention owing to its merits of high energy density, large voltage window, environmental friendliness, etc., when compared with other energy storage applications, such as nickel-metal hydride batteries, lead-acid batteries, supercapacitors, etc. ,2. Graph methods .−1 , low working potential, easy manufacture, and the saving of cobalt resources compared with Co3O4. However, low electronic conductivity and large volume expansion during cycling sets obstacles to the further utilization of CoO. Therefore, extensive endeavors have been devoted to reinforce the electronic conductivity and suppress the large volume change during lithiation and delithiation process. Generally speaking, enhancing strategies have been adopted based on the following aspects: (1) diverse nanostructures construction, such as nanospheres, nanotarrays, nanofibers, nanosheets, nanocubes, and other complex porous structures, hierarchical structures, etc. Such intricate nanostructures could provide CoO with elevated electrochemical capability on account of decreased Li ion diffusion distance and vast specific surface area which are favorable for fast Li ion transport and adequate contact between active materials and electrolyte; (2) combination with other highly electronic conductive materials, such as carbon-based materials and other transition metal oxides. Combination with carbon-based materials and nanostructures design are usually employed simultaneously [2 atmosphere [Cobalt monoxide (CoO) has been regarded as a promising anode in lithium ion batteries because of a high theoretical capacity of 790 mA·ganeously ,11,12. Ianeously , carbon aneously , hierarcaneously , heterosaneously , and caraneously . In thismosphere , which c2·4H2O  was dissolved in the dimethyl formamide  and stirred for 0.5 h. Then polyvinylpyrrolidone  was added into the solution stirring for another 12 h to form the precursor sol. Then the sol was loaded into a syringe with a flow rate of 0.9 mL·h−1 under an applied electric field of 0.8 kV·cm−1. The as-electrospun nanofibers were then collected and pre-oxidized at 250 °C, 275 °C, and 300 °C for 0.5 h separately in air, and the three samples named as P-250, P-275, and P-300, respectively. The pre-heated powders were then carbonized at 600 °C in a N2 gas atmosphere. The powders were subsequently calcined for 0.5 h in air. All three samples were denoted as F-250, F-275, and F-300, respectively.The Co/CoO/C nanospheres were prepared by combining electrospinning with subsequent heat treatments. Briefly, Co(Ac)−1 from room temperature up to 700 °C under ambient atmosphere.X-ray structural analysis was performed by a diffractometer . The morphology of samples was characterized with field emission scanning electron microscopy . The thermogravimetry-differential scanning calorimetry  curve was recorded at a ramping rate of 20 °C·min6 in ethyl carbonate/dimethyl carbonate with a volume ratio of 1:1 as electrolyte , and a porous polypropylene separator  in an argon-filled glove box (with O2 and H2O levels below 1 ppm). A slurry mixing 80 wt% active materials, 10 wt% super P, and 10 wt% polyvinylidene fluoride (PVDF) in N-methyl pyrrolidinone (NMP) solvent was stirred homogeneously for 12 h. Then the mixture was coated onto a copper foil current collector and dried at 110 °C for 12 h in −0.1 MPa vacuum. Discharge and charge tests were galvanostatically performed at room temperature in a potential range of 0.01–3 V (vs. Li/Li+) by a battery test system . Cyclic voltammograms (CV) curves were obtained using a CHI760 potentiostat  at a scan rate of 0.1 mV·s−1 with the voltage range of 0.01–3 V (vs. Li/Li+). Electrochemical impedance spectra (EIS) were performed with an amplitude of 5 mV in the range of 0.01 Hz to 100 kHz for the frequency by an impedance analyzer .Electrochemical performance was tested by assembling CR2032 coin cells with prepared electrode material, lithium metal as a counter electrode, 1 M LiPFThe thermogravimetric (TG) and differential scanning calorimetry (DSC) curves of as-electrospun nanofibers are displayed in 3O4 phase in the process of calcining in air at a mere 500 °C. However, only Co peaks appeared in the sample F-250. Furthermore, a gradual slope between 20° and 30°, which was attributed to the carbon residing in the samples. After the carbonization under N2 atmosphere and following post-heat treatment, the CoO and Co phase were still observed, notably, after the post-heat treatment, the CoO peaks were detected in the F-250 sample, which indicates that post-heat treatment in air helps to transfer more Co metal into the CoO phase.The XRD patterns of three samples experiencing the pre-oxidization process and the whole heat treatments were shown in the The morphology of samples after the pre-oxidization process by scanning electron microscopy (SEM) was shown in The morphology of final products experiencing the calcination under nitrogen atmosphere and following calcination in air is shown in the −1 after 95 cycles at a current density of 100 mA·g−1 with a coulombic efficiency of approximately 100% for every cycle. From the charge-discharge curves in From −1, respectively (the first three cycles at 100 mA·g−1 for the activation process). The F-275 delivers a high discharge capacity of around 523 mAh·g−1, and retains a stable specific discharge capacity of around 489 mAh·g−1 after 400 cycles, showing a capacity retention of about 93.5%, about 0.016% loss per cycle. Additionally, no apparent capacity decline was observed during 400 cycles. However, the pure Co3O4 sample shows obvious and rapid capacity decline in the initial few cycles and attains a relatively low capacity. Furthermore, F-250 and F-300 display distinct capacity loss in the initial cycles and the discharging capacities are much lower than that of F-275, which could result from the unfavorable morphology for lithium ion diffusion. The rate performance of three samples at various current densities from 100 mA·g−1 to 2000 mA·g−1 was shown in 3O4 in the −1 at 100, 200, 500, 1000, and 2000 mA·g−1 respectively, and returning to 710 mAh·g−1 at 100 mA·g−1. Additionally, the F-275 displays the reversible capacities of around 685, 600, 515, 439, and 354 mAh·g−1 at 100, 200, 500, 1000, and 2000 mA·g−1, and back to 716 mAh·g−1. However, the rate performance of F-250 sample shows very lower discharge capacity of only about 85 mAh·g−1 at 2000 A·g−1, and remarkable capacity decline as the increment of current densities. Pure Co3O4 delivers an inferior discharge capacity and unstable capacity retention, likewise, as shown in + [Rct is a symbol of charge transfer resistance. Noticeably, Rct of F-250, F-275, and F-300 are 87.98, 89.78, and 99.08 Ω, respectively, which are much slower than that of pure Co3O4 (142.1 Ω). Such noticeable difference displays that the Co/CoO/C nanospheres electrodes have better charge transfer capability, and the generation of cobalt and carbon could enhance the conductivity of electrodes remarkably. The F-275 displays the smallest Rct, which is consistent with the better electrochemical performance of F-275 than that of F-250 and F-300. The F-275 sample displays the most uniform nanosphere microstructure without obvious agglomeration like that in F-250 and F-300. The uniform nanospheres structure is beneficial for the electronic and lithium ions conductivity. Additionally, the microstructure of F-275 in electrodes after 500 cycles at 1000 mA·g−1 were further analyzed by SEM in own in + ,23. Diff−1. It shows that the unique Co/CoO/C nanosphere structure has a positive influence on enhancing the electrochemical performance of CoO electrode materials.3O4 electrodes. Such remarkable enhanced electrochemical performance shows that the in-situ generation of cobalt and carbon can not only improve the electronic conductivity but also stabilize the mechanical integrity during cycling process. The facile fabrication process without any complex and demanding reduction conditions shows significance for the fabrication of other metal and metal oxide composite electrodes.In summary, well-dispersed Co/CoO/C nanospheres were prepared successfully using a simple electrospinning process assisted with a pre-oxidization process and carbonization process. The unique Co/CoO/C nanosphere structure with generation of cobalt and carbon in situ renders the electrodes enhanced cycling stability and better rate capability when compared with the pure Co


Piscirickettsia salmonis-resistant phenotypes, the genome of AY3800B, an oxytetracycline-resistant isolate bearing a multidrug resistance plasmid, is presented here.The amount of antibiotics needed to counteract frequent piscirickettsiosis outbreaks is a major concern for the Chilean salmon industry. Resistance to antibiotics may contribute to this issue. To understand the genetics underlying  Salmon farming is one of the main economic activities of Chile. However, diverse epizootics have constantly been hampering its sustainability and development. Piscirickettsiosis, also known as salmonid rickettsial septicemia (SRS), deserves special attention, since it has shown remarkable resilience to being controlled. The disease has high incidence and mortality in seawater-reared salmonids . Due to Piscirickettsia salmonis, and triggered the selection of resistant types  during an SRS outbreak in 2013, in southern Chile. Sequencing was performed at Macrogen, Inc.  using the Pacific Biosciences (PacBio) SMRT cell 8Pac version 3 and DNA polymerase binding kit P6 version 2 for library preparation. An additional Nextera library was prepared to be run on an Illumina HiSeq 2000 platform. A total of 57,096 PacBio mapped reads  with an N50 of 23,273 bp from a total of 60,443 (107× coverage) were de novo assembled into a circular chromosome of 3,187,887 bp, and four plasmids of 60,086 bp (p1PS10), 33,556 bp (p2PS10), 45,767 bp (p3PS10), and 188,301 bp (p4PS10) using SMRT Analysis version 2.3.0.1 (PacBio). The sequences were checked for errors by mapping Illumina paired-end reads  over the genome built with PacBio reads. The data were annotated with the NCBI Prokaryotic Genome Annotation Pipeline using the best-placed reference protein set as the annotation method implemented in GeneMarkS+ revision 3.0 software resulting in 3,539 genes, 3,274 coding sequences (CDSs), 190 pseudogenes, six complete sets of rRNA genes, one small noncoding RNA (ncRNA), and 56 tRNAs. The chromosome depicts a G+C content of 39.73% (Isolate AY3800B, with an MIC to OTC of 256 µg/ml, was recovered from Atlantic salmon (f 39.73% .P. salmonis AY3800B belongs to the LF-89-like genogroup (Edwarsiella tarda (tet resistance determinant similar to the one found in the plasmid pRAS1, which conferred resistance to tetracyclines to a series of Aeromonas salmonicida isolates recovered in Norway (enogroup . Consistenogroup . Interesla tarda . Addition Norway . FurtherThis sequence will allow for an assessment of the epidemiology and management of this resistant phenotype.P. salmonis AY3800B have been deposited at DDBJ/EMBL/GenBank under the accession numbers listed in Chromosome and plasmid sequences of


Tmax, Cmax, t1/2, and AUC were calculated by noncompartment model. Compared with Radix Ginseng Rubra, SMS could significantly increase the content of ginsenosides Rh1 and Rg3 in the decocting process. Ginsenosides Rh1 and Rg3 following SMS treatment displayed higher Cmax, AUC(0–t), and AUC∞)(0– and longer t1/2 and tmax except for 20(R)-Rh1 in rat plasma. The results indicated SMS compound compatibility could influence the dissolution in vitro and the pharmacokinetic behaviors in vivo of ginsenosides Rh1 and Rg3, suggesting pharmacokinetic drug-drug interactions between ginsenosides Rh1 and Rg3 and other ingredients from Fructus Schisandrae and Radix Ophiopogonis. This study would provide valuable information for drug development and clinical application of SMS.Ginsenosides Rh1 and Rg3, as the main bioactive components from Ginseng, are effective for prevention and treatment of cardiovascular diseases. Sheng-Mai-San (SMS), a classical complex prescription of traditional Chinese medicines, is composed of Radix Ginseng Rubra, Fructus Schisandrae, and Radix Ophiopogonis. In this research, a sensitive and specific liquid chromatography-mass spectrometric method was developed and validated for stereoselective determination and pharmacokinetic studies of 20(R)- and 20(S)-ginsenoside Rh1 and 20(R)- and 20(S)-ginsenoside Rg3 epimers in rat plasma after oral administration of Radix Ginseng Rubra or SMS extracts. The main pharmacokinetic parameters including  Ginsenosides are glycosides with a dammarane skeleton and classified as protopanaxadiol or protopanaxatriol compounds (except ginsenoside Ro). Ginsenosides are the major bioactive ingredients of Ginseng and exhibit many pharmacological activities including vasorelaxation, antioxidation, anti-inflammation, and anticancer . GinsengSheng-Mai-San (SMS), a classical complex prescription of traditional Chinese medicine (TCM), contains Radix Ginseng Rubra, Fructus Schisandrae, and Radix Ophiopogonis . It was Radix Ginseng Rubra was from Jilin province in China. Radix Ophiopogonis was purchased from Mianyang of Sichuan province. Fructus Schisandrae was from Liaoning province. The three herbs were authenticated by Professor Min-jian Qin . Digoxin  was purchased from the National Institute for the Control of Pharmaceutical and Biological Products . 20(R)- and 20(S)-ginsenoside Rh1 and 20(R)- and 20 (S)-ginsenoside Rg3 (purity > 98%) were from Nanjing Zelang Medical Technological Co. Ltd. . D101 macroporous resin was provided by Chemical Plant of Nankai University . Ammonium chloride, ammonium acetate, and acetic acid were purchased from Nanjing Reagent Company . Acetonitrile  and methanol  were of HPLC grade.A Shimadzu 2020 LC-MS system, with an electrospray ionization interface (ESI) source and Shimadzu LCMS LabSolution Workstation software for data processing, was utilized to perform the analytical procedures. The system includes two Shimadzu LC-20AD pumps, a Shimadzu SIL-20A autosampler, a Shimadzu CTO-20A column oven, a CBM-20A system controller, and a Shimadzu DGU-20A3 online degasser. A Q-array-octapole-quadrupole mass analyzer was used as the detector.18 column  with the mobile phase containing 0.5 mmol/L ammonium chloride in double-distilled water (A) and acetonitrile (B) at a flow rate of 1.0 mL/min. A diverter valve was used to direct 0.3 mL/min HPLC elute to MS detector and 0.7 mL/min elute to a waste container. A gradient elution program was used as follows: 0.01→6.0 min, 38% B; 6.0→6.5 min, 38→50% B; 6.5→15.0 min, 50% B; 15.0→15.5 min, 50→38% B. The column temperature was kept constant at 40°C.The chromatographic separation was carried on a Diamonsil C− of 20(R)-ginsenoside Rh1 and 20(S)-ginsenoside Rh1 at m/z 673.45, 20(R)-ginsenoside Rg3 and 20(S)-ginsenoside Rg3 at m/z 819.60, and digoxin (IS) at m/z 815.60 were monitored in selected ion monitoring (SIM) mode.The negative-ion electrospray ionization mode was utilized under the following source conditions: curved desolvation line (CDL) temperature, 250°C; block temperature, 400°C; interface temperature, 350°C; nebulizing gas flow rate, 1.5 L/min; drying gas flow rate, 10 L/min. CDL voltage, 0 V; Q-array DC (direct current) voltage, 0 V; interface voltage and RF (radiation frequency) voltage were fixed as those in tuning. The chlorinated adducts of molecular ions [M + Cl]Radix Ginseng Rubra and Radix Ophiopogonis were cut into slices and Fructus Schisandrae was scrunched into powder before use. Three drugs were mixed in the ratio of 1 : 3 : 1.5 and the total weight was 110 g . The mixμm membrane filter and stored at 4°C. Calibration standards of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3  were obtained by spiking appropriate amount of working solutions to blank plasma. For quality control (QC) samples, four ginsenosides were prepared at concentrations of 1.0, 10.0, and 90.0 ng/mL in plasma, respectively. The standard spiked plasma samples were stored at −20°C until analysis.The stock solutions of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 were prepared in methanol and serially diluted with methanol to prepare working solutions. A 100 ng/mL internal standard (IS) solution was similarly prepared by diluting 0.2 mg/mL stock solution of digoxin in methanol. All solutions were passed through 0.45 μL plasma sample was transferred into a centrifuge tube together with 10 μL of IS solution. After vortex-mixing for 30 s, the analytes and IS were extracted from plasma with n-butanol for three times . Then the organic layer was transferred into another centrifuge tube and evaporated to dryness at 37°C. Finally, the residue was reconstituted in 20 μL methanol and centrifuged at 12,000 ×g for 10 min; 10 μL aliquot was injected into chromatographic system for analysis.An aliquot of 300 Male Sprague-Dawley rats, 193–215 g in weight, were supplied by Shanghai Slater Experimental Animal Feeding Center . The rats were kept in an air-conditioned animal quarter at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%. The animals were acclimatized to the facilities for four days and then fasted with free access to water for 12 h prior to experiment. Animal experiments were carried out in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of China. All animal protocols were approved by the Ethics Committee on the Care and Use of Laboratory Animals of China pharmaceutical University.μL blood samples were collected in heparinized Eppendorf tube via the oculi chorioideae vein before dosing and subsequently at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h following oral administration of Radix Ginseng Rubra or SMS extracts. After being mixed gently and centrifuged at 3,000 ×g for 10 min, the plasma sample was obtained and kept at −20°C until analysis.Rats were divided randomly into two groups (5 rats in each time point per group) and given an oral dose of 1.35 mL/100 g Radix Ginseng Rubra and SMS extracts, respectively, which was derived from the dose in clinical practice with correction. About 0.5 y = ax + b. For the sensitivity determination, a series of different diluted plasma standard samples were prepared. The LLOQ was estimated using a signal-to-noise ratio of 10 : 1. Intra- and interday precision and accuracy were assayed by determining QC samples at high, middle, and low concentration levels on three different validation days. The extraction recoveries of analytes at three QC levels were evaluated by comparing the peak areas obtained from QC samples with those from the standard solutions at the same concentration. The stability of analytes in rat plasma was investigated by analyzing QC samples at three concentrations stored for 12 h at ambient temperatures and after three freeze (−20°C) thaw (room temperature) cycles. The matrix effect was determined by comparing the peak response ratio of each analyte in blank plasma spiked with QC working solutions at three levels with that of each analyte in the corresponding concentration of prepared QC working solutions using pure water instead of blank plasma. For each concentration level, three duplicates were operated.The method was validated by linearity, the lower limit of quantification (LLOQ), intra- and interday precision, accuracy, extraction recovery, and stability. Plasma samples were quantified by using the ratio of the peak area of analyte to that of IS as the assay parameter. Standard curves representing peak area ratios versus analyte concentrations were described in the form of 0–t) was calculated using the linear-trapezoidal rule, with extrapolation to infinity (AUC∞0–) from the last detectable concentration using the terminal elimination rate constant (ke) calculated by linear regression of the final log-linear part of the drug concentration-time curve. Apparent elimination half-life (t1/2) was calculated as t1/2 = 0.693/ke. Ginsenoside AUCC value is derived from AUC∞0–/concentration ratio (SMS/Radix Ginseng Rubra). Differences between groups were evaluated by unpaired Student's t-test. Differences were considered statistically significant at P < 0.05.Data are expressed as the mean ± standard deviation (SD). Pharmacokinetic parameters were estimated by noncompartment model using WinNonlin 5.3 program package . The area under the curve -Rh1, 20(S)-Rh1, 20(R)-Rg3, 20(S)-Rg3, and digoxin to ESI were evaluated by measuring the full scan mass spectra in both positive and negative ionization modes. To obtain the maximum sensitivity, we investigated the effects of pH and additives with various mobile phases on the ionization efficiency of analytes. Compared with ammonium acetate and acetic acid, ammonium chloride as mobile phase additive showed the maximum sensitivity of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, 20(S)-Rg3, and digoxin in negative mode.n-butanol displayed less interference and thus was selected for sample pretreatment in our study. In addition, in view of the trace concentration of ginsenosides Rh1 and Rg3 in rat plasma following oral administration of SMS and Radix Ginseng Rubra extracts, only applying HPLC-MS method could not meet the analysis requirement in this study. For the plasma samples, concentrating the extract of LLE is another good choice for raising the concentration of analytes. In our study, 300 μL sample plasma was liquid-liquid extracted by n-butanol, evaporated to dryness, and then reconstituted by 25 μL methanol. The concentration of the analytes was condensed 12-fold around, and the sensitivity of the method was obviously improved.Various plasma sample pretreatment procedures were evaluated, including protein precipitation and liquid-liquid extraction (LLE). Conventional and simple liquid-liquid extraction procedure with − of Rh1, Rg3, and digoxin were not detected. The predominant protonated molecules found for Rh1, Rg3, and digoxin were [M + Cl]− m/z 673.45, 819.60, and 815.60, respectively. The mass spectrometric parameters were optimized to obtain the higher signal for the selected ion [M + Cl]−, which also showed less internal interference. The typical chromatograms of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 were presented in The full scan mass spectra of Rh1 and Rg3 after direct injection in mobile phase are presented in y) versus analytes concentration in spiked plasma samples (x). The linear range and regression equations for quantification of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 were presented in The linear regression analysis of analytes was constructed by plotting the ratio of peak area of analytes to that of IS (The extraction recoveries of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 in rat plasma were shown in μm nylon membrane filter before injection. The results are given in The contents of ginsenosides Rh1 and Rg3 in Radix Ginseng Rubra and SMS extracts were determined under the above chromatographic conditions. About 1 mL of herbal extract was diluted 5 times with water, and then 1 mL of the diluent was dissolved in 4 mL methanol and ultrasonicated for 30 min. The solution was filtered through 0.45 Cmax, AUC(0–t), and AUC∞)(0– and longer t1/2 and tmax except for 20(R)-Rh1. The significant increase in AUC and Cmax indicates that ginsenosides Rh1 and Rg3 were absorbed better in the intragastric administration of SMS than the Radix Ginseng Rubra extract. In addition, long t1/2 and tmax infer a delayed absorption and slow elimination of four ginsenosides after oral administration of SMS. It suggests that compound compatibility of SMS, that is, Radix Ginseng Rubra combined with Fructus Schisandrae and Radix Ophiopogonis, would affect the pharmacokinetics of ginsenosides Rh1 and Rg3 including promoting the absorption in vivo, inhabiting the elimination, and improving the bioavailability. However, the pharmacokinetic behavior and parameters of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 were different from the literature reported [The plasma concentrations of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 in rats were determined successfully following oral administration of Radix Ginseng Rubra and SMS extracts. The administration doses of four ginsenosides from SMS and Radix Ginseng Rubra extracts were different in this study. AUC value of each ginsenoside was converted to AUCc according to their doses due to AUC value proportional to dose for linear pharmacokinetics . Main phreported , 18. ThiTCM has been applied in clinical practice for more than 2000 years. Complex herbal prescriptions are the commonly used forms of TCM. Unlike chemically synthetic drugs of high purity, herbal medicines either as single herb or as combination of two or more herbs in composite formula contain hundreds of phytochemicals. However, only the constituents absorbed to blood and their metabolites possess the probability to exert pharmacological activities through the oral administration way. Generally, the compound compatibility of TCM influences the absorption, distribution, metabolism, and excretion in vivo of active ingredients , 9. SyneGinsenosides are regarded as the principal ingredients responsible for the pharmacological activities of SMS. However, pharmacokinetic studies of ginsenosides are challenging, especially for ginsenosides epimers. Firstly, because of the UV end absorption of ginsenosides, HPLC-UV method detection performed at 200~205 nm usually caused high level of baseline noise. Secondly, the amount of ginsenosides in SMS is relatively low. Furthermore, the weak bioavailability of ginsenosides following oral administration results in trace plasma concentration . These sGinsenosides Rh1 and Rg3 are main bioactive components of Radix Ginseng Rubra and confirmed as main effective constitutes in some compound prescriptions of TCM, such as Sheng-Mai-San. Ginseng-herb interactions were previously reported –26. Explt1/2 of 2.94 ± 0.76 h, 2.85 ± 0.69 h, 3.70 ± 0.43 h, and 4.14 ± 1.18 h for 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3, respectively. The elimination of ginsenoside Rh1 was significantly postponed (t1/2 increased to 3.69 ± 0.87 h for 20(R)-Rh1, 4.21 ± 1.05 h for 20(S)-Rh1, 4.45 ± 0.98 h for 20(R)-Rg3, and 6.38 ± 1.72 h for 20(S)-Rg3) when it was coadministrated with Fructus Schisandrae and Radix Ophiopogonis. Furthermore, coadministration with SMS extracts markedly increased the systemic exposure level of 20(R)-Rh1, 20(S)-Rh1, 20(R)-Rg3, and 20(S)-Rg3 in vivo with AUCc 86.90→271.76, 159.20→677.02, 29.50→53.82, and 95.00→212.87, respectively. Double peak phenomenon was observed in rats following oral administration of SMS extract compared with those in Radix Ginseng Rubra, which might be caused by hepatoenteral circulation, transformation from other ginsenosides, or other reasons. Under catalysis by gastric acid and intestinal bacteria in biological conditions, both protopanaxadiol- and protopanaxatriol-type ginsenosides undergo sequential deglycosylation metabolism to form secondary metabolites and finally aglycones [In pharmacokinetic study, ginsenosides Rh1 and Rg3 in Radix Ginseng Rubra showed quick elimination in vivo with short glycones –32. It sglycones , 34. It glycones , 36. Furglycones . These pCmax, AUC(0–t), and AUC∞)(0– and longer t1/2 and tmax except for 20(R)-Rh1 in rat plasma. The results indicated SMS compound compatibility could influence the dissolution in vitro and the pharmacokinetic behavior in vivo of ginsenosides Rh1 and Rg3, involving increasing the absorption, inhibiting the elimination, and improving the bioavailability, which suggested pharmacokinetic drug-drug interactions between ginsenosides Rh1 and Rg3 and other ingredients from Fructus Schisandrae and Radix Ophiopogonis in SMS. This study would provide valuable information for drug development and clinical application of SMS.In the present study, a rapid, sensitive, and specific LC-MS was developed and validated for stereoselective determination and pharmacokinetic studies of 20(R)- and 20(S)-ginsenoside Rh1 and 20(R)- and 20(S)-ginsenoside Rg3 epimers in rat plasma after oral administration of Radix Ginseng Rubra or SMS extracts. A simple liquid-liquid extraction procedure was used to prepare the samples with high recovery. With respect to the mobile phase, 0.5 mmol/L ammonium chloride in water was preferred to obtain abundant and stable signals. The established assay was applied to the pharmacokinetic study of compound compatibility of SMS. Compared with Radix Ginseng Rubra, SMS could significantly increase the dissolution of ginsenosides Rh1 and Rg3 in the decocting process. Ginsenosides Rh1 and Rg3 following SMS treatment displayed higher


The goal of this study was to assess mitochondrial function, energy, and purine metabolism, protein synthesis machinery from the nucleolus to the ribosome, inflammation, and expression of newly identified ectopic olfactory receptors (ORs) and taste receptors (TASRs) in the frontal cortex of typical cases of dementia with Lewy bodies (DLB) and cases with rapid clinical course (rpDLB: 2 years or less) compared with middle-aged non-affected individuals, in order to learn about the biochemical abnormalities underlying Lewy body pathology.Real-time quantitative PCR, mitochondrial enzymatic assays, and analysis of β-amyloid, tau, and synuclein species were used.The main alterations in DLB and rpDLB, which are more marked in the rapidly progressive forms, include (i) deregulated expression of several mRNAs and proteins of mitochondrial subunits, and reduced activity of complexes I, II, III, and IV of the mitochondrial respiratory chain; (ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; (iii) abnormal expression of nucleolar proteins, rRNA18S, genes encoding ribosomal proteins, and initiation factors of the transcription at the ribosome; (iv) discrete inflammation; and (v) marked deregulation of brain ORs and TASRs, respectively. Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from Alzheimer’s disease. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes, and absence of soluble tau oligomers are common in DLB and rpDLB. Low levels of soluble β-amyloid are found in DLB. However, increased soluble β-amyloid 1–40 and β-amyloid 1–42, and increased TNFα mRNA and protein expression, distinguish rpDLB.Molecular alterations in frontal cortex in DLB involve key biochemical pathways such as mitochondria and energy metabolism, protein synthesis, purine metabolism, among others and are accompanied by discrete innate inflammatory response. Dementia with Lewy bodies (DLB) is the second most common neurodegenerative dementia in the elderly, clinically manifested by fluctuating cognition with pronounced variation in attention and alertness, recurrent visual hallucinations which are typically well formed and detailed, and spontaneous motor features of parkinsonism; repeated falls, syncope, transient loss of consciousness, systematized delusions, hallucinations in other modalities, and neuroleptic sensitivity are not uncommon –4. TheseDementia with Lewy bodies is pathologically characterized by Lewy bodies and Lewy neurites in the brainstem, limbic system, and cortical areas , 7, 8. TWhether the amount of α-synuclein pathology  in cerebral cortex is predictable of dementia in LBDs is a matter of controversy –23. CholHowever, deficits in neurotransmission are not limited to dopaminergic and cholinergic systems. Synapses are primarily damaged in the neocortex in DLB –38. SynaAdditional molecular alterations converge in the pathogenesis of DLB, including impaired autophagy and ubiquitin-proteasome system of protein –50, as wThe average survival time for typical DLB from the beginning of symptoms is about 5–8 years . HoweverOur hypothesis is that alterations of several metabolic pathways converge in the pathogenesis of DLB and that impaired mitochondria and energy metabolism, purine metabolism, protein synthesis, and inflammation may be important factors in the pathogenesis of DLB. In the same line, although neuropathological studies have not shown differences between DLB and rpDLB , biochemThe present DLB-centered study was undertaken to analyze (i) levels of selected mRNAs and proteins of subunits of the five mitochondrial complexes and genes linked to energy metabolism; (ii) expression of genes encoding enzymes involved in purine metabolism; (iii) mRNA and protein expression of selected nucleolar proteins, rRNAs, and genes encoding ribosomal proteins, and protein expression of initiation and elongation factors of protein transcription at the ribosome; (iv) cytokines and mediators of the inflammatory response; and (v) gene expression of particular brain receptors involved in olfaction and taste known to be altered in other neurodegenerative diseases with abnormal protein aggregates. All these pathways were assessed in the frontal cortex of cases with typical course (DLB) and in cases with rapid course (rpDLB) compared with middle-aged (MA) individuals to identify molecular factors linked to Lewy body pathology.2O2. DLB cases (n = 13) were neuropathologically categorized following current staging classifications for LBD  or rapid DLB  on the basis of the natural clinical course. rpDLB here was defined as 2 years or less of disease duration from the first symptom to death  had not suffered from neurologic, psychiatric, or metabolic diseases (including metabolic syndrome) and did not have abnormalities in the neuropathological examination excepting stages I–II of NFT pathology and phases 1–2 of β-amyloid plaques.Brain tissue was obtained from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank and the Hospital Clinic-IDIBAPS Biobank following the guidelines of Spanish legislation on this matter  and approval of the local ethics committees. Processing of brain tissue has been detailed elsewhere , 60. The for LBD , 61, 62, for LBD , 63, and for LBD , and a f for LBD . Neuropato death . Cases wQuantification of β-amyloid burden was carried out with a Nikon Eclipse E800 microscope . The cortical total Aβ burden was calculated as the percentage of the area of Aβ deposition in plaques with respect to the total area in 9–10 pictures taken at random from frontal cortex in every case. β-Amyloid quantification was assessed using the Adobe Photoshop CS5 software .A summary of cases is shown in Table Purification of RNA from the right frontal cortex area 8 was carried out using RNeasy Lipid Tissue Mini Kit  following the protocol provided by the manufacturer combined with DNase digestion to avoid extraction and later amplification of genomic DNA. The concentration of each sample was obtained from A260 measurements with NanoDrop 2000 spectrophotometer . RNA integrity was tested using the Agilent 2100 BioAnalyzer  . Values Bivariate analyses were carried out to detect association of our variables with potential confounding factors  using Spearman or Pearson correlations for quantitative variables. Stastical analysis was performed with GraphPad Prism version 5.00 and SPSS 19. Postmortem delay had no effect on RIN values in the present series.® 9700 PCR System thermocycler (Applied Biosystems). A parallel reaction for one RNA sample was processed in the absence of reverse transcriptase to rule out DNA contamination.Retrotranscription reaction of RNA samples was carried out with the High-Capacity cDNA Archive kit  following the guidelines provided by the supplier, and using Gene AmpReal-time quantitative PCR (RT-qPCR) assays were conducted in duplicate on 1,000 ηg of cDNA samples obtained from the retrotranscription reaction, diluted 1:20 in 384-well optical plates  utilizing the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Parallel amplification reactions were carried out using 20× TaqMan Gene Expression Assays and 2× TaqMan Universal PCR Master Mix (Applied Biosystems) . Genes aThe selection of probes was based on criteria covering a larger project geared to analyzing similar expression of the same molecules related to inflammation, mitochondria, energy metabolism, purine metabolism, protein synthesis and selected receptors in several diseases including AD, PD, Creutzfeldt-Jakob’s disease, certain tauopathies, in addition to DLB, in all cases using the same probes and methods in an attempt to learn about commonalities and differences among these diseases in a particular brain region. Our idea is that all these pathways are altered in most neurodegenerative diseases with abnormal protein aggregates, but alterations are region-, stage-, and disease-specific.GUS-β), X-prolyl aminopeptidase (aminopeptidase P) 1 (XPNPEP1), AARS , and HPRT (hypoxanthine-guanine phosphoribosyltransferase) probes for normalization. The selection of these housekeeping genes was based on previous data showing low vulnerability in the brain of several human neurodegenerative diseases . The fold change was determined using the equation 2−ΔΔCT.Parallel assays for each sample were carried out using β-glucuronidase  at 200 mA/membrane for 40 min. Non-specific bindings were blocked by incubation with 5% milk in Tris-buffered saline (TBS) containing 0.1% Tween for 1 h at room temperature. After washing, the membranes were incubated at 4°C overnight with one of the primary antibodies  in TBS containing 3% albumin and 0.1% Tween. These membranes were incubated for 1 h with the appropriate HRP-conjugated secondary antibody , and the immune complexes were revealed with a chemiluminescence reagent . Monoclonal anti-β-actin antibody diluted 1:30,000  was blotted to control protein loading.Tissue was processed as reported elsewhere . A totalg for 10 min. The supernatant (S1) was conserved. The pellet was washed with two volumes of IB and centrifuged again under the same conditions. This last supernatant (S2) was combined with S1. Centrifugation at 10,000 × g for 10 min at 4°C resulted in the mitochondria-enriched pellet. The pellet was finally re-suspended in one volume of IB and stored at −80°C. Protein concentration was measured by Smartspect™ plus spectrophotometer  using the Bradford method . Twenty-five micrograms of mitochondria was loaded into each well. Activity of citrate synthase was evaluated following validated protocols . Isolation of mitochondria was carried out as reported in detail elsewhere . Brieflyrotocols  with slirotocols .g for 1 h at 4°C. The supernatant was the soluble fraction used for amyloid quantification, and the protein of this fraction was measured with BCA. The detection and measurement of β-amyloid 1–40 (Aβ40) and β-amyloid 1–42 (Aβ42) were carried out by enzyme-linked immune-absorbent assay with the corresponding detection kits , following the instructions of the supplier. TBS-soluble Aβ40 and Aβ42 levels were normalized to the total amount of protein from each individual sample  with a cocktail of protease inhibitors , and ultra-centrifuged at 100,000 × l sample .g for 30 min at 4°C. The pellet was re-suspended in 2% SDS and centrifuged at 14,000 × g for 30 min at 4°C. The supernatant was membrane-associated Aβ and the protein of this fraction was measured with BCA method . Proteins were separated in SDS-polyacrylamide gel electrophoresis. Thirty-five micrograms of protein was loaded onto a precast NuPAGE 4–12% Bis-Tris gel system  with MES buffer . The proteins were transferred to nitrocellulose membranes, 200 mA/membrane, for 90 min. Then membranes were boiled with PBS 1× for 15 min, and non-specific bindings were blocked by incubation in 5% non-fat dry milk in TBS containing 0.2% Tween for 1 h at room temperature. After washing, the membranes were incubated at 4°C overnight with primary antibodies to human amyloid-beta protein, clone: 4G8  and 6E10  in TBS containing 5% albumin and 0.2% Tween. After washing, the membranes were incubated at 4°C overnight with the primary antibody to human amyloid-beta protein, clone: 4G8  in TBS containing 5% albumin and 0.2% Tween. Membranes were then incubated for 1 h with the appropriate HRP-conjugated secondary antibody , and the immune complexes were visualized with a chemiluminescence reagent . β-Actin was used as a control of protein loading (Frozen samples were homogenized in TBS with a cocktail of protease and phosphatase inhibitors (Roche Molecular Systems). Homogenates were centrifuged at 14,000 ×  loading . Using tg for 20 min at 4°C (Ultracentrifuge Beckman with 70Ti rotor), supernatants were quantified for protein concentration (BCA), mixed with SDS-PAGE sample buffer, boiled, and separated to 8% SDS-PAGE gels. The proteins were transferred to nitrocellulose membranes, 200 mA/membrane, for 90 min. The membranes were blocked with 5% non-fat milk in TBS containing 0.2% Tween for 1 h at room temperature. After washing, the membranes were incubated at 4°C overnight with the primary antibody anti-tau-5  in TBS containing 5% albumin and 0.2% Tween. Membranes were then incubated for 1 h with the HRP-conjugated secondary anti-mouse antibody , and the immune complexes were visualized with ECL. β-Actin was used as a control of protein loading (Frozen samples were homogenized in lysis buffer: 100 mM Tris (pH 7.0), 100 mM NaCl, 10 mM EDTA, 0.5% NP-40, and 0.5% sodium deoxycholate plus protease and phosphatase inhibitors (Roche Molecular Systems). After centrifugation at 14,000 ×  loading . No atteg at 4°C for 10 min. The pellet was discarded and the resulting supernatant was ultra-centrifuged at 43,000 × g at 4°C for 1 h. The supernatant (S2) was kept as the PBS-soluble fraction. The resulting pellet was re-suspended in a solution of PBS, pH 7.0, containing 0.5% sodium deoxycholate, 1% Triton and 0.1% SDS, and this was ultra-centrifuged at 43,000 × g at 4°C for 1 h. The resulting supernatant (S3) was kept as the deoxycholate-soluble fraction. The corresponding pellet was re-suspended in a solution of 2% SDS in PBS and maintained at room temperature for 30 min. Afterward, the samples were centrifuged at 133,000 × g at 25°C for 1 h and the resulting supernatant (S4) was the SDS-soluble fraction. Equal amounts of each fraction were mixed with reducing sample buffer and processed in parallel for 10% SDS-PAGE electrophoresis and western blotting. Membranes were incubated with anti-α-synuclein oligomer-specific antibody  at a dilution of 1:1,000. The protein bands were visualized with the ECL method , sonicated, and centrifuged at 2,700 × L method .post hoc Tukey’s range test for multiple comparisons was used for normal variables. Statistical analysis was performed with GraphPad Prism version 5.01  and Statgraphics Statistical Analysis and Data Visualization Software version 5.1 . Differences between groups were considered statistically significant at p-values: *p < 0.05, **p < 0.01, and ***p < 0.001.The normality of distribution of the mean fold-change values obtained by RT-qPCR for every region and stage between controls and DLB cases was analyzed with the Kolmogorov–Smirnov test. The non-parametric Kruskal–Wallis test was performed to compare groups when the samples did not follow a normal distribution, whereas the one-way ANOVA with Because of the large number of parameters and regions analyzed, multiple comparison tests and false discovery rate might be employed , 76. Howpost hoc Tukey’s range test for multiple comparisons. Differences were considered statistically significant at p-values: *p < 0.05, **p < 0.01, and ***p < 0.001.Densitometry of western blot bands was assessed with the TotalLab program  and then subsequently analyzed with GraphPad Prism, Statgraphics Statistical Analysis and Data Visualization Software version 5.1  by one-way ANOVA with t-test using GraphPad Prism version 5.01  and Statgraphics Statistical Analysis and Data Visualization Software version 5.1 . In all experimental procedures the significance level was set at *p < 0.05, **p < 0.01, and ***p < 0.001.The enzymatic activities for each mitochondrial complex were expressed as a rate of nanomoles per minute per milligram of mitochondrial protein per protein concentration normalized with the mitochondrial complex activity rate of citrate synthase activity. Data were presented as mean ± SEM for all the experiments. All the data were analyzed with Student’s Brain weight and neuropathological characteristics in the frontal cortex including neuron loss, astrocytic gliosis, microgliosis, spongiosis, diffuse plaques, senile plaques, β-amyloid angiopathy, and α-synuclein aggregates (Lewy bodies and Lewy neurites) were assessed in DLB and rpDLB cases. Thal phase of β-amyloid deposition, Braak stages of NFT pathology, CERAD global, ABC classification, and LBD stage were also considered to frame AD- and LBD-related pathology in every case. Results are summarized in Table No differences in the amount of fibrillar β-amyloid, considering diffuse and senile plaques, were observed between the two groups; the total plaque burden varied from absent to frequent in both groups. The average percentage of Aβ40 of the total β-amyloid in plaques was 7.24% in DLB and 6.03% in rpDLB, and the percentage of Aβ42 was 68.49% in DLB and 67.70% in rpDLB. NFT pathology varied from stage I to stage VI in DLB and from stage 0 to stage V in rpDLB. NFTs in frontal cortex occurred in two DLB and two rpDLB cases.Astrocytic gliosis was variable from one case to another and varied from mild to moderate in DLB and rpDLB excepting one DLB in which astrocytic gliosis was considered severe.A remarkable observation was the discrete microglial response, as revealed with Iba1 and CD68 antibodies, in the frontal cortex in DLB and rpDLB.Neuropathological findings are summarized in Table ATP5G2 and ATP5H involved in mitochondrial complex (p < 0.05). Deregulation of energy metabolism genes included decrease in ATP6VOB and increase in ATP4A and SLC6A6 (p < 0.05). Regarding rpDLB, the expression levels of ATP6VOB and TOMM40, involved in energy metabolism, was decreased in rpDLB   when compared with MA cases and using β-actin for protein normalization, expression of which was maintained in DLB and rpDLB when compared with MA cases. NDUFS8 protein expression was not modified in DLB when compared with MA. Protein levels of SDHB (complex II), UQCRC2 (complex III), MTCO1 (complex IV), and ATP5A and ATP50 (complex V) were also significantly decreased in DLB when compared with MA (p < 0.05) using β-actin for protein loading normalization . However, the expression of several subunits showed a trend toward decrease in rpDLB; therefore, significant differences between DLB and rpDLB were restricted to NDUFB8, SDHB, ATP5A, and ATP50 (p < 0.01) . NDUFA7, NDUFS8, and ATP50 levels were similar in DLB and MA cases using VDAC for normalization. Curiously, expression levels of all the assessed subunits were preserved in rpDLB when normalized with VDAC. As a result, levels of NDUFA10, NDUFB10, NDUFB8, SDHB, UQCRC2, ATP5A, and ATP50 were significantly higher in rpDLB when compared with DLB   expression was reduced in DLB and less markedly in rpDLB when compared with MA thus suggesting a reduced number of mitochondria or reduced mitochondria size. Nevertheless, NDUFA10, NDUFB10, NDUFB8, UQCRC2, MTCO1, and AT5A protein expression was also reduced in DLB cases when using VDAC for normalization (p < 0.05)  Figure .ADA, AK1, ENTPD1, NME6, and PNP (p < 0.05); NME1, NME3, NME4  and PRUNE (p < 0.001) were upregulated in rpDLB when compared with MA cases   mRNA was significantly decreased in DLB when compared with MA (p < 0.05) (Table Nucleoplasmin 1 (5) Table .rRNA 18S expression was increased in rpDLB (p < 0.01) when compared with MA and also when compared with DLB (p < 0.01) (Table 1) Table .RPL7, RPL21, RPL30, and RPS5 , but RPL22 was downregulated in DLB when compared with MA (p < 0.05)  and the small (S) subunits were upregulated in DLB when compared with MA control cases including 5) Table .RPL7, RPL21, RPL23A, RPL30, RPL31, RPS5, RPS6, and RPS13 were upregulated in rpDLB in comparison to MA ; RPS16 was downregulated (p < 0.05)  in rpDLB when compared with DLB (Table p < 0.05). However, the expression levels of initiation factors eIF2α and eIF5 were significantly decreased in rpDLB when compared with MA cases . eIF2α, p-eIF2α, and eIF3η were significantly decreased (p < 0.05) in rpDLB when compared with DLB  and CST7 (p < 0.05) was found in rpDLB cases when compared with MA  , whereas the levels of Iba-1 were maintained when compared with MA cases   Figure .OR2D2, OR4F4, OR11H1, and OR52H1 was observed in DLB when compared with MA cases (p < 0.05). Upregulation of OR2D2 and OR2T33 was significantly higher in rpDLB compared with MA (p < 0.05). Significant differences between DLB and rpDLB were restricted to OR2T33 and OR4F4  (Table Increased expression of y) Table .TAS2R4, TAS2R5, and TAS2R14 were upregulated in DLB when compared with MA . TAS2R4, TAS2R5, TAS2R13, and TAS2R14 expression was significantly increased in rpDLB in comparison to MA cases  (Table 1) Table .p < 0.05 and p < 0.01, respectively)  involved in the uptake of gamma-aminobutyric acid (GABA)  . Whetherd (GABA) . ATP6V0Bneration . TOMM40 chondria . TogetheADA, AK1, ENTPD1, NME1, NME3, NME4, NME6, PNP, and PRUNE. However, only three genes, ENTPD2, NME3, and PRUNE, are significantly upregulated in rpDLB when compared with DLB.Several genes encoding enzymes linked to purine metabolism are upregulated in DLB and rpDLB although significant values are only obtained when comparing rpDLB with MA cases. These include NME gene family encodes nucleotide diphosphate kinases, which are involved in the phosphorylation of nucleotide diphosphates to form nucleotide triphosphates. These enzymes are enriched in synapses. Increased NME mRNA expression in DLB contrasts with its decrease in AD (NME in response to reduced synapses in DLB. The product of PRUNE participates in the metabolism of guanosine pentaphosphate and tetraphosphate and is linked to NME in memory conservation (DGUOK) phosphorylates purine deoxyribonucleosides in the mitochondrial matrix  deregulated expression of several mRNAs and proteins of mitochondrial subunits and reduced activity of complexes I, II, III, and IV of the mitochondrial respiratory chain; (ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; (iii) abnormal expression of certain nucleolar proteins, rRNA18S, genes encoding ribosomal proteins and initiation factors of the transcription at the ribosome; (iv) discrete inflammation; and (v) marked deregulation of brain ORs and TASRs. Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from AD. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes, and absence of soluble tau oligomers are common in DLB and DLB with rapid progression. Increased soluble β-amyloid 1–40 and β-amyloid 1–42, and increased TNFα mRNA and protein expression, distinguish DLB with rapid progression from typical DLB. Since high levels of soluble β-amyloid species are toxic for nerve cells and TNF-α.PG-E carried out RT-qPCR, western blots, and enzymatic activities. IL-G performed the studies of beta-amyliid and tau oligomers. OG-R participated in the selection of cases and in the neuropathological study. MG-G and AK helped PG-E. FL and SZ helped in molecular studies. MC helped in the processing of samples. JR helped in the design of molecular studies in rapid dementias. IZ helped in the design of the study of rapid dementias. EG carried out the neuropathological and supervised the clinical and pathological relations. IF directed the study, supervised the work, wrote the advanced version of the manuscript, which was circulated among the authors for comments and suggestions, and prepared the final version for submission.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.


Vascular complications in homocystinuria have been known for many years, but there have been no reports to date on involvement of the ascending aorta.We conducted a cross-sectional study of patients with homocystinuria, known to a single metabolic centre, and evaluated in 2016 with a transthoracic echocardiogram. Aortic root dilation was defined as Z-score ≥ 2.0 SD, and graded mild (Z-score 2.0–3.0), moderate (Z-score 3.01–4.0) and severe (Z-score > 4.0).The study population included 34 patients, median age of 44.3 years (IQR 33.3–52.2), 50% males, 69% diagnosed aged <18 years and 29% pyridoxine-responsive. Eight (24%) had a history of hypertension. Seven patients (21%) were found to have a dilation of the aortic root, mild in two cases (6%), moderate in four (12%) and severe in one (3%). None had dilation of the ascending aorta. Significant aortic regurgitation, secondary to moderate aortic root dilation, was documented in two patients. A single patient had significant mitral regurgitation due to prolapse of both valve leaflets, as well as mild aortic root dilation. Comparing patients with a dilation of the aortic root to those without, there were no significant clinical, laboratory or echocardiographic differences, with the only exception being that the diameter of the ascending aorta was larger in the group with a dilated aortic root, albeit within normal limits.A subset of patients with homocystinuria have isolated dilation of the aortic root similar to that observed in Marfan syndrome.The online version of this article (10.1007/s10545-017-0094-7) contains supplementary material, which is available to authorized users. Homocystinuria  is a rare autosomal recessive genetic disease caused by cystathionine β-synthase  deficiency Mudd . The pheThromboembolic disease is a major cause of morbidity and mortality in HCU. In an untreated cohort of HCU patients, 50% experienced a thromboembolic event by the age of 30 and thromboembolism was reported as a significant factor in 80% of deaths , the John Radcliffe Hospital, Oxford (N = 1) and Southmead Hospital, Bristol (N = 1) who underwent a transthoracic echocardiogram in 2016. The biochemical diagnosis of HCU was based on plasma total homocysteine (Hcy) levels, according to the current recommendations  + [(age at last follow up – age at diagnosis) * mean Hcy during follow up].p-value <0.05 was considered significant. Statistical analyses were carried out using IBM SPSS Statistics for Windows, version 24.0 .Continuous variables are reported as median (interquartile range), categorical values as number (percentage). Continuous variables were compared using Mann-Whitney test and categorical variables compared using Fisher’s exact test. The relationship between cumulative Hcy exposure and Z-score was assessed using Spearman’s rank-order correlation. A CBS mutations 1566del]). This gene panel included the ACTA2, COL3A1, FBN1, FBN2, FLNA, GATA5, MFAP5, MYH11, MYLK, NOTCH1, PRKG1, SKI, SLC2A10, SMAD3, SMAD4, TGFB2, TGFB3, TGFBR1 and TGFBR2 genes. Of the four patients with moderate aortic root dilation, two lifted weights regularly in the gym and one of the two also took protein powder supplements.The study population consisted of 34 patients, with a median age of 44.3 years (IQR 33.3–52.2), 50% of them males. Seventy percent of patients had been diagnosed before the age of 18, and 29% were pyridoxine-responsive. Patients who were not pyridoxine-responsive were advised not to consume excessive protein, but as a restricted protein diet is difficult to initiate and sustain in older patients, only four patients followed a formal prescribed low protein diet with specific HCU-appropriate amino acid supplementation. Twenty-seven (79%) had at least one of the known complications of HCU, the most frequent being lens dislocation (present in 62%). Vascular complications were venous in all cases, no patient had a history of arterial thrombosis. Eight patients (24%) had a history of hypertension. Clinical, laboratory and echocardiogram findings of the overall population are reported in Table n = 12), no correlation was found between cumulative Hcy exposure and aortic root Z-score , a disease caused in most cases by mutations in the FBN1 gene, that encodes fibrillin-1, but to a lesser degree. By forming a lattice around elastic fibres, fibrillin-1 plays an important structural role in the extracellular matrix. However, haploinsufficiency leading to a weakening of connective tissue does not explain all components of the MFS phenotype, such as long bones  have been recorded by indwelling catheter during weight lifting  was 0.04%


Tai-Kadai (TK) is one of the major language families in Mainland Southeast Asia (MSEA), with a concentration in the area of Thailand and Laos. Our previous study of 1234 mtDNA genome sequences supported a demic diffusion scenario in the spread of TK languages from southern China to Laos as well as northern and northeastern Thailand. Here we add an additional 560 mtDNA genomes from 22 groups, with a focus on the TK-speaking central Thai people and the Sino-Tibetan speaking Karen. We find extensive diversity, including 62 haplogroups not reported previously from this region. Demic diffusion is still a preferable scenario for central Thais, emphasizing the expansion of TK people through MSEA, although there is also some support for gene flow between central Thai and native Austroasiatic speaking Mon and Khmer. We also tested competing models concerning the genetic relationships of groups from the major MSEA languages, and found support for an ancestral relationship of TK and Austronesian-speaking groups. The geography of Thailand encompasses both upland and lowland areas, and Thailand is one of the most ethnolinguistically-diverse countries in Mainland Southeast Asia (MSEA). With a census size of ~68 million in 2015, there are 70 different recognized languages belonging to five different major language families: Tai-Kadai (TK) (90.5%), Austroasiatic (AA) (4.0%), Sino-Tibetan (ST) (3.2%), Austronesian (AN) (2.0%), and Hmong-Mien (HM) (0.3%) [The recorded history of the CT people or Siamese started with the Sukhothai Kingdom, around the 13th century A.D . HoweverHere we extend our previous study by adding 560 new complete mtDNA genome sequences from 22 groups (mostly from CT) speaking TK, AA, and ST languages; when combined with the previous data , there aSamples were analyzed from 560 individuals belonging to 22 populations classified into four groups: 1) the central Thais (CT) (seven populations: CT1-CT7); (2) the Mon (two populations: MO6-MO7); (3) the TK speaking groups from northern Thailand, including Yuan (four populations: YU3-YU6), Lue (four populations: LU1-LU4) and Khuen (TKH); and (4) the ST speaking Karen  (Table  the centwww.mbio.ncsu.edu/BioEdit/bioedit.html). A multiple sequence alignment of the sequences and the Reconstructed Sapiens Reference Sequence (RSRS) [We generated complete mtDNA sequences from genomic libraries with double indices and mtDNA enrichment based on protocols described previously , 15. Thee (RSRS)  was obtae (RSRS) . The 560Φst, pairwise difference), permutation test, analyses of molecular variance (AMOVA), and a Mantel test of the correlation between genetic and geographic distances were also carried out with Arlequin [Φst matrix by Arlequin [Haplogroup assignment was performed with the online tools Haplogrep  and MitoArlequin . Three tArlequin . To get Arlequin .Φst distance matrix was visualized by a multi-dimensional scaling plot (MDS) using STATISTICA 10.0 . A Discriminant Analysis of Principal Components (DAPC) was employed using the dapc function within the adegenet R package [www.fluxus-engineering.com) and visualized in Network publisher 1.3.0.0.The  package . Median- package  of haplo−8 and 9.883 × 10−8 for data partitioned between coding and noncoding regions [Bayesian skyline plots (BSP) per population and maximum clade credibility (MCC) trees per haplogroup, based on Bayesian Markov Chain Monte Carlo (MCMC) analyses, were constructed using BEAST 1.8.0 . BEAST i regions  and TracAn approximate Bayesian computation (ABC) approach was utilized to test different demographic scenarios concerning the origin of CT populations and the relationships between SEA language families. For the maternal origin of CT populations, we considered the same three demographic scenarios tested in our previous study for the origins of North/Northeastern Thai and Laos populations:  demic dih = 0.83 ± 0.08; haplogroup diversity = 0.73 ± 0.09; S = 99), although this was also the group with the lowest sample size  and Mon from central Thailand (MO7) (MPD = 39.32 ± 17.70 and π = 0.0024 ± 0.00119)  than a geographic classification , indicating that language family seems to be a better indicator than geography of the genetic structure of Thai/Lao populations, however there is much more variation among populations within the same group for both classifications , resistance distance  and least-cost path distance , further supporting the limited impact of geography on the genetic structure of Thai/Lao populations. Furthermore, a DAPC analysis showed that clustering groups by language family resulted in more discrimination among groups than clustering by geographic criteria  and Seak (SK), as found previously  and in tFourteen of the 174 haplogroups occur in at least ten individuals and together account for 33.92% of the 560 sequences; these are F1a1a, B6a1a, F1f, B5a1a, F1a1a1, C7a1, C7a, M*, M12a1a, M21a, M7b1a1a3, R9b1a1a, R9b1a3, and B5a1b1 (Supplementary Table The remaining haplogroups (66.08%), which occur in lower frequency, tend to be more widely distributed, e.g., G2a1 and basal M sublineages in MO7 and subhaplogroups F (x F1a1a and F1a1a1), M7b1a1 and B4 in Lue (LU) and Khuen (TKH) at varying frequencies  are prevalent in almost all groups , with the exception of some AA groups , Karen, and CT groups; these groups have other widespread haplogroups, e.g., D, M12-G, M , A, C, and N (xN9a) Fig. . Apart fFor the combined dataset, we estimated coalescence ages of SEA haplogroups and their sublineages. We analyzed haplogroups that have additional sequences from the present study and have more than five sequences in total Table . The ageThere are many lineages with ages older than 30 kya found in our Thai/Lao samples, e.g. B4, B5, D, F1, F3, M7, M*, M12, M13, M17, M21, M71, M73, M74, M91, R9, R22, N10, and U . In doing so, it is important to keep in mind that we are not testing the relationships of these language families, as that would require linguistic data. However, determining the best-fitting model based on genetic relationships may help discriminate among hypotheses concerning the language family relationships that make predictions about the genetic relationships of populations speaking those languages. We tested five models of the language family relationships Fig. . The obsThe present study adds to our previous study of Thai/Lao mtDNA genome sequences by including 22 additional groups from Thailand, including the AA-speaking Mon (MO), ST-speaking Karen, and several TK speaking groups, especially the CT. Similar to our previous mtDNA study , geneticThe Mon, who were a previously dominant group in MSEA located in present-day southern Myanmar and central Thailand since the 6 to 7th century A.D. , have beThe Karen in Thailand are refugees who migrated from Myanmar starting from the 18th century A.D. due to the influence of Burmese . HoweverThe present results emphasize the common maternal ancestry of CT and other TK speaking groups in MSEA, e.g., Laos and Southern China. Demic diffusion is still the most probable scenario for TK-speaking populations Fig. , possiblFinally, we used simulations to test hypotheses concerning the genetic relationships of groups belonging to different language families. We found that Starosta’s model  providedSpecifically, the best-fitting model suggests that after separation of the prehistoric TK from AN stocks around 5–6 kya in Southeast China, the TK spread southward throughout MSEA around 1–2 kya by a demic diffusion process, accompanied by population growth but with at most minor admixture with the autochthonous AA groups. Meanwhile, the prehistorical AN ancestors entered Taiwan and dispersed southward throughout ISEA, with these two expansions later meeting in western ISEA. The lack of mtDNA haplogroups associated with the expansion out of Taiwan in our Thai/Lao samples has two possible explanations: either the Out of Taiwan expansion did not reach MSEA ; or, if the prehistoric AN migrated through this area, their mtDNA lineages do not survive in modern Thai/Lao populations. Ancient DNA studies in MSEA would further clarify this issue. Moreover, although mtDNA analyses are informative in elucidating genetic perspectives in geographically and linguistically related populations, they have an obvious limitation in that they only provide insights into the maternal history of populations. Future studies of Y chromosomal and genome-wide data will provide further insights into the genetic history of Thai/Lao populations and the role of factors such as post-marital residence patterns and migration in shaping the genetic structure of the region.Supplementary InformationSupplementary Table S2


HOXB9, as a HOX family transcription factor, playing a significant role in embryonic development and cancer progression. However, the function of HOXB9 and its precise mechanism in regulating endometrial cancer progression remains unknown. Here, we demonstrated that the expression of HOXB9 was increased in endometrial cancer, and associated with histological grade and lymph node metastasis. In addition, elevated HOXB9 predicts a poor prognosis in endometrial cancer patients. Interestingly, bioinformatics analysis of TCGA cancer database showed that HOXB9 expression is positively correlated with E2F3 expression. Moreover, HOXB9 promoted E2F3 expression by directly targeting to its promoter. Furthermore, we found that knocking down E2F3 abolished the ability of HOXB9 in enhancing cell migration. Taken together, for the first, we demonstrated the function and mechanism of HOXB9 in regulating endometrial cancer progression, and indicated HOXB9 may be a novel prognostic marker of endometrial cancer. The patients present with high-grade tumors are aggressive, and frequently diagnosed with tumors spread beyond the uterus4. Surgery is the major treatment for EC, while it is quite important to develop new therapeutic strategies for EC5. The investigation of new molecular mechanisms in EC may be useful to identify new diagnostic and therapeutic targets.Endometrial cancer (EC) is one of the leading causes of gynecologic malignancies8. During embryogenesis, sequential HOX expression from 3ʹ to 5ʹ along the anteriorposterior (AP) axis could control body segmentation according to the rules of spatial and temporal collinearity11. In particular, HOXB9 together with other HOX genes controls the specification of thoracic skeletal elements and mammary gland development13.The HOX genes, encode a group of transcription factors, share a highly conserved homeobox domain. In vertebrates, the whole 39 HOX genes are identified and grouped into four clusters 17. In the previous study, we found HOXB9-regulated lung adenocarcinoma progression by directly targeting JMJD618. In addition, upregulation of HOXB9 showed poor overall survival in invasive human breast cancer, and promoted epithelial-to-mesenchymal transition (EMT)21. However, increased expression of HOXB9 showed better overall survival in colon adenocarcinoma, pancreatic ductal adenocarcinoma and gastric carcinoma patients17, indicating HOXB9 played an opposite role in these cancer progression.In addition to its key roles in embryo development, HOXB9 was also found to be participated in the regulation of numerous human cancers23. As an important member of E2F family, E2F3, encodes two isoforms, E2f3a and E2f3b, whose expression is driven by distinct promoters24. Overexpressed E2F3 expression has been reported in ovarian, prostate and lung cancers27. In the thyroid cancer mouse model, a role for E2F3 has been found in the metastasis of tumor cells to the liver and lung28. Using conditional knockout approaches, Trikha et al. reported that ablation of E2F3 in tumor-associated macrophages (TAMs) results in decreased pulmonary metastasis without affecting the tumor growth29. Tsuruta et al. reported that the expression of E2F3 was upregulated in 28.6% (20 of 70) of EC cases30. However, the role of E2F3 in EC progression remains unclear.The E2F family of transcription factors is conserved from nematodes to mammals, which were identified as factors that control the cell cycle, differentiation, apoptosis and stress responsesAlthough HOXB9 has been extensively investigated in some cancer types, its role in EC has never been reported. In this report, we demonstrated that HOXB9 is overexpressed in EC, and is correlated with EC cell migration. Moreover, HOXB9 promotes EC progression by targeting oncogenic protein E2F3.P = 0.0196; Fig. P = 0.0081) and lymph node metastasis status  and high (score of 2 or 3). The representative images of “low” and “high” staining, as well as negative controls, in endometrial carcinoma, normal proliferative endometrium and atypical hyperplasia tissues were shown in Fig. http://gepia.cancer-pku.cn/)31 and the LinkedOmics database (http://www.linkedomics.org)32, showed that HOXB9 expression was significantly overexpressed in human EC tissues compared with normal tissues , there was no difference in the cell proliferation rate in the three groups Fig. . Then, wWe then tested the EC cell migration mediated by HOXB9. Using transwell assays, we found that cell migration ability was decreased when knocked down HOXB9 in Ishikawa and RL95-2 cells Figs. . In contr value > 0.2 or < –0.2) with HOXB9 in TCGA from the LinkedOmics database (http://www.linkedomics.org)  Figs. . Next, wg) Figs. . Meanwhig) Figs. .Fig. 3Th35. E2F3 was reported overexpressed in multiple tumors, and correlated with cancer metastasis. When we examined the mRNA expression levels of several tumor metastasis-related genes after knocking down HOXB9 (data not shown), we only found E2F3 mRNA expression levels were decreased significantly . All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C with 5% COFlag-HOXB9 expression plasmid was constructed by subcloning the HOXB9 full-length complementary DNA (cDNA) fragments into 3× flag vector. All constructs were confirmed by DNA sequencing. The following antibodies were used: HOXB9 (Santa Cruz: sc-398500 and sc-133671) and E2F3 (Abcam: ab50917).Cell proliferation assays were performed using the CellTiter 96®AQueous One Solution Cell Proliferation Assay kit according to manufacturer’s instructions (Promega). Briefly, EC cells were plated in 96-well plates at a density of 900 cells per well. In all, 15 μl CellTiter 96®AQueous One Solution reagent was added to the cells per well, and incubated for 1 h at 37 °C. Then cell growth was measured in a microplate reader at 490 nm.Colony formation assay was used to determine the function of HOXB9 in promoting cell proliferation. In brief, 2000 cells were seeded onto six-well culture plates. After incubation for 2 weeks, cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde, following with staining by 0.5% crystal violet solution. The colonies were counted using a light microscope.6 Ishikawa stable cells. Tumor sizes were measured at the indicated time. After 24 days when the tumor reached to approximately 1 cm in diameter, the tumors were dissected, and the tumor weight was measured.Balb/c nude mice was injected subcutaneously into the flank with 1 × 105 cells/ml was seed into the upper chamber in serum-free media. The lower wells contains 20% (v/v) FBS. After 8-h migration at 37 °C, cells on the upper surface of the membrane filter were removed. The migrated cells through the inserts were fixed with 4% formaldehyde and stained by crystal violet, and counted.Cell suspension containing 1 × 10The EC cell lines were cultured until a monolayer forms. Artificial wounds were created on the cell monolayer, then capture the images of wound healing at 0, 6 and 12 h, and compare the images to quantify the cell migration rate.Total RNA was isolated from cells using Trizol reagent (Invitrogen) and cDNA was synthesized. Two-step real-time polymerase chain reaction (PCR) was performed using the SYBR Green Mix (Roche) and a LightCycler®96 detection system (Roche) according to manufacturer’s instructions. Specific primers for HOXB9 and E2F3 were designed as follows:HOXB9 forward primer, 5ʹ-CCATTTCTGGGACGCTTAGCA-3ʹ; HOXB9 reverse primer, 5ʹ-TGTAAGGGTGGTAGACGGACG-3ʹ; E2F3 forward primer, 5ʹ-AAGAAGAAGTCTAAAAACAACGTCCAA-3ʹ; E2F3 reverse primer, 5ʹ-CTTGACACTGGGCCAGCAT-3ʹ.GAPDH gene.All mRNAs were normalized to the mRNA level of 48. The ChIP DNA complex were precipitated and subjected to RT-PCR with the indicated primers.The EC cell lines were crosslinked with 1% formaldehyde for 10 min, and subjected to ChIP assays as described beforehttp://gepia.cancer-pku.cn/)31 and the LinkedOmics database (http://www.linkedomics.org)32, which are web-based tools to deliver fast and customizable functionalities based on TCGA data. GEPIA can deliver fast and customizable functionalities based on TCGA data, and provides key interactive and customizable functions including differential expression analysis, correlation analysis and patient survival analysis. The LinkedOmics web allows flexible exploration of associations between a molecular or clinical attribute of interest and all other attributes, providing the opportunity to analyze and visualize associations between billions of attribute pairs for each cancer cohort.The results of TCGA database were mainly analyzed by the web-based tools, GEPIA , with the loss of polarity and marked nuclear atypia, also presenting atypical mitoses. The glands were extremely irregular with respect to shape and size, arranged “back-to-back”. However, the endometrial carcinoma were characterized by a glandular proliferation marked with intra-luminal papillary projections, with the presence of outgrowths and branches that realize a confluent pattern. These are lined by stratified neoplastic epithelia with a thin fibrovascular axis.2O2. The slides were incubated with primary antibody at 4 °C overnight, then the two-step plus poly-HRP anti-rabbit/mouse IgG detection system was applied. The immunohistochemical staining results were assigned a mean score considering both the intensity of staining and the proportion of cells with an unequivocal positive reaction. Each section was independently assessed by two pathologists without prior knowledge of patient data. Positive reactions were defined as those showing brown signals mainly in the cell nucleus. A staining index  was determined by the staining intensity and positive area. The scores (0–3) were defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. For statistical analysis, scores of 0–1 were considered low expression and scores of 2–3 considered high expression.Immunohistochemical staining was performed using 5-mmol/L thick sections. Tissue sections were deparaffinized and rehydrated gradually, and antigen retrieval was performed in pH 6.0 citrate buffer for 10 min. To quench endogenous peroxidases, peroxide blocking was performed for 30 min with 3% Ht-test.Chi-square tests were used to determine category variable differences. Overall survival distributions were estimated using Kaplan–Meier analysis. For other experiments, statistical significance was determined by Student’s FigureS1Supplementary Information