PIK3CA  gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the “clean” cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples  were derived from cultured tissues obtained from six PROS patients  whose spectrum of PIK3A-related overgrowth included HHML , CLOVES , and MCAP . We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients’ derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets . The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors  in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity  and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients.Postzygotic mutations of the The online version of this article (10.1007/s10048-018-0540-1) contains supplementary material, which is available to authorized users. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha  gene-related overgrowth  syndromes comprise a heterogeneous group of rare, congenital, segmental overgrowth phenotypes underlying somatic activating mutations of genes in the PI3K/AKT/mTOR pathway  (on chromosome 14q32.33) [AKT2 (on chromosome 19q13.2) [AKT3 (on chromosome 19q43-q44) [PTEN [phosphatase and tensin homolog] gene (on chromosome 10q23.31)   , 45, thuThis 6-year-old girl is the third child of a healthy 42-year-old woman and a non-consanguineous 52-year-old healthy man, whose family history was unremarkable. She was conceived naturally. She was born at term after a normal pregnancy: her fetal ultrasound scans were normal. Birth weight was 3.800 g (75th centile), length 51 cm (75th centile), and head circumference 36 cm (75th centile). At birth, macrodactyly of the I and II toe of the left foot with partial syndactyly between the 2nd and 3rd toes was recorded. Based on these clinical data, she was suspected to have Proteus syndrome. At the age of 1 month, a subcutaneous mass in her left abdominal region was observed. General physical examination at age 2 months showed that her weight was 5600 g (90th percentile), length 56 cm (50th percentile), and head circumference 38.5 cm (50th percentile). She presented macrodactyly of the I and II toes of the left foot with increased growth of the left leg and a subcutaneous mass in the left abdominal region; magnetic resonance imaging (MRI) of the abdomen revealed that the mass was compatible with a subcutaneous lipoma, which was later confirmed by histological examination of a sample of biopsied tissue. The girl was first referred to one of our institutions at age 4 months and followed up at age 9 months, 3 years and 10/12 months, and 5 years; she is still under follow-up at our institutions. Cognitive development is normal. During her last diagnostic work-up and follow-up controls, she underwent surgery for reduction of the abdominal mass and for removal of the first toe and transposition of the second toe to replace the first toe. Skin biopsies from the affected  regions were obtained during these procedures , length 52 cm (75th percentile), and head circumference 33 cm (50th percentile). Postnatally, increased growth of the trunk, widespread cutaneous capillary malformations, and gigantism with exadactyly of the right hand became evident, suggestive for a complex lymphatic-arteriovenous overgrowth malformation. Upon monthly follow-up visits, weight and length growth were regular; however, subcutaneous masses Fig. A, 1B andThis 17-year-old boy was the only child born to non-consanguineous parents. His father and grandfather showed diffuse angiomatosis. He was conceived naturally. His fetal ultrasound scan showed IUGR. At birth, a diffuse capillary malformation involving the trunk and limbs was recorded. At age 3 months, he was noted to have left asymmetric overgrowth. During his follow-up visits, cardiac and abdominal ultrasound scans were repeatedly normal. Early developmental milestones were delayed, and at age 7 months, a brain MRI scan revealed focal hemimegalencephaly with right perisylvian polymicrogyria. From age 5 years, he started to manifest episodes of generalized tonic-clonic seizures, which proved to be refractory to antiepileptic therapy. He was severely cognitively impaired and developed an attention deficit disorder. On physical examination, at age 13 years and 8/12 months, his weight was 41.5 kg (10th–25th percentile), height 154.2 cm (10th–25th percentile), and head circumference 50.2 cm (2 SD < 3rd percentile). He had left asymmetric overgrowth, involving the face, trunk, and limbs (mainly the legs) with diffusely soft and thick irregularly marbled skin and prominent capillaries and veins on the trunk, abdomen, and limbs. His 2nd and 3rd left toes were significantly larger than the contralateral and showed proximal cutaneous syndactyly. Besides the increased growth, he had dysmorphic features including malar hypoplasia, long philtrum and high palate, and S-shaped scoliosis. Skin biopsies from the affected  skin regions were obtained.This was the only child of healthy unrelated parents. His family history was unremarkable. Pregnancy was normal and repeated prenatal ultrasound evaluations were within normal limits. He was born at term through cesarean section because of breech presentation. Birth weight, length, and head circumference were at the 50th percentiles. Apgar scores were 10/10 at 5 and 10 min. Since birth multiple skin haemangiomas and diffuse capillary malformations were evident on the trunk, upper, and lower limbs. Mild facial asymmetry (right > left) was evident  signed  and tissue samples using the QIAamp Mini Kit , according to the manufacturer’s instructions, and quantified on a Bio Spectrometer Plus . The entire coding region of the s report .PIK3R1, PIK3R2, PIK3CA, PTEN, PDK1,PDK2, KRAS, AKT1, AKT2, AKT3, RICTOR, MAPKAP1, MLST8, MTOR, IRS1, GAB1,GAB2, THEM4, MAPK8I1, PTPN11, and RAPTOR) was used according to our previous report [The Ion AmpliSeq Custom Panel of the 21 genes involved in the PI3K/AKT/mTOR pathway  and to the BED file designed using Ion AmpliSeq Designer. Alignments were visually verified with the software Alamut® v2.8.0 (Interactive Bio software) . Reads were aligned to the hg19 human reference genome from the UCSC Genome Browser (re) Fig. .The mean average read depth and the percentage of reads mapping on the ROI out of the total number of reads (reads on target) were calculated using the Coverage Analysis plugin . For each sample, the percentage of ROI with a minimum coverage of 100× was calculated using the amplicon coverage matrix file Table .https://ionreporter.lifetechnologies.com/ir/). Common single nucleotide variants , exonic synonymous variants, and intronic variants were removed from the analysis, while exonic non-synonymous, splice site, and loss-of-function variants were analyzed.Variant calling was performed with the Variant Caller plugin configured with somatic high stringency parameters. Variants were annotated using the Ion Reporter 5.0 software  was used to interpret variants. Online databases, including dbSNP (database the single nucleotide polymorphism database), 1000 Genomes, ClinVar, EXAC (exome aggregation consortium), COSMIC , ESP (exome sequencing project) were used. The pathogenicity prediction programs such as PolyPhen2, SIFT, Mutation Taster, and splice prediction programs were used to evaluate variants not previously described.2 avoiding confluence at any time. For phosphorylation studies, cells were grown in RPMI 10% fetal bovine serum or transferred to serum free medium for 6 h prior to testing the drugs. Wortmannin (10 μM) and rapamycin (100 nM) were purchased from Sigma-Aldrich  and LY294002 (25 μM) from Selleckchem . ARQ 092 was synthesized by ArQule, Inc. Woburn, MA.Patient-derived primary fibroblasts were isolated according to our previous report (11) and grown in RPMI supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1% L-glutamine in a humidified incubator at 37 °C and 5% CO3 cells/well 1 day before treatment. The end point of the cell culture experiments was collected before cells reached confluence. Supernatants (containing dead/floating cells) were collected and the remaining adherent cells detached by trypsin/EDTA (Sigma-Aldrich). Cell pellets were re-suspended in 1× PBS and 10 μl was mixed with an equal volume of 0.01% trypan blue solution. Trypan blue exclusion test was performed to determine viable  vs dead cells . The percentages of dead cells were calculated. Cell numbers were counted with Countess II Automated Cell Counter (Life Technologies). Experiments were independently repeated at least three times.The number of primary cells was determined by counting. Cells were seeded into 24-well plates at 4.6 × 103 cells/well 1 day before treatment. The end point of the cell culture experiments was before reaching a confluent state. After 24, 48, 72, or 96 h of drug (or DMSO) exposure, 10 μl of the Cell Proliferation Reagent WST-1 was added to each well and incubated at 37 °C in a humidified incubator for 1 h. The absorbance was measured on a microplate reader  at 450/655 nm. Each assay was performed in three replicates, and the experiment was repeated three times. The cell proliferation was calculated as the ratio of WST-1 absorbance of treated cells to WST-1 absorbance of control cells of the same experimental group.The anti-proliferative effect of ARQ 092 and of PI3K/AKT/mTOR inhibitors  was evaluated on PROS cells derived from the six patients enrolled in the study using the Cell Proliferation Reagent WST-1  according to manufacturer’s instructions. Cells were seeded into 96-well plates at 3 × 10Immunoblotting analyses were performed according to the instructions of Cell Signaling Technology .For each treatment, all cells grown on plates were collected and homogenized in 1× lysis buffer  supplemented with protease and phosphatase inhibitors . Protein quantification was performed with MicroBCA™ Protein Assay Kit . Protein extracts of 20 μg from each sample were denatured in 5× Laemmli sample buffer. Proteins were separated in SDS-polyacrylamide gel and transferred to nitrocellulose membranes, using Trans-Blot® Turbo™ Mini Nitrocellulose Transfer .The blocking agents used were 5% BSA  for membranes incubated with anti-phospho antibodies and 5% nonfat dry milk  for membranes incubated with anti-total antibodies.Western blots were performed using primary antibodies at the dilution of 1:500 using 5% BSA for anti-phospho and 5% nonfat dry milk for anti-total, instead anti-ACTB was used at the dilution of 1:5000 with 5% nonfat dry milk. The antibodies used were as follows: polyclonal anti-ACTB , monoclonal anti-phospho-AKT (Thr308) , polyclonal anti-phospho-AKT (Ser473) , polyclonal anti-AKT , monoclonal anti-phospho-AKT1S1 (Thr246) , monoclonal anti-AKT1S1 , polyclonal anti-phospho-RPS6KB1 (Ser371) , polyclonal anti-RPS6KB1 , monoclonal anti-phosho-RPS6 (Ser235/236) . Western blots were developed with the Clarity TM Western ECL substrate chemiluminescence reagent . The densitometric evaluation was performed by the ImageJ software.t test. A value of P < 0.05 was considered statistically significant.Statistical significance of the results was analyzed using the Student’s The datasets generated during and/or analyzed during the current study are not publicly available due to data and privacy protection considerations but may be available upon justified request.In a previous study , we estamax between 1 and 1.5 μM. It is rationale to use 1 μM for long-term treatment. Thus, we used ARQ 092 at 1 μM for long-term treatment. Primary fibroblasts were treated with ARQ 092 at 1 μM in the presence or absence of serum for 24, 48, 72, and 96 h. ARQ 092 reduced phosphorylated AKT (Ser473 and Thr308) and AKT1S1 after 24 h of treatment. ARQ 092 did not change the levels of total AKT and AKT1S1  as compared to control cells when both were grown with or without serum  harboring the oncogenic mutation PIK3CA p.His1047Arg with 50% mutant allele frequency, we evaluated the phosphorylation status of AKT (Ser473 and Thr308) and its downstream target, pAKT1S1 (Thr246), pRPS6 (Ser235/236), pRPS6Kβ1 (Ser371)  harboring the mutation PIK3CA p.Glu726Gly and patient 4 (MCAP) harboring the mutation PIK3CA p.His1047Tyr with mutant allele frequencies of 37 and 26%, respectively. As shown in Fig. PIK3CA-related overgrowth spectrum (PROS) represents a heterogeneous group of disorders, including congenital segmental overgrowth phenotypes with somatic PIK3CA mutations characterized by overlapping clinical features with variable tissue specificity. Some of these phenotypes are associated with pleiotropic and more severe manifestations [PIK3CA mutations in PROS patients are identified in affected tissues at a variable frequency but are generally absent in blood and unaffected tissues [PIK3CA mutations, often strongly oncogenic, are frequently observed in several common human tumor types for which intensive efforts are under way to develop new drugs for use in common cancer therapy. PIK3CA alterations result in activated PI3K/AKT signaling [stations –6. PIK3C tissues , 2, 11. ignaling , 32–34. ignaling –45. The ignaling . We haveAs a critical node connecting the PI3K and mTOR pathways, targeting AKT has the advantage to inhibit the PI3K pathway and circumvent activation of additional pathways dependent on multiple classes and isoforms of PI3K kinases . To date, the clinical anticancer efficacy of many PI3K inhibitors, apart from a few exceptions, is limited, because of an emerging resistance mechanism that determines reactivation of PI3K pathway or activation of complementary pro-survival pathways and significant toxicity for treated patients . It is iAlthough rapamycin (sirolimus) has been tested in a phase II trial against PROS (NCT02428296), accumulated studies have shown that Rapalogs (such as sirolimus) caused only partial inhibition of mTORC1 but had no effect on mTORC2, which induces persistent survival promoting AKT signaling. In addition, such weak inhibition of mTORC1 releases negative feedback leading to rebound activation of upstream signaling . At thisFigure S1Example of reads alignment views with Alamut software of the two index cases (patient 6 and 1). Alamut displays varying level of data detail depending on the zoom level. The position of the mismatch respect to the reference is delimited with blue bars and in the gray box frequency data and reads number are shown. (PDF 52 kb)Table S1(DOC 109 kb)


During 2012, approximately 69% of children aged 5–17 years had a dental visit in the past 6 months; among children aged 2–4 years, the percentage with a dental visit was 45%. Approximately 19% of those aged 5–17 years and 12% of those aged 2–4 years had a visit >6 months to ≤1 year before. Approximately 40% of those aged 2–4 years and 5% of those aged 5–17 years had not had a dental visit in >2 years or had never seen a dentist.Source: Bloom B, Jones LI, Freeman G. Summary health statistics for U.S. children: National Health Interview Survey, 2012. Vital Health Stat 2013;10(258).Reported by: Lindsey Jones, MPH, izf4@cdc.gov, 301-458-4548; Barbara Bloom, MPA; Gulnur Freeman, MPA.


Brucella spp. are intracellular gram-negative bacteria including several virulent species, which cause a chronic zoonotic infection in a wide range of mammalian hosts known as brucellosis. A new Brucella species, Brucella microti, was recently isolated from wild rodents and found to be highly pathogenic in mice. Using this species-specific model, it was previously found that CD8+ T cells are required to control this infection. In order to find out the role of TLR-mediated responses in the control of this pathogen, the course of infection of B. microti was analyzed over 3 weeks in wild-type (WT) and TLR knock out (KO) mice including TLR2−/−, TLR4−/−, TLR9−/−, TLR2×4−/− and TLR2×4×9−/−. WT and single TLR2, TLR4 and TLR9 KO mice similarly control infection in liver and spleen. In contrast, bacterial clearance was delayed in TLR2×4−/− and TLR2×4×9−/− mice at 7 and 14 days post-infection. This defect correlated with impaired maturation and pro-inflammatory cytokine production in B. microti-infected dendritic cells from TLR2×4−/− and TLR2×4×9−/− mice. Finally, it was found that Tc cells from TLR2×4−/− and TLR2×4×9−/− mice showed reduced ability to inhibit growth of B. microti in macrophages, suggesting the involvement of TLR2 and 4 in the generation of specific Tc cells. Our findings indicate that TLR2 and TLR4 are required to control B. microti infection in mice and that this effect could be related to its participation in the maturation of dendritic cells and the generation of specific CD8+ Tc cells.Toll-like receptors (TLRs) recognize pathogen-derived molecules and play a critical role during the host innate and adaptive immune response.  Brucella are Gram-negative facultative intracellular pathogens capable of infecting a variety of hosts, causing the worldwide zoonosis known as brucellosis , followed by a chronic plateau phase and a declining phase that ends by the clearing of the bacteria in both organs. The duration of these phases depends on the route and the dose of the inoculum and the chronic phase can last more than 20 weeks  by pattern recognition receptors (PRRs) is the first line of defense involved in the generation of an immune response against infection. This process activates intracellular signaling cascades that culminate in gene activation and production of inflammatory cytokines, chemokines, and co-stimulatory molecules , 4 (TLR4−/−), 9 (TLR9−/−), 2 and 4 (TLR2×4−/−) and 2, 4, and 9 (TLR2×4×9−/−) were kindly provided by Marina Freudenberg from the Max-Planck-Institute for Immunobiology and Epigenetics, Freiburg and bred at Animal facilities of the CITA. Their genotypes were analyzed as described  , and mouse strains deficient for Toll Like Receptor 2  of B. microti was injected intraperitoneally (ip) and, at different time points, mice were euthanized by CO2 asphyxiation and blood, spleen, and liver samples were collected aseptically. Organs were weighed, homogenized, serially diluted in PBS, plated onto tryptic soy agar and incubated 48 h at 37°C for determining the number of viable Brucella organisms.A sublethal dose (10B. microti (105 CFU) ip. After 7 days CD8+ (Tc) cells were positively selected from spleen using anti-CD8-MicroBeads and autoMACS . Finally, Tc cells were resuspended in DMEM supplemented with 10% of inactivated fetal calf serum (FCS) containing 30 μg/ml of gentamicin before using them in different assays.WT B10 mice and mice deficient in TLR 2, TLR 4, and TLR 9 were infected with a sublethal dose of Intracellular expression of granzyme B in MACS-enriched Tc cells was analyzed by FACS as previously described  as previously described  of 25:1 and incubated for 45 min at 37°C, in a 5% CO2 atmosphere. At this time point enriched Tc from WT or KO mice were added at 5:1 ratio. Subsequently, medium was removed, cells were washed with PBS and further incubated with complete DMEM medium containing 30 μg/ml of gentamycin. After 24 h macrophages were lysed with 0.1% of Triton-X100 and Brucella CFU number was determined by dilution plating.For replication experiments −/−, TLR4−/−, TLR9−/−, TLR2×4−/−, TLR2×4×9−/− and MyD88−/− mice, in RPMI 1640 medium containing 10% of FCS serum, 100 U/ml of penicillin/streptomycin, 50 mM of 2-ME, and 10% of supernatant of X63Ag8653 cell cultures as source of GM-CSF . On days 2 and 4, the cell medium was refreshed and, on day 6, cells showed differentiated morphology and expressed the DC markers CD11c+, MHC class II (MHCII) low and CD40 low (d∫ata not shown), confirming their identity as immature DCs.DCs were generated from bone marrow cells using wild-type C57BL/10, TLR2in vitro B. microti infection experiments 4 × 105 DC were seeded in 24 well plates and infected with B. microti at a MOIs of 25:1 for 45 min at 37°C, 5% CO2. Subsequently, medium was removed, cells were washed with PBS and further incubated with complete RPMI medium containing 30 μg/ml of gentamycin. After 1.5, 24, and 48 h DC were lysed with Triton-X 0.1% and CFU number was determined. At each time supernatants were collected and the levels of TNFα and IL-6 were measured employing commercial ELISA kits (eBiocience).For E. coli-derived LPS (1 μg/ml) or heat killed B. microti . Subsequently, the expression of MHCII, CD40, CD86 and CD80 was analyzed on CD11c+ cells using MHCII-APC, CD40-APC, CD86-FITC, CD80-FITC and CD11c-PE antibodies (Miltenyi Biotec). Samples were fixed with 4% PFA and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences).To analyze DC maturation, BMDC were stimulated for 24 h with P < 0.05. Biological replicates are considered as the number of individual mice.Statistical analysis was performed using GraphPad Prism software. The difference between means of three or more independent groups was performed using one-way ANOVA test with Bonferroni's post-test. The results are given as the confidence interval (p), and are considered significant when B. microti infection, WT B10, TLR2−/−, TLR4−/− and TLR2×4−/− mice were infected ip with a sublethal dose (105 cfu) of B. microti and bacterial load was determined in spleen and liver 3, 7, 14, and 21 days after infection. As shown in Figure −/− mice compared with WT B10 mice. The absence of either TLR2 or TLR4 did not affect bacterial clearance.In order to analyze the role of TLR2 and TLR4 in the control of −/− mice in spleen and liver after 7 days, time after which wild type animals began to clear infection. The differences between the two groups were less pronounced, but still significant, after 14 days. No differences were found anymore between the different groups after 21 days.By day 3, the time of maximum bacterial load, no differences were found between the different animal groups. However, significant differences were found between WT B10 and TLR2×4in vivo both TLR2 and 4 are required for the early control of B. microti replication. Previous works have shown that TLR9 is involved in the early control of B. ovis and B. abortus infection. Thus, we decided to analyze the role of TLR9 in the control of B. microti infection. In this experiment bacterial replication was only analyzed after 7 and 14 days of infection, times after which significant differences were found between WT B10 and TLR2×4−/− mice.Our results indicate that −/− and TLR2×4×9−/− mice compared to WT B10 mice. No differences were found between WT B10 and TLR9−/− mice. In addition, bacterial replication was similar in TLR2×4−/− and TLR2×4×9−/− mice, indicating that TLR9 does not play any role in the control of B. microti infection.As shown in Figure Brucella spp.  and cell surface expression of CD40, CD80, CD86 and MHC-II was analyzed on CD11c+ cells. In comparison with control non-stimulated cells, BMDC from WT mice stimulated with LPS or HKBM showed enhanced expression of CD40  of B. microti and, 7 days later, CD8+ cells from spleen were enriched by MACS and incubated with B. microti-infected macrophages. As shown in Figure + T cells from WT, TLR2−/−, TLR4−/− or TLR9−/− deficient mice. In contrast, bacteria replicated significantly higher in macrophages incubated with CD8+ T cells from TLR2×4−/− or TLR2×4×9−/− mice, confirming a defect in the anti-bacterial activity of CD8+ T cells in the absence of TLR2 and TLR4.Finally, we analyzed if impaired maturation of DC from TLR2, 4 and 9 deficient mice, correlated with a reduced ability to activate  doses 10 CFU ip o+ T cells since cells from WT and TLR KO mice expressed similar levels of the cytotoxic activation marker granzyme B  bacterial pathogens  expressed by classical B. microti infection? Our group has previously found that CD8+ Tc cells are also required to control B. microti infection  in CD8+ T cells requires three independent signals: (a) Ag presentation, (b) co-stimulation, and (c) IL-12 or adjuvant (Curtsinger et al., B. microti in BMDM might be related to a lower generation of B. microti-specific Tc cells due to impairment in DC maturation.What would the impact be of TLR2 and TLR4 deficiency on the generation of adaptive immune responses during B. microti infection in mice by a mechanism that seems to be dependent on DC maturation and generation of specific B. microti CD8+ Tc cells. This finding provides more insights into the control of a novel emerging species of the Brucella genus and advises caution when interpreting the mechanisms involved in host-pathogen interaction, using non-specific pathogens of this bacterial genus.In conclusion, TLR2 and TLR4 cooperate to control Conceived and designed the experiments: MJ, JP. Performed the experiments: MA, LS, SC-R, PJ-S. Analyzed the data: MA, LS, SC-R, PJ-S. Contributed reagents/materials/analysis tools: MF, MJ, JP. Wrote the paper: MA, LS, MJ, JP.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.


Targeted therapies in HER2-positive metastatic breast cancer significantly improve outcomes but efficacy is limited by therapeutic resistance. HER2 is an acutely sensitive Heat Shock Protein 90 (HSP90) client and HSP90 inhibition can overcome trastuzumab resistance. Preclinical data suggest that HSP90 inhibition is synergistic with taxanes with the potential for significant clinical activity. We therefore tested ganetespib, a HSP90 inhibitor, in combination with paclitaxel and trastuzumab in patients with trastuzumab-refractory HER2-positive metastatic breast cancer.2) on days 1, 8, 15, and 22 of a 28-day cycle with escalating doses of ganetespib  on days 1, 8, and 15. Therapy was continued until disease progression or toxicity. The primary objective was to establish the safety and maximum tolerated dose and/or recommended phase II dose (RP2D) of this therapy. The secondary objectives included evaluation of the effects of ganetespib on the pharmacokinetics of paclitaxel, and to make a preliminary assessment of the efficacy of the combination therapy.In this phase I dose-escalation study, patients with trastuzumab-resistant HER2-positive metastatic breast cancer received weekly trastuzumab (2 mg/kg) and paclitaxel , including prior pertuzumab in 9/9 patients and ado-trastuzumab emtansine (T-DM1) in 8/9 patients. The most common grade 1/2 adverse events (AEs) were diarrhea, fatigue, anemia, and rash. There were no grade 4 AEs related to ganetespib. The overall response rate was 22%  and stable disease was seen in 56% (5/9 patients). The clinical benefit rate was 44% (4/9 patients). The median progression-free survival was 20 weeks (range 8–55).2 in combination with weekly paclitaxel plus trastuzumab. The combination was safe and well tolerated. Despite prior taxanes, pertuzumab, and T-DM1, clinical activity of this triplet regimen in this heavily pretreated cohort is promising and warrants further study in HER2-positive metastatic breast cancer.The RP2D of ganetespib is 150 mg/mNCT02060253. Registered 30 January 2014.ClinicalTrials.gov  HER2-positive disease accounts for 15–20% of breast cancers, and traditionally has an aggressive clinical course and inferior survival outcome . ClinicaHSP90 belongs to a class of molecular chaperone proteins that help modulate cellular responses to environmental stress . In partA novel approach to the treatment of metastatic breast cancer is the combination of HSP90 inhibitors and taxanes. Taxanes disrupt an essential structural component (microtubules) of mitosis, and HSP90 inhibitors impact the regulatory (checkpoint) proteins controlling progression through the cell cycle . In addiThe primary objective of this study was to establish the safety, tolerability, maximum tolerated dose (MTD), and/or recommended phase II dose (RP2D) of ganetespib plus paclitaxel in conjunction with trastuzumab in patients with HER2-positive metastatic breast cancer. The secondary objectives included evaluation of the possible effects of ganetespib on the pharmacokinetics (PK) of paclitaxel, and to make a preliminary assessment of the efficacy of the combination.9/L, platelets ≥ 100 × 109/L, bilirubin ≤ 1.5 upper limit of normal (ULN), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 ULN, and serum creatinine ≤ 1.5 ULN). Patients must have received prior trastuzumab and those with estrogen receptor (ER)-positive disease must have received prior endocrine therapy. Any number of prior lines of chemotherapy in the metastatic setting was allowed. Progression on prior treatment with pertuzumab and T-DM1 was required  and/or T-DM1 (2/2013)).Patients were eligible if they were aged ≥ 18 years, had locally advanced or metastatic HER2-positive disease (defined as FISH ratio ≥ 2.0 or immunohistochemistry (IHC) 3+), ECOG ≤ 2, measurable disease per RECIST 1.1 , and adePatients were excluded if they were pregnant or lactating, had prior grade 3 hypersensitivity to cremophor or trastuzumab, had prior HSP90 inhibitor therapy, had active central nervous system metastases, New York Heart Association (NYHA) class III/IV congestive heart failure requiring active treatment, left ventricular ejection fraction (LVEF) < 50% at baseline, baseline QTc > 470 milliseconds, or grade ≥ 2 peripheral neuropathy, were on any medications known to prolong QTc, had preexisting left bundle branch block (LBBB), history of uncontrolled dysrhythmias, or a requirement for antiarrhythmics, had myocardial infarction (MI) or ischemic heart disease within 6 months, or had known active infection with HIV or hepatitis B or C viruses.The study was approved by the institutional research ethics board of Memorial Sloan Kettering Cancer Center and New York University Langone Medical Center. All participants gave informed consent before they entered the study.2) followed by ganetespib. If the patient’s last dose of trastuzumab was >21 days before enrollment, they received a loading dose of trastuzumab at 4 mg/kg over 90 minutes. Ganetespib was then administered intravenously over 60 minutes. The starting dose of ganetespib was 100 mg/m2, and if there were no DLTs the next cohort would escalate to 150 mg/m2. A further dose level of 125 mg/m2 was incorporated in the circumstance of good tolerance of the 100 mg/m2 but poor tolerance of the 150 mg/m2 dose levels. There was no dose escalation for paclitaxel and trastuzumab. Therapy was continued until disease progression or unacceptable toxicity.Patients received intravenous infusions of trastuzumab and paclitaxel with ganetespib on days 1, 8, and 15 and of trastuzumab and paclitaxel on day 22 of a 28-day cycle. The sequence of administration was trastuzumab (2 mg/kg) followed by paclitaxel (80 mg/mhttp://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40). Patients were evaluated for DLT during cycle 1. DLT was defined as any drug-related grade ≥ 4 nonhematologic adverse events (AEs) or any grade 3 nonhematologic AEs not improving to baseline or grade ≤ 1 by day 14; grade 4 neutropenia lasting ≥ 7 days, or febrile neutropenia, grade 4 thrombocytopenia, or any grade 3 thrombocytopenia that has not recovered to grade ≤ 2 by day 7; or any treatment-related toxicity prompting a dose reduction of ganetespib during the DLT observation period.Patients were examined and assessed for toxicities during and prior to each cycle. Toxicity was graded according to National Cancer Institute (NCI) CTCAE version 4.0  after start of paclitaxel infusion. Blood samples were also collected at the following time points after starting ganetespib infusion: 1.5, 2, 4, 7, 21, 24, 27, and 31 hours. Each blood sample (3–5 ml) was collected in sodium heparin tubes and transferred into two polypropylene tubes (1 ml each) and stored at –80 °C. PK were performed in collaboration with Synta Pharmaceuticals, Inc. PK parameters of paclitaxel (such as the area under the curve (AUC) and the maximum serum concentration (Cmax)) were examined descriptively to evaluate the effect of ganetespib on these measures.2 and six patients at 150 mg/m2. Patient demographics are presented in Table Nine patients were enrolled into two dose cohorts: three patients at 100 mg/m2 in this patient. No patients on study required ganetespib dose reductions. There were no deaths on study.All nine patients were included in the safety analysis. No patients voluntarily withdrew from the study and no patients were taken off study due to toxicities. There were no grade 4 AEs related to ganetespib. The most common drug-related AEs were diarrhea grade 1/2, 78%), fatigue , anemia , rash , elevated AST or ALT , and nausea   of 22%. Five additional patients achieved SD (56%), and duration of SD ranged from 11 to 29 weeks. The CBR was 44% (4/9 patients). Representative CT scans prior to and after this triplet regimen in a patient with chest wall soft tissue metastasis who achieved PR are shown in Fig. Of the nine patients enrolled, confirmed partial tumor responses were achieved in two of the nine patients, both in the 150 mg/m2. For paclitaxel, the AUC was 6280 h*ng/ml, the elimination half-life (T1/2) was 13.6 hours, the Cmax was 3750 ng/ml, the clearance was 13.4 L/h/m2, and the mean resident time (MRT) was 9.0 hours  in patients with heavily pretreated trastuzumab-refractory HER2-positive metastatic breast cancer. Additionally, five patients achieved SD (56%) with a CBR of 44% (4/9 patients). Notably, in our previous phase II single-agent ganetespib trial, the ORR rate of ganetespib was 15% with two PRs in trastuzumab-refractory ER+/HER2+ metastatic breast cancer. There were seven SDs of which six were seen in patients with HER2-positive metastatic breast cancer and one in a patient with TNBC.Aside from ganetespib, there are a number of other second-generation HSP90 inhibitors that are in preclinical or clinical testing, including resorcinol derivatives , purine derivatives , and other inhibitors including SNX-5422, NVP-HSP990, and XL888 –42. Some127I), which was replaced with the PET radionuclide 124I to result in the imaging agent, 124I-PU-H71 [124I-PU-H71 in patients with advanced solid tumors and lymphoma not only determined the microdose biodistribution of PU-H71 but also ensured tracer avidity of tumors [124I-PU-H71 PET to determine tumor PK and the intratumoral drug concentration. Findings from this study showed close concordance between intratumoral drug concentrations as determined by tumor biopsies with estimated measurements from 124I-PU-H71 PET [124I-PU-H71 PET as a biomarker to visualize PU-H71 uptake and to estimate intratumoral concentrations of the inhibitor.Another heavily studied area in the field of HSP90 inhibitors is the identification of biomarkers and companion diagnostic assays which are crucial to identify patients most likely to respond to therapy. Our group conducted a retrospective study to explore potential biomarkers in patients treated with HSP90 inhibitors. Among many potential candidates analyzed , HER2 was found to be the most important individual biomarker and the only one with correlation to response with HSP90 inhibitor therapy . While tI-PU-H71 . Importaf tumors . Tracer -H71 PET , highlig2 in this triplet therapy. Based on the clinical activity in this heavily pretreated population, this combination warrants further study in HER2-positive metastatic breast cancer. In addition, further exploration of biomarkers that predict sensitivity to ganetespib is a critical step for the optimal clinical development of this and other HSP90 inhibitors.The combination of ganetespib with paclitaxel plus trastuzumab is well tolerated, safe, and active with a RP2D of ganetespib at 150 mg/m


A complete genomic sequence of Japanese encephalitis virus (JEV) was detected by viral metagenome analysis on aborted piglets. A phylogenetic analysis of this genome reveals that it is highly similar to previously reported India JEV genomes. The complete JEV sequence is 10,718 nucleotides long. Flaviviridae, genus Flavivirus, is a single-stranded RNA virus and has a diameter of about 50 nm. The genomes of this viurs are about 11 kb in size and encode for the capsid, membrane, and envelope structural proteins, as well as for seven nonstructural proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5 , which belongs to the family Culex sp. mosquitoes, pigs, and birds  was identified in brain samples from aborted piglets in Sichuan suspected of having Japanese encephalitis–associated disease. Phylogenetic analysis based on the nearly full-length genome indicated that the sequence of JEV strain JEV/SC/2016-1 was closely related to isolate 057434 (EF623988) from India belonging to genotype III.The genome of JEV strain JEV/SC/2016-1 was generated with reverse transcriptase (RT)-PCR using four pairs of primers, which were designed using Primer Premier version 5.0 software from alignments of available JEV genomes. RNA was extracted and reverse transcribed using an RT kit (TaKaRa). The RT-PCR products were purified using a TIANgel midi purification kit (Tiangen). The target DNA fragments were cloned into pMD18-T vectors (TaKaRa) and sequenced by TSINGKE Biological Technology .The genome of JEV strain JEV/SC/2016-1 has a length of 10,718 nucleotides (nt). The open reading frame  encodes a polyprotein processed into three structural proteins, C (127 amino acids [aa]), prM/M (167 aa), and E (500 aa), and seven nonstructural proteins, NS1 (415 aa), NS2a (164 aa), NS2b (131 aa), NS3 (619 aa), NS4a (267 aa), NS4b (137 aa), and NS5 (905 aa). The complete genome of the strain showed a high nucleotide homology (98%) to reference strain 057434 (EF623988). When the amino acid sequences of C, prM/M, and E were analyzed, strain JEV/SC/2016-1 had nine changes , compared with the reference strain 057434. This indicated that JEV strain JEV/SC/2016-1 showed a close relationship to strain EF623988 and that JEV had undergone an evolution in its continuous transmission. These data will provide valuable information about the evolutionary characteristics and genetic diversity of JEV.KX779521.The genome sequence of JEV strain JEV/SC/2016-1 was deposited in GenBank under the accession number


In vitro, we found that chemerin suppresses hCG-induced progesterone production in cultured follicle and corpus luteum and that this effect is attenuated significantly by anti-GPR1 MAB treatment. Furthermore, when the phosphoinositide 3-kinase (PI3K) pathway was blocked, the attenuating effect of GPR1 MAB was abrogated. Interestingly, PGF2α induces luteolysis through activation of caspase-3, leading to a reduction in progesterone secretion. Treatment with GPR1 MAB blocked the PGF2α effect on caspase-3 expression and progesterone secretion. This study indicates that chemerin/GPR1 signaling directly or indirectly regulates progesterone synthesis and secretion during the processes of follicular development, corpus luteum formation, and PGF2α-induced luteolysis.Chemerin, a chemokine, plays important roles in immune responses, inflammation, adipogenesis, and carbohydrate metabolism. Our recent research has shown that chemerin has an inhibitory effect on hormone secretion from the testis and ovary. However, whether G protein-coupled receptor 1 (GPR1), the active receptor for chemerin, regulates steroidogenesis and luteolysis in the corpus luteum is still unknown. In this study, we established a pregnant mare serum gonadotropin-human chorionic gonadotropin (PMSG-hCG) superovulation model, a prostaglandin F2α (PGF2α) luteolysis model, and follicle and corpus luteum culture models to analyze the role of chemerin signaling through GPR1 in the synthesis and secretion of gonadal hormones during follicular/luteal development and luteolysis. Our results, for the first time, show that chemerin and GPR1 are both differentially expressed in the ovary over the course of the estrous cycle, with highest levels in estrus and metestrus. GPR1 has been localized to granulosa cells, cumulus cells, and the corpus luteum by immunohistochemistry (IHC).  TIG2) and retinoic acid receptor responder protein 2 (RARRES2) , G protein-coupled receptor 1 (GPR1), and chemokine (C-C motif) receptor like 2 (CCRL2), as an endocrine, paracrine, and autocrine signaling molecule. All three receptors are seven transmembrane domains -stimulated testosterone secretion. It was recently reported that many chemokines play important roles in fertility and reproduction . Tena-SePrevious research has shown that chemerin inhibits gonad hormone secretion from testis and ovary  were obtained from Guangdong Medical Laboratory Animal Center. All procedures related to animal use were approved by the Committee on the Use of Live Animals for Teaching and Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.For estrous cycle staging, vaginal smears were performed daily in the morning at the same time each day using cotton swabs wetted with PBS; smears were placed on glass slides and cytology was evaluated under a microscope. Estrus was determined by the presence of cornified cells. Metestrus was scored by the presence of large round cells with an irregular border. A high density of leukocytes indicated the stage of diestrus, whereas small nucleated cells indicated proestrus   to stimulate follicular development, followed by injection of 5IU of hCG (Sigma) 48h after PMSG injection to induce ovulation.Animals were anaesthetized before blood were collected by removal of eyeball and then killed by cervical dislocation before the ovaries samples were collected at 24 and 48h after PMSG administration and 24, 48, 72, and 96h after hCG administration. Serum samples were send to Beijing North Institute of Biological Technology  to measure hormone level of estradiol, progesterone and testosterone. Ovaries were either put in RNAiso Plus (Takara Bio) and stored at −80°C for total RNA isolation or fixed in Bouin’s solution and embedded in paraffin for histological examination.Six- to 8-week-old females were housed with males, the occurrence of copulatory plugs was verified by visual examination at each day morning. Females on the third, fourth, and fifth postpartum day were injected s.c. with 25μg/100µL PGF2α, control group injected s.c. with saline. Ovaries and blood samples  were taken at 12h after PGF2α and saline injected. Serum was send to Beijing North Institute of Biological Technology to measure hormone level of estradiol, progesterone and testosterone. Ovaries were either snap frozen in RNAiso Plus and stored at −80°C for RNA or fixed in Bouin’s solution and embedded in paraffin for histological examination.Twenty-five-day-old immature female mice were injected i.p. with 1mg/kg mouse GPR1 antibody and 5IU of PMSG, followed by the injection of 1mg/kg mouse GPR1 antibody and 5IU of hCG 48h after PMSG, another group injected 1mg/kg rat IgG as control, 96h after hCG, injected PGF2α to induce luteolysis, another group injected saline as control. Ovaries and blood samples  were taken at 6h after PGF2α and saline injected. Serum was send to Beijing North Institute of Biological Technology to measure hormone level of estradiol, progesterone and testosterone. Ovaries were either snap frozen in RNAiso Plus and stored at −80°C for RNA or fixed in Bouin’s solution and embedded in paraffin for histological examination.Chemerin , Gpr1 , steroidogenic acute regulatory protein , cytochrome P450 cholesterol side-chain cleavage , 3β-hydroxysteroid dehydrogenase , and β-actin .Total RNA from tissues was extracted using RNAiso Plus and subjected to qPCR analysis. RNA samples (0.5μg) were reverse transcribed into cDNA according to the manufacturer’s instructions . The PCR mixtures contained 10μL SYBR Premix Ex Taq II (Toyobo), 1μL of each primer, 1μL cDNA, and 7μL DNase-free water to a final volume of 20μL. Cycle conditions were 10 s at 95°C, followed by 45 cycles at 95°C for 5s, at 60°C for 30s, and at 72°C for 30s. The reaction was completed with a dissociation step for melting point analysis at 50–95°C (in increments of 0.5°C for 10s each). The primers were designed on the basis of the published sequences of −ΔCT method, where CT was the cycle threshold , mouse StAR , and mouse caspase-3 (Abcam), diluted 1:100 in PBS with 1% BSA. The secondary antibodies were horseradish peroxidase (HRP)-donkey-anti-rat (Abcam) to GPR1 and HRP-anti-rabbit  to caspase 3 and StAR, diluted 1:200 in PBS with 1% BSA. Staining was visualized using a DAB Substrate Kit for peroxidase , and slides were counterstained with hematoxylin. Control sections were immunostained with a nonspecific IgG to check for nonspecific staining.50=3nM) phosphoinositide 3-kinase (PI3K) signaling pathway inhibitor wortmannin (Invitrogen). For groups receiving antibody treatment, follicles were precultured for 1–2h with 0.5μg/mL mouse GPR1 antibody (Stanford Brian’s laboratory) before drug added. Other groups added 0.5μg/mL rat IgG (Abcam) as control. Plates were incubated at 37°C, 5% CO2  to stimulate follicular development. Forty-eight hours after PMSG injection, mice were killed, and both ovaries were removed. A 1mL syringe needle was used to remove fat and mesangial tissue around the ovary, and then eye tweezers and needles were used to mechanically isolate follicles under a stereomicroscope. Follicles isolated from the same ovary were added to a single well of a 24-well plate with 1mL DMEM F12, cocultured with 100nM recombinant mouse chemerin (R&D Systems), 0.01IU/mL hCG (Sigma) and 15nM  to stimulate follicular development, followed by injection of 5IU of hCG (Sigma) 48h after PMSG injection to induce ovulation. Seventy-two hours after hCG injection, mice werekilled, ovaries were removed, fat and mesangial tissue were removed from around the ovary using a 1mL syringe needle, and luteal tissue was mechanically separated under a stereomicroscope using eye tweezers and needles. Luteal tissue isolated from the same ovary were added to a single well of a 24-well plate with 1mL DMEM F12, cocultured with 100nM recombinant mouse chemerin (R&D Systems) and 0.01IU/mL hCG (Sigma). For groups receiving antibody treatment, follicles were precultured for 1–2h with 0.5μg/mL mouse GPR1 antibody (Stanford Brian’s laboratory) before adding drug. Other groups added 0.5μg/mL rat IgG (Abcam) as control. Plates were incubated at 37°C, 5% CO125I] RIA Kits (Beijing North Biotechnology Research Institute). The sensitivity of the progesterone and estradiol RIA assays was 20ng/mL. The intra-assay error and inter-assay error were <10 and <15%, respectively.Progesterone and estradiol levels in conditioned media were measured using commercial iodine [s.e.m. and statistical significance was assessed by either one-way ANOVA followed by Fisher’s least significant difference test for post hoc comparisons or the Student’s t-test (GraphPad Prism). A P value of <0.05 was considered to be statistically significant.All data are presented as mean±Chemerin and Gpr1 mRNAs were found to be expressed in mouse ovary during the estrous cycle  . Theca cin vitro cultured human ovarian granulosa cells. Some cytokines also have an effect on granulosa cells: insulin-like growth factor (IGF) can promote estrogen secretion by granulosa cells  can cause female infertility or first trimester miscarriage  to J V Z, Guangdong Grant 2015A020212030 to P G R, Shenzhen Grant .J V Z and Y L Y conceived and designed the experiments; C H, B B W, J C, and T X X performed the experiments; Y L Y, L F S, and L R R analyzed the data; L R R, B A Z, and P G R contributed reagents/materials/analysis tools/housing animals; Y L Y, J V Z, and P G R wrote and revised the manuscript.


RASSF1A, APC, BVES, TIMP3, GSTP1, and HOXA9 were selected as the candidate genes. The MethyLight method was used to assay promoter methylation statuses. The diagnostic performances of markers were assessed by constructing receiver operating characteristic (ROC) curves. The prevalences of methylation for RASSF1A, APC, BVES, HOXA9, GSTP1, and TIMP3 were 52.04%, 36.73%, 29.59%, 20.41%, 17.35%, and 11.22%, respectively. APC methylation completely overlapped with RASSF1A methylation. The area under the curve (AUC) for RASSF1A methylation (0.718) was better than the corresponding AUC for AFP (0.609) in distinguishing HCC from CHB. When RASSF1A, BVES, HOXA9, and AFP were combined, the AUC was 0.852 , and the sensitivity and specificity were 83.7% and 78.9%, respectively. In conclusion, an assay that combines methylation of the RASSF1A, BVES, and HOXA9 gene promoters in serum and AFP could significantly improve HBV-related HCC diagnoses.This study sought to evaluate the diagnostic value of the methylation of multiple gene promoters in serum in hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). A total of 343 participants were enrolled, including 98 patients with HCC, 75 patients with liver cirrhosis (LC), 90 patients with chronic hepatitis B (CHB), and 80 healthy individuals.  Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors and causes an estimated 50 million deaths per year worldwide , 2. ChroCpG island methylation in the gene promoter is an important epigenetic mechanism that is often involved in carcinogenesis. Certain tumor suppressor genes and other pivotal genes that regulate cell signaling pathways are frequently silenced in tumor tissues due to promoter hypermethylation –5. TheseRASSF1A), adenomatous polyposis coli (APC), blood vessel epicardial substance (BVES), TIMP metallopeptidase inhibitor 3 (TIMP3), glutathione S-transferase pi 1 (GSTP1), and homeobox A9 (HOXA9) were selected as candidate targets; these genes are frequently methylated during carcinogenesis of digestive tract carcinoma [In this study, RAS association domain family protein 1A . In detail, the 98 patients with HCC had been diagnosed via liver ultrasound, computed tomography (CT), serum AFP level, and ultimately histological examination. The 75 patients with LC had been diagnosed via liver ultrasound and CT and exhibited LC accompanied by portal hypertension and hypersplenism. The 90 patients with CHB satisfied diagnostic criteria based on guidelines for the prevention and treatment of chronic hepatitis B (2010 version) issued by the Chinese Society of Hepatology and the Chinese Society of Infectious Diseases of the Chinese Medical Association. Additionally, the HCC patients, LC patients, and CHB patients had HBV surface antigen- (HBsAg-) positive serum. Subjects who presented with other liver diseases, such as autoimmune hepatitis, alcoholic hepatitis, and infection with another type of hepatitis virus, were excluded from the study. Eighty healthy individuals were obtained from the Physical Examination Center of Hangzhou First People's Hospital. All subjects provided written informed consent, and this study was approved by the ethics committee of Hangzhou First People's Hospital.μl of serum. Serum DNA was modified via sodium bisulfite treatment and purified using the EpiTect Bisulfite Kit . The aforementioned operations were performed in accordance with the protocols recommended by the manufacturer.Five-milliliter samples of peripheral blood were drawn from the patients and healthy subjects. Samples were centrifuged at 2000 ×g for 10 min. Subsequently, 2 ml of serum was collected from each sample via centrifugation at 12000 ×g for 5 min and stored at −80°C until use. A serum DNA extraction kit  was used to extract DNA from 600 in vitro methylation. The 20 μl reaction system consisted of 2 μl of 10 × NEBuffer, 1 μl of genomic DNA (15 μg/l) from umbilical cord blood from a healthy fetus, 2 μl of S-adenosylmethionine (SAM) (1600 μM), 1 μl of CpG methyltransferase (M.SssI) (4 U/μl) , and 14 μl of nuclease-free water. This mixture was incubated at 37°C for 1 h, and the reaction was then stopped at 65°C for 20 min. Methylated genomic DNA was treated and purified using the sodium bisulfite modification approach described above.One reaction was performed for ACTB) was used as the internal reference gene to correct for differences in DNA template quantities among samples. The PCR mixture had a final volume of 20 μl and contained 1 μl of bisulfite-treated DNA, 0.15 μl of each primer (10 μM), 0.1 μl of each probe (10 μM), 9.6 μl of nuclease-free water, and 10.0 μl of 2 × PCR Buffer , which consisted of Taq DNA polymerase, reaction buffer, and a deoxynucleotide triphosphate mixture. PCR was performed using an ABI 7500 Sequence Detection System . The PCR program included an initial denaturation step at 95°C for 3 min followed by 45 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 1 min. M.SssI-treated DNA, normal lymphocyte DNA, and nuclease-free water were used as a positive control, a negative control, and a blank control, respectively. Each sample was assessed in duplicate, with the average of the two duplicates used for analysis. Gene promoter methylation statuses are presented as percentage of methylated reference (PMR) values [The methylation status of each gene was examined using methylation-specific quantitative PCR (MethyLight). The sequences of the primers and probes for MethyLight were previously described –22; thes) values . A PMR ≥) values –26.U test was used to examine differences in nonparametric variables. Associations between methylation and clinicopathologic parameters were determined using chi-square (χ2) tests. Diagnostic efficacies were presented as areas under ROC curves (AUCs). P values <0.05 were regarded as statistically significant. All data analyses were performed using SPSS software, version 21 .The Mann–Whitney P < 0.05). In particular, relative to the healthy control group, the HCC, LC, and CHB groups had higher ALT, TBIL, and AFP levels but lower ALB and PLT levels .General clinical information was collected for 343 subjects. Serum levels of alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL), AFP, and blood platelet (PLT) count significantly differed among the four groups  but infrequently observed in serum from healthy subjects (3.75%). The other 5 genes showed low methylation rates in LC patients (2.67%–5.33%) and no detected methylation in healthy subjects.Rates of hypermethylation of RASSF1A methylation in serum was greater than the sensitivities of the other diagnostic indicators, whereas the sensitivities of BVES, APC, TIMP3, GSTP1, and HOXA9 methylation in serum were all lower than the sensitivity of AFP (≥20 ng/l) , BVES methylation (0.636), AFP (≥20 ng/l) (0.609), and HOXA9 methylation (0.521) indicated that these metrics were the top 4 indicators for distinguishing between HCC and CHB.For discriminating between HCC and CHB, the sensitivity of 20 ng/l) . For allRASSF1A, BVES, and HOXA9 in serum and AFP (≥20 ng/l) to distinguish HCC from CHB, ROC curves were constructed. The results showed that when methylation statuses of RASSF1A, BVES, and HOXA9 in serum were utilized together, the AUC was 0.834  and the sensitivity and specificity were 73.5% and 91.1%, respectively. For a combination of the methylation statuses of RASSF1A, BVES, and HOXA9 in serum and AFP (≥20 ng/l), the AUC was 0.852  and the sensitivity and specificity were 83.7% and 78.9%, respectively  were selected as candidates. These genes are involved in a variety of cellular functions and signaling pathways, such as cell proliferation, invasion and adhesion , metastasis and angiogenesis (TIMP3), detoxification (GSTP1), and cell differentiation (HOXA9) [RASSF1A , followed by APC , and that APC methylation completely overlapped with RASSF1A methylation. Notably, methylated RASSF1A and methylated APC were both sometimes detected in LC and CHB. These results imply that methylation of RASSF1A and APC may be a common aberrant epigenetic change during the development of HCC and could even be involved in early stages of hepatocarcinogenesis.In this study, a magnetic bead method was used to isolate and purify serum DNA, and the MethyLight method was used to perform the DNA methylation assay. These methods guaranteed the reliability of the study results. Six tumor-associated genes ( (HOXA9) –15. We fRASSF1A methylation, BVES methylation, AFP (≥20 ng/l) and HOXA9 methylation were the top 4 biomarkers for distinguishing HCC from CHB but that only RASSF1A methylation exhibited better sensitivity (52.0%) and specificity (91.5%) than AFP . Cell-free methylated RASSF1A exhibited large discrepancies with respect to diagnostic performance, including wide ranges for sensitivity (0.27 to 0.94) and specificity (0.38 to 0.95); this phenomenon was likely due to HCC heterogeneity and the selected methylation assay method [RASSF1A, BVES, and HOXA9 in serum and AFP (≥20 ng/l) exhibited an improved AUC (0.852), sensitivity (83.7%), and specificity (78.9%). Recently, Lu et al. [APC, COX2, RASSF1A, and miR-203) for diagnosing HBV-related HCC using a high-throughput approach. In that study, the 4 biomarkers were combined to form a plasma methylation predictive panel that achieved a sensitivity of 84.2%, a specificity of 83.0%, and an AUC of 0.87 with respect to discriminating between HBV-related HCC and noncancerous control samples. These data illuminated the high diagnostic potential of methylated markers in cell-free DNA from HCC patients. In addition, several literature reports have demonstrated correlations between clinicopathological characteristics of HCC, including clinical prognosis, and such markers [Comparisons indicated that tivity 0. to 0.94 u et al.  screenedtivity 0. to 0.94  markers .ADAMTS5) polymorphism was identified to be a useful marker for aflatoxin B1- (AFB1-) related HCC diagnosis and prognosis [Currently, molecular pathologic epidemiology (MPE) is an emerging field of epidemiology based on molecular classification of cancer . MPE resrognosis . Hence, rognosis . Neverthrognosis .RASSF1A, BVES, and HOXA9 gene promoters in serum and AFP could significantly improve HCC diagnoses for patients with chronic HBV infection.In summary, an assay that combines methylation of the


Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp301, the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce3+ or Ca2+. The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed.Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of  Actinobacteria, Spirochaetes, Alpha-, Beta-, Gamma-, and Deltaproteobacteria, of the Phyla Firmicutes, Bacteroidetes, Chloroflexi, Acidobacteria, Nitrospirae, Verrucomicrobia, Cyanobacteria, and Planctomycetes, and even of the domain Archaea  was retrieved from a distance within 1 m of the sampled specimen. Approximately 5 cm4 (1:10 w:v). Epiphytic suspensions and soil dilutions in 10 mM MgSO4 were inoculated on plates (1.6% agar) of methanol mineral salts medium  containing 0.5% methanol; 6.89 mM K2HPO4; 4.56 mM KH2PO4; 0.228 mM CaCl2; 0.811 mM MgSO4; 1.71 mM NaCl; 3.7 μM FeCl3; 3.8 mM (NH4)2SO4; 20 nM CuSO4; 41.5 nM MnSO4; 38 nM Na2MoO4; 0.163 μM H3BO3; 0.243 μM ZnSO4; and 21 nM CoCl2, and incubated at 30°C for 8–10 d. Isolated bacterial colonies were streaked in the same medium and incubated at 30°C until growth was observed. Isolated colonies were grown in the same medium and also in GP containing (L−1): Casein peptone 10 g, glycerol 10 g, and agar 15 g. DNA was extracted from cells growing in MMSM medium with the DNA Isolation Kit for Cells and Tissues  following the recommended instructions of the supplier.In order to isolate endophytes, approximately 2 mm of surface plant tissues including the cuticle were discarded under sterile conditions. The remaining plant material was macerated in a sterile mortar and resuspended in 10 mM MgSO4, resuspended in 10 mL of the same solution, and 5 μL of the suspension was inoculated in 5 mL of modified MMSM with 30 μM CaCl2 or lacking Ca2+ but with 30 μM CeCl3. Cells were incubated at 30°C under shaking for 5 d. Bacterial growth was assessed by absorbance at 600 nm 72, 96, 120, and 144 h after the inoculation. The cultures of three independent replicates grown in either Ca2+ or Ce3+-MMSM broths were statistically compared by the unpaired t-test, P<0.05.Isolates were grown in GP plates at 30°C for 4 d. One loopful of bacterial cells was washed twice in 10 mM MgSOM. extorquens JCM2802, which had 100 ng. One microgram of U. maydis 207 was used as a negative control. One hundred nanograms of DNA 32P-labeled probes specific for mxaF and xoxF5 were used for hybridizations. These probes were obtained by the PCR amplification of Methylobacterium extorquens JCM2802 genomic DNA with the primers mxa f1003 and mxa r1561 . Prehybridization and hybridization were performed at 65°C for 12 h using Rapid Hyb buffer . The membranes were washed under high stringency conditions   .mxaFxoxFf916 and mxaFxoxFr1360  with the primers used for PCR amplification.16S rRNA genes were amplified with the primers B27F (5′-TAG AGT TTG ATC CTG GCT CAG-3′) and B1392R (5′-CAG GGG CGG TGT GTA-3′) using the following conditions: one initial denaturation at 95°C for 3 min, 26 cycles at 94°C for 30 s, 57°C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. Methanol dehydrogenase genes were amplified with the primers oxFr1360  designedSequence analyses were performed with MEGA 7.0 . The seqxoxF sequences under the accession numbers KT932117–KT932121, KT932123, KT932124, KT932126–KT932128 and KY884986–KY884988  were obtained using methanol as the sole carbon and energy sources ; six isolates were endophytic (three Alphaproteobacteria and three Gammaproteobacteria); and ten were rhizospheric , four Alphaproteobacteria, and three Gammaproteobacteria). The identity of methylotrophic bacteria from the plant surface, from inside the plant, or the rhizosphere were as follows: Arthrobacter, one epiphyte and two rhizospheric; Pedobacter, one rhizospheric, Microvirga, four epiphytes; Inquilinus, two rhizospheric; Methylobacterium, one epiphyte, and one rhizospheric; Rhizobium, one rhizospheric; Sphingomonas, one endophyte; Subaequorebacter/Geminicoccus, one endophyte; Massilia, four epiphytes; Acinetobacter, twelve epiphytes, three rhizospheric, and three endophytes; and Pseudomonas, one epiphyte  with in 34.2%  of the iin 34.2% . After tt region , Table 2mxaf and xoxF-targeted primers, eleven clearly hybridized with a xoxF5 probe from M. extorquens  were the most frequently isolated organisms. It has been reported that Acinetobacter uses methanol as a carbon source (301 has previously been detected in this genus (Proteobacteria and Actinobacteria as some of the most common taxa in the rhizosphere and soil from cacti and other plants from arid lands (The methylotrophic isolates from the environment of n source , 61 and is genus . Similarid lands , 13, 26.N. macrocephala include xoxF-like dehydrogenases-possessing microorganisms. Their ecological role in xerophytic plants warrants further study. Since the cultivation procedures employed in the present study do not necessarily produce a real picture of bacterial diversity, the future application of non-culture approaches will enrich knowledge on methylotrophic diversity in this environment. In future inoculation experiments, we intend to detect the isolates of methylotrophic bacteria that may stimulate the growth of N. macrocephala, particularly in the vulnerable juvenile stage.Methylotrophic bacteria are ubiquitous and have meaningful roles in ecosystems. Since water is mostly limited in arid environments, perennial plants from these environments show restrained growth, particularly throughout the dry season. The community of methylotrophic culturable bacteria associated with the semi-arid thriving cactus


Nardostachys chinensis Batalin (NCBAE) on blood pressure and cardiac hypertrophy using two-kidney one-clip (2K1C) hypertensive rats. The aim of this study was to investigate the effects of the aqueous extract of−1·d−1 for 6 weeks. Twelve weeks after surgery, rat SBP and echocardiographic parameters were measured, cardiac histopathology was assessed, serum NO and LDH were detected, and the expression of Bcl-2 and caspase-3 of left ventricular tissue was assessed by western blot. 2K1C rat models were set up by clipping the left renal artery. Sham-operated rats underwent the same surgical procedure except for renal arterial clipping. 2K1C hypertensive rats were orally given NCBAE at doses of 210, 420, and 630 mg·kg Treatment with NCBAE resulted in a decrease of SBP, LVPWd, LVPWs, IVSd, IVSs, LVW/BW ratio, and cardiomyocyte CSA, an increase of LVEF, and inhibition of 2K1C-induced reduction in serum NO and elevation of LDH compared with 2K1C group. NCBAE intervention also showed a significant increase of Bcl-2 expression and reduction of cleaved caspase-3 level dose-dependently in left ventricular tissue.−1·d−1. Our data demonstrate that NCBAE has an antihypertensive property and protective effect on 2K1C-induced cardiac hypertrophy especially at the dose of 630 mg·kg Natural medicinal plants could be an ideal and safe source to develop effective agents for hypertension and hypertensive cardiac disorders.Hypertension leads to many types of organ damage, including cardiac hypertrophy, which increases the risk of heart failure , 2, and Nardostachys chinensis Batalin , which belongs to the genus Nardostachys , is a well-known traditional herbal medicine, enriched with a series of aristolane, nardosinane, guaiane-type sesquiterpenes [terpenes . The rooterpenes , aromatiterpenes , stomachterpenes , 7, and terpenes –10. NCB terpenes  and is uterpenes , 11. Morterpenes  and showterpenes , 14. Recterpenes . It alsoterpenes . Its carIn the progression of hypertensive cardiac disorders, the disappearance of cells by apoptosis is an indispensable process in addition to the proliferation and hypertrophy of cardiac cells , 18. The Nardostachys chinensis Batalin (NCBAE) on systolic blood pressure (SBP), echocardiographic parameters, cardiomyocyte cross-sectional area (CSA), serum NO levels, and activity of serum lactate dehydrogenase (LDH) and assessed the involvement of cardiac expression of Bcl-2 and caspase-3 in its therapeutic effects in this study.In order to research the possibility of NCB on BP regulation and cardiac protection in 2K1C hypertensive rats, we examined the effects of the aqueous extract ofPlant Material. The roots and rhizomes of NCB were purchased from Xinjiang Bencaotang Chinese Herbal Pieces Co. Limited  and authenticated by Dr. Haiyan Xu, Institute of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi, China. A voucher specimen (XJMUCL-VSN-2014-001) of NCB was deposited in the herbarium of the Central Laboratory of Xinjiang Medical University .Extract. The air-dried medical material was crushed to a fine powder (60 mesh). The powder was dissolved in hot distilled water (100°C) and extracted twice with distilled water (tenfold volume) for 1 h at each time, and the decoctions were combined. The solution was filtered and evaporated to yield a brown extract. The yield of NCBAE was 13.90% (w/w). The resulting powder stored at 4°C was diluted according to the doses  needed with distilled water and filtered before use. Chemicals. Atorvastatin (Ator), which was obtained from Pfizer Inc. , was dissolved in distilled water as a positive control. The dose of Ator for administration was calculated and expressed as mg·kg−1 body weight (16.5 mg·kg−1·d−1). Rabbit anti-rat monoclonal antibody of anti-Bcl-2 was obtained from Abcam® Inc. . Polyclonal antibody of anti-caspase-3 was obtained from Cell Signaling Technology® Inc. . Polyclonal antibody of anti-β-actin was obtained from Bioworld Technology Inc. .High-Performance Liquid Chromatography (HPLC) Analysis. The standard compounds linarin (purity 98.94%) and β-sitosterol (purity 98%) were obtained from Chengdu PureChem-Standard Co., Ltd. . HPLC-grade reagents, acetonitrile, and methanol were obtained from Sigma-Aldrich . Analytical column used for separation was C18  from Dalian Elite Analytical Instruments Co., Ltd. . All other chemicals used in HPLC analysis were of reagent grade.β-sitosterol was performed by LC-20AT high-performance liquid chromatography  in the Central Laboratory of Xinjiang Medical University. In brief, a total of 10 mg of NCBAE and 5 ml of methanol were mixed and sonicated for 20 min (100 Hz). After the dissolution, add methanol to 10 ml, shake well, and filter through a 0.22 μm membrane for HPLC analysis. The standard solutions of linarin and β-sitosterol were prepared in methanol at a concentration of 0.045 mg/ml and 0.98 mg/ml. Gradient elution was performed with solution A (acetonitrile) and solution B (water) in the following gradient elution program: 0–10 min, 15% of solution A; 11–60 min, 95% of solution A; 61–70 min, 15% of solution A. The dried extract was dissolved in HPLC-grade methanol (1 mg/ml) and filtered through a sterile 0.22 μm syringe filter and 10 μL volume was injected to the HPLC. Flow rate was maintained at 1 ml/min and temperature was adjusted to 25°C.Determination of linarin and Mass Spectrometric Analyses. Mass spectrometric analyses were performed using an Acquity UPLC  coupled with a Micromass Quattro Ultima triple quadrupole mass spectrometer  equipped with an electrospray ion source (ESI) operating in positive mode. Data were acquired with Empower 3 software. Analytical column used for separation was UPLC BEH C18  from Waters Co. The chromatographic run was performed at 200 μL min−1 with a gradient elution of a mobile phase which consisted of solution A (water) and solution B (acetonitrile). Briefly, the experimental system was operated with an ESI interface in positive ionization mode. The cone and desolvation gas flow rates were set at 50 L h−1 and 500 L h−1, respectively. Capillary voltage was set at 2.98 kilovolts, source temperature was 120°C, and desolvation temperature was 300°C. The cone voltage of linarin and β-sitosterol was set at 40 and 50 volts, respectively.Normotensive male Wistar rats with a body weight ranging between 250 and 300 g were obtained from the Laboratory Animal Center of Xinjiang Medical University. The rats were kept at constant physical ambient conditions  and supplied with tap water and standard diet ad libitum.2K1C renovascular hypertension was induced as described previously –26. Brie−1·d−1 (NCBAE (L)), 420 mg·kg−1·d−1 (NCBAE (M)), and 630 mg·kg−1·d−1 (NCBAE (H)) in three 2K1C groups each, Ator was intragastrically administered at a dose of 16.5 mg·kg−1·d−1 in the 2K1C group, and distilled water was administered in 2K1C and sham animals for about 6 weeks. At the end of the experiment, all animals were deeply anesthetized with chloral hydrate and blood was drawn by abdominal aortic puncture. The blood samples were centrifuged at 3000 rpm at 25°C for 10 min, and serum was obtained and stored at −80°C for biochemical assays. The hearts were excised, washed with ice-cold saline, and blotted with filter paper and the left ventricles were divided into two halves. One half was fixed in 4% formaldehyde solution for histopathological study, and the other half was stored at −80°C for western blot analysis.Treatments were started 6 weeks after surgery and maintained for 6 weeks. Animals were randomly assigned to one of six groups: NCBAE was intragastrically administered at doses of 210 mg·kgSBP of all the animals was measured before the surgery and once a week after clipping by BP-6 Tail-Cuff Blood Pressure Measuring System  with rats under consciousness. All animals were kept calm and warmed for 15 min till pulsatory signals were displayed steadily before the SBP measurement. The mean of three measurements was obtained from each rat once a week until the end of the experiment.After six-week treatments, rats were weighed, and transthoracic echocardiography was performed in all rats in this study under anesthesia  with spontaneous respiration. The chest was shaved, and the animal was situated in the supine position. Left ventricle (LV) function was evaluated by echocardiography using Philips HD IIXE  with a 7.5 MHz probe (S12-4). Two-dimensionally (2D) guided M-mode echocardiographic images in the left ventricular long-axis level were taken. Measurements were averaged from five consecutive beats. The rats of all groups underwent echocardiography after six weeks of treatment. The following parameters were measured: left ventricular posterior wall at end diastole (LVPWd), left ventricular posterior wall at end systole (LVPWs), interventricular septal diameter at end diastole (IVSd), interventricular septal diameter at end systole (IVSs), left ventricular internal diameter at end diastole (LVIDd), left ventricular internal diameter at end systole (LVIDs), percentage of left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF). Echocardiographic examination was performed by a professional echocardiographer blinded to the study groups.After imaging was complete, hearts of rats in each group were removed immediately under anesthesia. Left ventricles were collected and weighed. The organ mass (mg) was normalized by the body weight (g) giving a left ventricular weight/body weight (LVW/BW) ratio index. The index was adopted to prevent variations among different animal sizes.μm thick) and stained with hematoxylin-eosin. The images were digitally captured (magnification: ×400) using a Leica DM3000 microscope . Cardiomyocyte stained cross-sectional areas of each group were measured and digitalized by Image-Pro Plus 6.0 software . CSA/control ratios of each group were calculated and analyzed [The rats' hearts were harvested and fixed in 4% formaldehyde solution for at least 24 h and embedded in paraffin. The paraffin-embedded specimens were cut into sections .μg of protein was loaded in each well. The proteins were then transferred onto PVDF membranes and blocked with blocking buffer  for 60 min. The membranes were incubated overnight at 4°C with the rabbit anti-rat monoclonal antibodies of anti-Bcl-2  and polyclonal antibodies of anti-caspase-3  and anti-β-actin , respectively. Then, the membranes were incubated with alkaline phosphatase goat anti-rabbit IgG . Immunoreactive proteins were visualized by using BCIP/NBT kit . Densitometric quantification was undertaken through using ImageJ Program.To analyze protein expression, left ventricular tissues were prepared and proteins extracted for western blotting analyses. The protein extracts were boiled in 4 × loading buffer at 100°C for 10 min. Protein extracts of each group were subjected to SDS-PAGE using a 12% polyacrylamide gel, and 40 P < 0.05 were considered statistically significant.All the data were expressed as mean ± SD. Multiple comparisons between groups were made with one-way analysis of variance (ANOVA) using SPSS (17.0) software package. Values of m/z ratios of linarin and β-sitosterol were 593.16 and 415.70, respectively. This data confirmed the existence of linarin and β-sitosterol in NCBAE . Treatment of NCBAE (H) and NCBAE (M) caused reduction in the development of SBP in 2K1C rats . Compared with the 2K1C group, NCBAE (H) treatment and Ator treatment decreased LVW/BW ratio significantly.As shown in P < 0.01, Figures P < 0.01, Figures As shown in P < 0.01). Treatment with NCBAE (H) and Ator for 6 weeks resulted in a significant increase in serum NO level compared with 2K1C group (P < 0.01, P < 0.01, As shown in P < 0.01, P < 0.01, Figures Western blotting analysis showed that 2K1C renovascular hypertension led to cleavage of caspase-3 and downregulated expression of Bcl-2 in left ventricular tissue of rats (β-sitosterol, nardosinanone F, nardosinanone I, kanshone E, narchinol A, narchinol B, and others [β-sitosterol in NCB aqueous extract, and these findings are consistent with the researches mentioned above. But there still are some other chemical components of the herbal extract that need to be further studied. A study demonstrates that nardosinanone F, nardosinanone I, nardosinonediol, kanshone E, narchinol A, and narchinol B protect rats' cardiomyocytes from H2O2 injury in vitro [In recent years, a number of chemical compounds from NCB were obtained and their structures were identified as acaciin, ursolic acid, octacosanol, kanshone A, nardosinonediol, nardosinone, d others , 29. In in vitro . Ursolicin vitro . HoweverIn our research, we used a recognized model of experimental hypertension, the 2K1C renovascular hypertensive rats , 31. In During six-week NCBAE treatments, we observed that the extract showed an antihypertensive effect, decreased SBP of hypertensive rats compared with 2K1C group. We also found that NCBAE reduced LVPWd, LVPWs, IVSd, IVSs, LVW/BW ratio, and cardiomyocyte CSA and increased LVEF of hypertensive rats compared with 2K1C group. These results indicate that NCBAE regulates BP and attenuates ventricular hypertrophic remodeling in 2K1C hypertensive rats. NO, as an endothelium-derived relaxing factor , 36, plaCardiac dysfunction is also associated with myocardial apoptosis , 43. ApoIn conclusion, increased BP, reduced NO level, increased activity of LDH, and imbalance of Bcl-2 and caspase-3 increase myocardial hypertrophy, which exacerbates cardiac dysfunction. This cardiac injury and pathological remodeling can be reversed to some degree by NCBAE treatment. As a traditional herbal medicine, NCBAE may become a potent therapy for hypertensive cardiac disorders. For the deeper recognition, further studies are required to elucidate specific mechanisms of NCBAE intervention on 2K1C hypertension-induced cardiac injury, and the relevant effective chemical compounds of the herbal extract need to be further explored as well.


Plant Architecture Related Traits (PATs) are of great importance for maize breeding, and mainly controlled by minor effect quantitative trait loci (QTLs). However, cloning or even fine-mapping of minor effect QTLs is very difficult in maize. Theoretically, large population and high density genetic map can be helpful for increasing QTL mapping resolution and accuracy, but such a possibility have not been actually tested.P1, pl1 and r1 underlying silk color demonstrated the map quality. After QTL analysis, a total of 51 loci were mapped for six PATs. Although all of them belong to minor effect alleles, the lengths of the QTL intervals, with a minimum and median of 1.03 and 3.40 Mb respectively, were remarkably reduced as compared with previous reports using smaller size of population or small number of markers. Several genes with known function in maize were shown to be overlapping with or close neighboring to these QTL peaks, including na1, td1, d3 for plant height, ra1 for tassel branch number, and zfl2 for tassel length. To further confirm our mapping results, a plant height QTL, qPH1a, was verified by an introgression lines (ILs).Here, we employed a genotyping-by-sequencing (GBS) strategy to construct a linkage map with 16,769 marker bins for 1021 recombinant inbred lines (RILs). Accurately mapping of well studied genes We demonstrated a method for high resolution mapping of minor effect QTLs in maize, and the resulted comprehensive QTLs for PATs are valuable for maize molecular breeding in the future.The online version of this article (10.1186/s12870-018-1233-5) contains supplementary material, which is available to authorized users. Studtb1 , qPH3.1  [qPH3.1 , and qph.1 [qph1 . HoweverPrevious QTL studies of agronomic traits in crops were mainly based on relatively small populations and a few hundred or less molecular markers. QTL intervals of these studies typically ranged from 2 to 50 cM –17, whicMarker density is another key consideration influencing the precision for recombinational breakpoints identification, and would also reduce the mapping resolution. Recent progress in high-throughput sequencing technologies has markedly enhanced and accelerated genotyping. The genotyping-by-sequencing (GBS) strategy, named “bin mapping”, has been successfully applied in rice , ArabidoZmCCT, an important photoperiod response gene in maize, was cloned by regional genotyping of key recombinants in 866 maize-teosinte BC2S3 lines and analysis of other diverse maize populations [Several QTL mapping studies have been performed using large populations and increased marker density in targeted regions in plant. ulations . Two larulations . Recent ulations . These sIn this work,we constructed a RIL population derived from Zhengdan958 (the most widely planted hybrid in China) of 1147 lines, generated a high density marker set using GBS strategy, constructed a bin map, and phenotyped the population in multiple environments. Fifty-one plant architecture related loci with small effect were mapped to relatively small confidence intervals, which led to a list of reasonable candidate genes. One of these QTLs was further verified with introgression lines. Thus we demonstrated a method for high resolution mapping of minor effect QTLs in maize, and the resulted comprehensive QTLs for PATs are valuable for maize molecular breeding in the future.7 plants and the parental lines were phenotyped at three locations in China, Sanyuan  and Shangzhuang  in Beijing city, and Xinxiang  in Henan Province. In Sanyuan, two replications and the randomized complete block design were applied, while for the remaining two environments, an augmented incomplete block design [lme4 package [h2 were calculated by R software as reported formula [The RILs population used here includes 1147 lines, which was derived by crossing of Zheng58 and Chang7–2  with remarkably higher PH were picked out and planted in Shangzhuang in summer of 2012. After checked by four Indel markers , 9_5 and 10_1, that harboring the qPH1a allele descend from Chang7–2 were found and crossed with Zheng58. In summer of 2013, the progenies (BC6F1) of 9_5 and 10_1 were phenotyped and genotyped, and 3 recombinants (BC6F1), G36–3, G40–7 and G41–8, were found and crossed with Zheng58 again. In the summer of 2014, progenies (BC7F1) of these 3 recombinants were planted in Shangzhuang, followed by measuring PH and genotyping by 4 Indel markers. A method named “recombinant-derived progeny testing strategy” [qPH1a.An ILs population constructed by Chang7–2 as donor parent, Zheng58 as recurrent parent was used for verifying the reliability of trategy”  was invoShoot tissues from five seedlings per genotype were bulked for total genomic DNA isolation and extraction using the CTAB protocol .Zheng58 and Chang7–2 were deeply sequenced using Illumina HiSeq2000 for 28.41  and 24.95  fold coverage of the maize genome respectively . DNA of SNP identification was conducted using a strict pipeline as reported , where BTo ensure the map quality, only RILs with sequencing depth greater than 0.02× and genotyped SNPs more than 8000 were retained, which excluded 18 RILs from the dataset.The reported sliding window method  with minest.rf. The R/qtl function est.map (“kosambi” method) was manipulated for linkage map calculation. A genome wide recombination ratio analysis was calculated by (linkage distance)/ in 1 Mb windows, and the filtered gene set gene density (B73 maize reference genome V2) and SNP density were recorded at the same time.The RIL population was further filtered by excluding the lines with greater than 15% residual heterozygous genotypes or with more than 360 breakpoints (91 RILs), to avoid material confounding, leaving a set of 1021 lines.Blocks without any breakpoints across the remaining population were grouped into bins, and bins with a physical length less than 5 kb were merged together. The allele frequency  of each bin was recorded to investigate instances of distorted segregation. Bins with a distorted segregation ratio larger than 2/1 or a heterozygous ratio larger than 15% were subsequently discarded  for further improvement of the linkage map.QTL mapping software for RIL populations does not generally accept heterozygous loci, so bins with heterozygous genotype were regarded as missing followed by the “argmax” imputation method in R/qtl. Pair-wise recombinational fractions for all bins were calculated by the R/qtl function p < 0.05) were used for defining QTL logarithm (base 10) of odds scores (LOD) threshold, 1.5 LOD-drop method was applied for defining QTL confidence interval, and linear QTL model was used for calculating QTL additive effect and phenotypic variation explained.The QTL study of the Zhengdan958 RILs was conducted using the R/qtl package as reported . BrieflyThrough deep sequencing of the two parents for above 24× coverage and identifying SNPs by a strict pipeline, 2,231,331 high-quality SNPs were detected between Zheng58 and Chang7–2. Approximately 67% of the SNPs were located in intergenic regions, 8% in exons, 12% in introns, and 14% within 2 kb upstream or downstream of genes , but not overall SNP density , but negatively correlated with intergenic regions , including 1710 bins (10.2%), with total length of 155.8 Mb which covered 7.6% of the genome  [rf2 (restorer of fertility2) [The 1021 RILs were produced from 6 generations of self pollination. Overall, theses RILs captured ~ 50,040 crossover events, resulting in an average of 4.9 per chromosome (Table ility32)  on chromtility2)  on chromP1 (pericarp color1) gene, which is known to contribute to the phlobaphene pigmentation of maize floral organs [pl1 gene, which was contributed to purple pigment in maize plant [r1 gene, which was known contributed to silk color variation in maize [P1, pl1 and r1 to relative small confidence intervals  demonstrated our map quality.The bin map quality and the QTL mapping pipeline were firstly tested using the silk color phenotype. The phenotypes were scored into 5 classes according to the intensity of red pigmentation , along with EH and EH/PH (r = 0.667) were strongly correlated, while a modest correlation was observed between PH and TL (r = 0.420), and ULN and EH/PH (r = − 0.360). The heritability estimates of PH, EH, EH/PH, TBN, and TL were high with all being greater than 0.7 , including 8 for PH, 9 for EH, 8 for EH/PH, 14 for TL, 6 for TBN, and 6 for ULN  and 239.92–249.10 Mb  on chromosome 1, 11.93–14.76 Mb  and 234.72–236.84 Mb  on chromosome 2, 176.73–180.82 Mb  and 199.25–202.15 Mb  on chromosome 3, 133.70–142.27 Mb  on chromosome 8. These regions could result from pleiotropy or linked genes. Thirty-one positive effect QTLs were inherited from Chang7–2.Totally, 51 plant architecture related QTLs scattering across 10 maize chromosomes were detected above the threshold , as adjacent two peaks were less than 25 Mb apart  was located in an interval encompassing ra1 (ramosa1) [qPH3a and qEP3a, LOD = 6.74 and 5.14, 2.20% and 5.03% variance explained for PH and EH/PH respectively) in a ~ 3 Mb region of chromosome 3 includes na1 (nana plant1) [qPH5, LOD = 7.56, 3.05% variance explained) co-localized with td1, which was involved in CLAVATA signaling pathway and harboring known impact on plant height variation in maize [qEP9a, LOD = 4.72, 1.72% variance expected) encompassing d3, a gene controls cytochrome P450-mediated biosynthesis of gibberellins that involved in maize dwarfing phenotype [qTL2a, LOD = 4.83, 1.99% variance expected) just overlapped with zfl2, which controls inflorescence development and architecture in maize [qEP1, LOD = 11.80, 3.92% variance expected) in a 3.9 Mb region of chromosome 1 contains br1  [qEP2, LOD = 3.88, 0.95% variance expected) in chromosome 2 co-localized with d5 locus  [With the aid of high resolution bin map and large RIL population, many QTLs were located to relatively small genomic intervals that included genes with known function Fig. . For TBNramosa1) , an impo plant1) , an impoin maize . The EH/henotype . A TL QTin maize . An EH/Pgdb.org) , which hgdb.org) , anotherzap1, shown to be allelic to ZmMADS3, a transcription factor that influences maize internode number [myb151 which is homologous to Arabidopsis AtMYB87, the latter could suppress longitudinal elongation of multiple organs [sbp21, homologous to Arabidopsis AtSPL10, which controls shoot-associated lateral organs development [In addition to genes that had been mapped or cloned in maize, several candidate genes inferred from homologues were also identified in our QTL intervals Fig. . The lare number . The EH e organs  that mayelopment , may conelopment . Since tqPH1a. In the summer of 2012, two ILs, named 9_5 and 10_1 (BC5F1), with heterozygous genotypes in the qPH1a region were chosen and backcrossed with Zheng58. In the summer of 2013, 132 and 113 progenies (BC6F1 generation ILs) of 9_5 and 10_1, were planted respectively, followed by measuring plant height and genotyping by 4 Indel markers  were significantly different , named G36–3, G40–7 and G41–8, in the progenies of 9_5 and 10_1 were picked out and tested by the same method. G36–3 harbored heterozygous genotype upstream of Indel_91 and homozygous genotype downstream in the qPH1a region, while G40–7 and G41–8 just the opposite  were identified in the region where only a single QTL were detected by Salvi et al. [Closely linked loci in coupling phase or repulsion phase can cause misevaluation or misdetection of QTLs, which is a major challenge in quantitative trait studies. In our study, there were 4 regions containing closely linked QTLs for the same trait (as adjacent two peaks were less than 25 Mb apart)  or regu1 [OsLG1 ) of impoAdditionally, Zhengdan958 is the most widely used maize hybrid in China. Their two parental lines, Zheng58 and Chang7–2, are the top breeding materials in our current breeding system. Zheng58, an improved Reid germplasm, is derived from 5003, which was derived from an elite hybrid from the US and have being the most important dwarf germplasm in China . Chang7–In this study, by combining large population and high-density bin map, we mapped a list of minor effect QTLs for PATs in maize. The interval sizes of these QTLs were greatly reduced as compared with previous studies using smaller size of population or smaller number of markers. A number of well studied genes with similar function were located in these QTL intervals, and a plant height QTL were further confirmed by an introgression lines. These QTLs represent a set of reliable resource for maize molecular breeding in the future, and the method used here demonstrated the power of combining large population with high-density markers for minor effect QTL mapping.Additional file 1: Figure S1.Figure S2. Sequencing depth profile of Zhengdan958 RILs. Figure S3. Distribution of false GBS SNPs in 1 Mb windows by parent. Figure S4. Pair-wise recombinational fractions (upper left) and LOD scores (lower right) of the bins. Figure S5. The distribution of segregation distortions across ten chromosomes.Segregation distortions were tested by Chi-test, and –log10 (PChi-test) were plotted against their physical positions. Threshold of no distortion  were showed as red dashed lines. Figure S6. The 5 classes of silk color. Figure S7. Phenotypic distributions of PH, EH, EH/PH, TNB, TL and ULN. Table S1. The 240 barcodes used in GBS. Table S2. GBS error rate for parental lines. Table S3. Pair-wise Pearson’s correlation coefficients (lower left) and p-values (upper right) among recombination, gene density, and SNP density in 1 Mb intervals by genomic region. Table S5. Pearson correlation coefficients among different traits in two environments.Lower left, the correlation coefficients; Upper right, p-values of correlation test. Table S6. Heritability of different traits. Table S7. Summary of mapped QTLs. Table S8. Markers used for verifying qPH1a. (PDF 934 kb)Distribution of SNPs polymorphic between Zheng58 and Chang7–2 in different genomic regions. Additionnal file 2: Table S4.Regions with 3 fold higher recombinant ratio than genome average. D_Downstream, D_Exon, D_Intron, D_Upstream, D_Intergenic: SNP density in 2 k–downstream, exon, intron, 2 k–upstream of genic regions and SNP density in intergenic regions. (XLSX 21 kb)