Frankliniella occidentalis for an empirical microsatellite survey and validation; 132,251 candidate microsatellites were identified, 92,102 of which were perfect. Dinucleotides were the most abundant category, while (AG)n was the most abundant motif. Sixty primer pairs were designed and validated in two natural populations, of which 30 loci were polymorphic, stable, and repeatable, but not all in Hardy–Weinberg equilibrium (HWE) and linkage equilibrium. Four marker panels were constructed to understand effect of marker selection on population genetic analyses: (i) only accept loci with single nucleotide insertions (SNI); (ii) only accept the most polymorphic loci (MP); (iii) only accept loci that did not deviate from HWE, did not show SNIs, and had unambiguous peaks (SS) and (iv) all developed markers (ALL). Although the MP panel resulted in microsatellites of highest genetic diversity followed by the SNI, the SS performed best in individual assignment. Our study proposes stringent criteria for selection of microsatellites from a large-scale number of genomic candidates for population genetic studies.Recent improvements in next-generation sequencing technologies have enabled investigation of microsatellites on a genome-wide scale. Faced with a huge amount of candidates, the use of appropriate marker selection criteria is crucial. Here, we used the western flower thrips  Molecular markers are useful tools for population genetics, linkage map construction, gene mapping, and phylogenetics analyses235Frankliniella occidentalis is an economically important invasive agricultural and horticultural pest. It is native to western North America, but has spread rapidly worldwide through the movement of horticultural material  since the late 1970s8910111214161718182022The western flower thrips (WFT) Although the development of microsatellite markers is now more accessible, the criteria for what constitutes good microsatellite markers are still controversial. Empirical studies have shown that selecting the most polymorphic markers, as many researchers often do, will generally overestimate genetic diversityA total of 132,251 candidate microsatellites were identified from 4,151 WFT genome scaffolds, 92,102 of which were of the perfect type. The total relative abundance was 322.01 loci/Mb. The total relative density was 9.07 Kb/Mb, i.e., ca. 1% of the WFT sampled genome is constructed of microsatellites. Dinucleotides were the most abundant category, having highest density, followed by penta-nucleotides and tri-nucleotides . The micThe initial QDD3 program output provided 5,279 primer pairs, of which 4,175 were tri-nucleotide microsatellites and 1,104 were tetra-nucleotide. Following our stringent filter, a total of 381 primers (Appendix S1) flanking perfect microsatellites were retained. We found 305 primer pairs flanking tri-nucleotide microsatellites, and 76 pairs flanking tetra-nucleotide microsatellites. We selected and synthesised 60 primer pairs from different scaffolds for amplification (Appendix S1). In our initial test, 30 primers generated polymorphic genotypes, 23 primers failed to generate amplification in any individual, and seven primers failed in more than two individuals (Appendix S1).HO) and expected heterozygosities (HE) ranged from 0.1 to 0.875 and 0.348 to 0.895, respectively  and expected heterozygosity (HE) values were higher for the MT population than for the YQ population in all cases, while both populations have quite similar FIS values . A significant difference was also observed in SNI versus SS in the YQ population . No significant differences were found between the remaining panel pairs in the YQ populations nor in all the MT population panel pairs.The allelic richness  showed an increasing trend in order of MP, SNI, ALL, and SS values. However, the differences were not statistically significant. In the STRUCTURE analyses, all panels obtained their highest Delta K at K  =  2, which suggests two distinct clusters. The average posterior probability (Q) was high in all cases . However, some individuals were not correctly assigned to their respective population. There were 1, 2, 4, and 4 individuals incorrectly assigned for the SS, ALL, MP, and SNI microsatellite markers, respectively  events in flanking regions or may result from an excess of base substitutionsFST accuracy33Amplification success rates were considered as an important criterion in our study. Failed amplifications may relate to null alleles , which cHE and AR than the stringently selected markers (the SS panel), while the SNI panel obtained higher polymorphism levels. However, similar FST values were estimated by different marker panels, indicating that the approach of microsatellite selection will not influence the analysis of population differentiation, as previously reportedIn our empirical study, the most polymorphic markers (the MP panel) resulted in higher We examined and developed a large number of microsatellite markers from the WFT at the genome-wide scale. Our analyses revealed several distinct characteristics regarding the distribution of microsatellites in the species. High-quality primers can feasibly be designed and selected as appropriate markers for further research by taking advantage of the abundant microsatellites that can be isolated from genome sequences. We found that stringent criteria considering amplification efficiency, SNI within loci, and population genetics parameters can obviously improve individual assignment analysis, through a comparison of the performance of different marker panels. Our study provides an example for the bulk discovery of microsatellites and the development of stringent marker selection criteria for population genetics studies.A total of 55 adult females of WFT from eight sampled sites were used in the study. Among the specimens, one individual from each of the eight collection sites was used for an initial examination of selected primer pairs, while 47 from two of the collection sites  near Beijing, China, were used for a population-level survey. All samples were stored in 98% alcohol, and frozen at −80 °C. Total genomic DNA was extracted from a single female adult using a DNeasy Blood and Tissue Kit .F. occidentalis genome scaffolds  submitted to GenBank by “the Frankliniella occidentalis whole genome shotgun (WGS) project”  for microsatellite discovery. Microsatellites were scanned for and identified using the program SciRoKo, with the default fixed penalty motif criteria36We used the 4,151 The QDD3 programGenotyping accuracy and stability are associated with target region motifs. Dinucleotide microsatellites are prone to polymerase slippage during the amplification process, which may lead to mistyping394143Imperfect (those with repeat region interruptions), compound, and multiple microsatellites within an amplified region may generate complex genotypes. Therefore, output primers were further filtered manually using the following, more stringent, criteria: (i) only perfect (without repeat region interruptions) loci were retained; (ii) the minimum distance between the 3′ end of two primer pairs and their target region had to be >10 bp; and (iii) primers using the ‘A’ design strategy that do not have multiple microsatellites, nanosatellites, or homopolymers in the amplicon were retained.et al.Sixty primer pairs were selected and synthesised for validation. A universal primer (CAGGACCAGGCTACCGTG) was added to the 5′ end of the forward primers following the method of Blacket We initially tested for primer effectiveness and polymorphism level using one individual from each of the eight different sampling sites. The amplifications were performed using the GoTaq Green Master Mix  in a final volume of 10 μl. The polymerase chain reaction (PCR) reaction mixture contained three primers: 0.08 μl forward primer, 0.16 μl reverse primer, and 0.32 μl universal primer labelled with fluorescence . Our PCR protocol follows: 5 min at 95 °C, followed by 35 cycles of 95 °C for 30 s, 56 °C for 40 s, and 72 °C for 40 s, with a final extension at 72 °C for 15 min. PCR products were analysed on an ABI 3730xl DNA Analyzer  using the GeneScan 500 LIZ size standard . Genotyping was conducted using GENEMAPPER 4.0 . Primer pairs that failed in more than two individuals, or that were monomorphic in eight individuals, or that produced more than two peaks were discarded. Those remaining primer pairs that generated polymorphic genotypes were further analysed in the next step.For the next step, 48 female adults from two populations were used to analyse the genetic characteristics of each primer pair. The PCR reaction mixture, cycling conditions, and genotyping methods were identical to those of the previous step. We repeated failed amplifications twice to eliminate occasional technical mistakes.HO) and expected heterozygosity (HE). Allelic richness and the inbreeding coefficient of an individual relative to the subpopulation (FIS) were obtained using FSTAT 2.9.3FST) were tested and/or calculated using GENEPOP 4.2.1FST outlier method employed in the program LOSITANFST” and “neutral mean FST.”.The genotyping data of two populations were analysed. The macros Microsatellite ToolsK value (from 1 to 5) with 200,000 Markov chain Monte Carlo iterations, after a burn-in of 100,000 iterations. The Delta (K) methodK values by submitting the Structure output to Structure Harvester Web 0.6.94K values inferred from the previous step were processed using CLUMPP 1.1.2We examined population genetic structure using STRUCTURE 2.3.4Six factors were taken into account for the selection of our microsatellite markers: HWE, LD, polymorphism, amplification fail rate, single nucleotide insertions (SNI), and peak patterns genotyped in GENEMAPPER. We constructed three microsatellite panels based on three criteria for each locus : (i) theHow to cite this article: Cao, L.-J. et al. Bulk development and stringent selection of microsatellite markers in the western flower thrips Frankliniella occidentalis. Sci. Rep.6, 26512; doi: 10.1038/srep26512 (2016).


Lymphomas arising in the liver are extremely rare. Here, we describe a case of Hepatitis C virus infection with primary hepatic lymphoma (PHL) presenting with hyperbilirubinemia. A 45-year-old African American male presented with abdominal pain, pruritus, and itching for two days. CT of abdomen and pelvis with contrast showed numerous masses in the liver. The liver biopsy was consistent with diffuse large B cell lymphoma (DLBCL). Conventional chemotherapy was avoided initially because of hyperbilirubinemia. Hence, radiation therapy was given initially to reduce his bilirubin levels and tumor size. The patient was able to complete six cycles of rituximab combined with cyclophosphamide, adriamycin, vincristine, and prednisone (R-CHOP) chemotherapy and achieved a complete response verified by positron emission tomography-computed tomography (PET-CT). PHL should be considered when there are numerous space occupying liver lesions seen on imaging. Hyperbilirubinemia may be a reason for delay in treatment for some of these patients. Hence, the role of radiation therapy prior to treatment with R-CHOP is an alternative to management for stage IV diffuse large B cell lymphoma. Lymphomas arising in liver are extremely rare. They can be primary  or secondary (secondary involvement of liver in systemic non-Hodgkin's lymphoma (NHL)). Primary hepatic lymphoma (PHL) comprises only about 0.016% of all cases of non-Hodgkin's lymphoma . BecauseA 45-year-old African American male with a past medical history of hypertension, HCV infection, and polysubstance abuse was admitted with epigastric and right upper quadrant abdominal pain, pruritus, and itching for two days. Symptoms are associated with nonbilious vomiting, fatigue, and a 9 kg unintentional weight loss over three months. He denied any history of Hepatitis B in the past. Ten months before, he had a similar complaint where evaluation at an outside facility showed a hepatic mass. A liver biopsy at that time showed acute inflammation suggestive of an abscess with extensive chronic inflammation and fibrosis adjacent to the necrosis, moderate to severe acute and chronic periportal inflammation with portal lymphoid aggregates. Cultures from the biopsy were negative for any organisms but the patient was treated with an empiric course of vancomycin and piperacillin/tazobactam for four weeks. His social history was significant for cigarette smoking, alcohol, and intravenous illicit drug use of heroin and cocaine. The family history was significant for a brother with gastric lymphoma, now in remission. Physical exam was significant for skin excoriations, scleral icterus without any lymphadenopathy, hepatomegaly, and tenderness along the epigastrium and right upper quadrant areas.3, and platelet count of 240 × 103. His metabolic panel showed normal renal function tests with a calcium of 9.4 mg/dL. His comprehensive metabolic panel revealed a total bilirubin of 6.1 mg/dL, direct bilirubin of 4.1 mg/dL, aspartate aminotransferase (AST) of 150 U/L, alanine aminotransferase (ALT) of 142 U/L, alkaline phosphatase (ALP) of 282 IU/L, gamma-glutamyl transferase (GGT) of 385 IU/L, total protein of 7.4 g/dL, and albumin of 3.6 g/dL. His prothrombin time was 13.7 seconds with an International Normal Ratio (INR) of 1.04. Amylase and lipase levels were within normal limits. Hepatitis B panel showed immunity to Hepatitis B. His HCV RNA viral load was 422131 IU/mL (5.63 log). HCV genotype is 1b. HIV by ELISA was negative. His alpha-fetoprotein levels (AFP) were 2.2 ng/mL. Blood cultures were negative. Triphasic computed tomography (CT) of the abdomen and pelvis with contrast showed multiple masses in the liver (largest being 8.8 cm) without any cholelithiasis or extrahepatic bile duct dilatation  CT showed decreased metabolic activity compared with normal adjacent liver parenchyma, suggesting a positive response to treatment and no extranodal disease. Meanwhile, total bilirubin levels fell to 3.4 mg/dL  and alloCaccamo et al.  defined It is rare for a patient with PHL to present with very high bilirubin levels. Jaundice in PHL patients can be due to the following reasons: (a) patients having a concomitant history of cirrhosis, (b) patients presenting with fulminant liver failure due to massive sinusoidal infiltration by lymphoma, and (c) lymphoma causing intra- or extrahepatic biliary obstruction. The patient that we described above had a progressive increase in total bilirubin reaching a peak value of 23.3 mg/dL. To the best of our knowledge, this is the first report described in literature where a patient with HCV infection had such a high bilirubin level due to PHL.60% of the patients with PHL tested for HCV were positive . Some ofHistological subtypes of PHL described in the literature include diffuse large B cell lymphoma (DLBCL), Burkitt's lymphoma, mucosa associated lymphoid tissue (MALT) lymphoma, mantle cell lymphoma, and T cell lymphoma with the most frequent one being DLBCL 96%) . PHL has% . PHL hThe treatment of patients with PHL reported in the literature has not been consistent. Patients were treated with various modalities like surgery, combination chemotherapy, radiation therapy, or a combination of these. Localized PHL could be cured by resection, or the tumor burden could be reduced before or after surgery with chemotherapy . SurgicaA complete remission (CR) rate of 83.3% was achieved in PHL when systemic combination chemotherapy was used as the main modality of treatment . SimilarAdriamycin (one of the agents in CHOP regimen), an anthracycline, is predominantly excreted in bile. Patients with cholestasis have delayed clearance of doxorubicin and its metabolites and can lead to greater systemic toxicity , 18. DesWhen multiple space occupying lesions are seen on imaging of the liver without any primary lesion or any other organ involvement with normal alpha fetoprotein, CA19–9, and carcinoembryonic antigen (CEA) levels, PHL should be considered in the differential diagnosis of these lesions and biopsy should be strongly considered in patients with HCV infection. PHL can cause significant hyperbilirubinemia in patients with HCV infection. If PHL is causing hyperbilirubinemia in a patient with HCV infection, localized radiation therapy can be considered to reduce bilirubin levels within an acceptable range where full dose of chemotherapy can be initiated in avoiding serious life-threatening complications.


The poly(styrene-methyl methacrylate) latex particles as potential physical shale stabilizer were successfully synthesized with potassium persulfate as an initiator in isopropanol-water medium. The synthesized latex particles were characterized by Fourier transform infrared spectroscopy (FT-IR), particle size distribution measurement (PSD), transmission electron microscopy (TEM), and thermal gravimetric analysis (TGA). FT-IR and TGA analysis confirmed that the latex particles were prepared by polymerization of styrene and methyl methacrylate and maintained good thermal stability. TEM and PSD analysis indicated that the spherical latex particles possessed unimodal distribution from 80 nm to 345 nm with the D90 value of 276 nm. The factors influencing particle size distribution (PSD) of latex particles were also discussed in detail. The interaction between latex particles and natural shale cores was investigated quantitatively via pore pressure transmission tests. The results indicated that the latex particles as potential physical shale stabilizer could be deformable to bridge and seal the nanopores and microfractures of shale to reduce the shale permeability and prevent pore pressure transmission. What is more, the latex particles as potential physical shale stabilizer work synergistically with chemical shale stabilizer to impart superior shale stability. In the past decade, shale gas, as a clean and unconventional energy, has become progressively important in the energy landscape worldwide –5. In drHistorically, emulsion polymerization was widely used in the preparation of polymer materials, especially nanomaterials, but the water resistance and surface smoothness of polymers were influenced inevitably by residual emulsifiers , 18. TheIn this work, the emulsifier-free latex particles as potential physical shale stabilizer in water-based drilling fluids were synthesized by emulsifier-free emulsion polymerization of styrene (St) and methyl methacrylate (MMA) using solvothermal method with potassium persulfate as an initiator in isopropanol-water medium. The newly synthesized latex particles were characterized in detail and the influencing factors on particle size distribution (PSD) of latex particles, such as isopropanol volume, reacting temperature, and initiator concentration, were also discussed. Moreover, the interaction between latex particles and natural shale cores was investigated quantitatively via pore pressure transmission tests.The monomers of styrene (St) and methyl methacrylate (MMA), obtained from Sinopharm Chemical Reagent Co. Ltd. (China), were distilled under vacuum before use. The initiator, potassium persulfate, was obtained from Aladdin Reagent Co. Ltd. (China) and was recrystallized for purification. Isopropanol and sodium chloride, AR grade, were obtained from Sinopharm Chemical Reagent Co. Ltd. (China) and used as received. The distilled water was used throughout the experiments.The chemical shale stabilizer, SDCS, is an aluminum complex self-developed to impart shale stability. The aluminum complex could completely dissolve in the water-based drilling fluids when pH of the fluids is maintained above 11. When interacting with formation water of low pH, it could precipitate in the shale matrix and decrease the shale permeability, thus providing a barrier to pore pressure transmission. In addition, the chemical shale stabilizer could withstand high temperature and salt concentration.2/g, respectively. The cylindrical shale cores were used in the pore pressure transmission tests with a diameter of 2.54 cm and a length of 0.80 cm.The shale samples, here, were obtained from the Sichuan Basin, China. The main clay minerals of shale samples were determined to be illite/smectite and illite by X-ray diffraction analysis , with caThe P(St-MMA) latex particles were prepared by the emulsifier-free polymerization of styrene and methyl methacrylate using solvothermal method with potassium persulfate as an initiator in isopropanol-water medium. The styrene, methyl methacrylate, potassium persulfate, isopropanol, and distilled water of characteristic concentration were successively added into a hydrothermal synthesis reaction kettle. Then, the mixture above was stirred using electromagnetic stirring at room temperature for 15 min and at 90°C for another 2.5 h. The P(St-MMA) latex particles with different reaction conditions were prepared using similar methods. The recipes for synthesis of P(St-MMA) latex particles were shown in −1. The purified latex particles were dried under vacuum at 80°C, and mixture of latex particles samples and potassium bromide (KBr) was pressed into pellets for FT-IR analysis. The transmission electron microscopy (TEM) images of latex particles were taken with JEOL JEM-2100UHR TEM using an accelerating voltage of 200 kV. The particle size distribution (PSD) of P(St-MMA) latex particles was analyzed with dynamic light scattering using Mastersizer 3000 . The samples of TEM and PSD were diluted before testing. The thermal gravimetric analysis (TGA) was conducted with a simultaneous thermal analyzer  at a heating rate of 0~50°C/min under nitrogen atmosphere. It should be pointed out that the latex particles used for characterization were synthesized using S-3 recipe , scanning from 4000 to 400 cm3 recipe .The pore pressure transmission test was developed using the pressure transmission technique to characterize the hydraulic properties of shale and the pore pressure transmission tests in this paper were performed on the simulation equipment for hydramechanics coupling of shale, developed by China University of Petroleum (East China) , and basDuring the pore pressure transmission test, shale cores were subjected to hydraulic or osmotic gradients or both when exposed to upstream and downstream fluids. The confining pressure and axial pressure were all 5.0 MPa and upstream pressure was maintained at 3.0 MPa. The initial downstream pressure was 1.0 MPa. The pore pressure transmission tests were performed at 70°C. The downstream pressure, namely, pore pressure, was monitored throughout the tests. With no chemical potential difference between upstream and downstream fluids, the downstream pressure would become equal to the applied constant fluids pressure at the upstream because of nonzero permeability of shale cores. But, when there is chemical potential difference present, any difference between the downstream pressure and the applied constant fluid pressure could be measured in response to osmotic pressure.K is the permeability of shale cores, μm2; μ is the viscosity of fluids, mPa.s; β is the static compression ratio of fluids, MPa−1; V is the enclosed volume of downstream fluids, cm3; L is the length of shale cores, cm; A is the cross-sectional area, cm2; t is total experimental time, s; Pm is the upstream pressure, MPa; P0 is the pore pressure, MPa; P is the real-time downstream pressure, MPa.The permeability of shale cores could be calculated using formula  24]. Co. Co24]. σ is the membrane efficiency of shale cores, %; ΔP is the actual osmotic pressure, MPa; R is the ideal gas constant, 8.314 J·mol−1·K−1; T is the test temperature, K; VW is the partial molar volume of water, 18 cm3·mol−1; awsh is the water activity of pore water; awdf is the water activity of drilling fluids.Shale could act as a nonideal semipermeable membrane, and membrane efficiency was defined as the ratio of actual osmotic pressure and ideal osmotic pressure to characterize its nonideality . ConsideIn addition, the pore structure of shale cores before and after tests was characterized by Hitachi S-4800 field-emission scanning electron microscope (SEM) analysis.−1, corresponding to the C=O stretching band, and two characteristic absorption peaks at around 1236 cm−1 and 1142 cm−1, corresponding to the C–O–C symmetric stretching band. The stretching vibrations of benzene skeleton were presented at around 1601 cm−1, 1489 cm−1, and 1454 cm−1, and 754 cm−1 and 700 cm−1 were characteristic bending vibrations of single substitution benzene ring. It was also observed from −1, 3065 cm−1, 3020 cm−1, and 2996 cm−1 [The FT-IR spectrum of purified latex particles  showed a996 cm−1 , 27. BecThe weight loss observed up to 200°C was attributed to the desorption of physically absorbed water and dehydration of the hydrated cations. The organic compounds were decomposed between 200 and 500°C . From thAs can be seen from When it comes to reducing shale pore pressure transmission, particle size distribution is one of the most important factors influencing plugging mechanism and efficiency of pore throats and microfractures , 32, andIt can be seen from The effect of reacting temperature on D90 value of latex particles is shown in aw = 0.998), and the upstream fluids were 3% sodium chloride solution , 20% sodium chloride solution , 3% sodium chloride solution with 3% SDPS , 20% sodium chloride solution with 2% SDPS, and 1% SDCS , respectively.During the pore pressure transmission tests, the P(St-MMA) latex particles were used as physical shale stabilizer (SDPS) and self-developed aluminum complex was used as chemical shale stabilizer (SDCS). The downstream fluid was 3% sodium chloride solution  to 1.01 × 10−8 μm2. Because there is no chemical potential difference between USFL-1/USFL-3 and DSFL, the effect of preventing or reducing pore pressure transmission is mainly related to internally bridging and sealing shale microfractures of deformable P(St-MMA) latex particles.The curves of pore pressure transmission tests are shown in aw = 0.875), the pore pressure (downstream pressure) would be less than upstream pressure due to chemical potential difference between USFL-2 and DSFL. After interacting with USDL-4, the downstream pressure decreased gradually and offset the hydraulic pressure. It is concluded that shale acts as a semipermeable membrane, and the hydraulic pressure could be offset by the backflow caused by the development of osmosis when there exists chemical potential difference. According to formula  latex particles have been successfully prepared by emulsifier-free emulsion polymerization of St and MMA, using potassium persulfate as an initiator in isopropanol-water medium. The latex particles possessed unimodal distribution from 80 nm to 345 nm with the D90 value of 276 nm, and the particle size was influenced significantly by isopropanol volume, reacting temperature, and initiator concentration. The latex particles could disperse uniformly in the water-based drilling fluids as potential physical shale stabilizer and they could be deformable to internally bridge and seal pore throats and microfractures of shale. What is more, a cooperative action was observed in P(St-MMA) latex particles  and aluminum complex  to reduce pore pressure transmission and increase membrane efficiency of shale, thus improving shale wellbore stability. The newly synthesized latex particles were alternatives to conventional plugging agents in water-based drilling fluids for shale gas.


Three months later, SR was obtained in 83/91 or 80/89 cases from WK or sotalol groups (P = 0.762). By another analysis, SR was obtained in 86/90 or 77/90 cases from 131I or ATD groups (P = 0.022). Then, we randomly assigned the successfully SR-reverted patients into three groups: WK, sotalol, and control (no antiarrhythmia drug was given) groups. After twelve-month follow-up, PAF recurrence happened in 1/54, 2/54, and 9/55 cases, respectively. Log-Rank test showed significant higher PAF recurrent rate in control patients than either treatment (P = 0.06). We demonstrated the same efficacies of WK and sotalol to assist SR reversion from hyperthyroidism-caused PAF. We also showed that either drug could maintain SR in such patients.We aimed to compare effectiveness of Wenxin Keli (WK) and sotalol in assisting sinus rhythm (SR) restoration from paroxysmal atrial fibrillation (PAF) caused by hyperthyroidism, as well as in maintaining SR. We randomly prescribed WK (18 g tid) or sotalol (80 mg bid) to 91 or 89 patients. Since it was not ethical not to give patients antiarrhythmia drugs, no control group was set. Antithyroid drugs were given to 90 patients ;  Successful management of hyperthyroidism could result in restoration of sinus rhythm (SR) in up to two-thirds of patients [Atrial fibrillation is the most common cardiac rhythm disturbance, increasing in prevalence with age. By definition, atrial fibrillation is a supraventricular tachyarrhythmia characterized by uncoordinated atrial activation with consequent deterioration of atrial mechanical function –3. Clinipatients . Mechanipatients –12. It i Nardostachys chinensis Batal. extract (NcBe), Codonopsis, notoginseng, amber, and rhizoma polygonati. Burashnikov and colleagues [Wenxin Keli (WK) is a pure Chinese herb medicine. It has been reported to be useful in the treatment of atrial fibrillation –15, ventlleagues  recentlylleagues . In factTherefore, in this open label and randomized study, we aimed to prospectively compare the effectiveness between WK and sotalol in assisting SR reversion from hyperthyroidism-caused PAF. We also intended to study their effectiveness in the maintenance of SR. Sotalol was chosen as a comparing drug, because it was proven to have efficacy to restore and maintain SR from atrial fibrillation. And sotalol possessed both class II and class III antiarrhythmic effects , 3.From January 2011 till January 2013, a series of 180 hyperthyroidism patients (diagnosed as Graves' disease), who came to either Nuclear Medicine Department or Endocrinology Department, were consecutively enrolled in this prospective study. All of the patients had symptomatic PAF. There were 98 males (55.48 ± 12.02 years old) and 82 females (56.12 ± 9.98 years old). Entry criteria included PAF due to hyperthyroidism; electrocardiographic evidence of atrial fibrillation; symptoms such as palpitations, light headedness, chest pain, and dyspnoea in association with PAF; good compliance. Exclusion criteria were PAF due to other reasons, recent myocardial infarction, heart failure, inflammation such as pneumonia and diarrhea, unstable hepatic or renal function, poor compliance, and other major medical problems that would leave the patient with a life expectancy of less than two years. All enrolled patients gave their informed consent. This study was approved by the Institutional Review Board of Tianjin Medical University General Hospital .The diagnosis of PAF was made according to the American College of Cardiology Foundation/American Heart Association Task Force guideline definition; briefly, PAF had episodes that were generally less than 7 days (most less than 24 h), yet it was usually recurrent , 2.This study was designed as a prospective, open label, and randomized investigation. Generally, patients eligible for the study were allocated to one of the treatments using a computer generated random number algorithm. As reported , the cliInitially, baseline demographic data were obtained from the subjects. Relevant symptoms, cardiac diagnoses, and medical history were noted. Physical examination, 24-hour ambulatory electrocardiograph and/or regular 12-lead electrocardiograph, and serum biochemical tests  were carried out. All electrocardiographic recordings were reviewed by at least two experienced observers.131I was also given to 90 patients . Due to the similar ethical reason, no control group was set. ATD-treated patients were given methimazole . 131I therapeutic procedure was performed according to our protocol [131I (about 74 kBq) by a nuclear multifunctional instrument . Then 131I effective half-life time (T1/2eff) and maximum uptake in thyroid were calculated. Thyroid ultrasonography was performed by using a color doppler ultrasound machine . Thyroid volume was calculated with the following formula: volume (cm3) = (width × length × thickness of left lobe) + (width × length × thickness of right lobe). Thyroid weight (g) = 0.479 × volume (cm3). Serum thyroid hormones were tested by an immunofluorometric assay, including free triiodothyronine , free thyroxine , and thyroid stimulating hormone . The therapeutic dose of 131I was calculated as the following formula [T1/2eff (days) × maximum uptake (%)). Absorption dose = 100 Gy/g thyroid tissue; 0.67 is a rectified factor. Participants visited our outpatient department every month. At each scheduled follow-up visit, physical examination and routine laboratory tests were done. And, at the end of the third month, ambulatory electrocardiograph and/or regular 12-lead electrocardiograph were repeated; all relevant symptoms were documented. Disappearing of PAF was defined as restoration of SR.In the first part of the study, we randomly prescribed WK (18 g tid) or sotalol (80 mg bid) to 91 patients  or 89 patients , respectively. This part of the study compared the effectiveness of WK and sotalol to restore SR from PAF. In this investigation, it was not ethical not to give the patients any antiarrhythmia drugs. So, we did not design control; we just compared WK and sotalol. Antithyroid drugs (ATD) were given to 90 patients , and protocol , 20. Thy formula , 20: dosIn the second part of the study, we randomly assigned the successfully SR-reverted patients into one of the following three groups: 54 cases were given WK (9 g tid), 54 cases were given sotalol (40 mg bid), and 55 cases served as control. In this part of the study, the control patients did not take any antiarrhythmia drug. Since patients recruited at this stage had much better improved thyroid status, and all of them were in SR when entering this investigation, it was ethically approved by our Institutional Review Board not to give the control patients any antiarrhythmia drugs. If patients were still in hyperthyroidism status, appropriate dose of methimazole was given to maintain euthyroidism. If the patients were in posttherapeutic hypothyroidism status, appropriate dose of levothyroxine was given to maintain euthyroidism. For hypothyroid patients who had already restored SR, WK and sotalol were stopped. Participants were asked to visit our outpatient department every three months. At each scheduled or sometimes unscheduled follow-up visit, physical examination and routine laboratory tests were repeated. And, at the end of the twelfth month, ambulatory electrocardiograph and/or regular 12-lead electrocardiograph were done; all relevant symptoms were documented. Time-point of PAF recurrence, its frequency, and related symptoms were collected as well.Participant flow chart was presented in t-test. Differences between multiple groups were analyzed by one-way analysis of variance (ANOVA), and then least significant difference (LSD) test was used for multiple comparisons among the groups.  χ2 test was adopted to determine case number changes of patients after different treatments.  χ2 test was also used to check whether sex had a significant influence on the intergroup differences. Kaplan-Meier analysis by Log-Rank χ2 test was used to estimate the cumulative recurrent rate of PAF in different groups. P value not exceeding 0.05 was considered statistically significant.All data were presented as mean ± SD. Statistics were performed with SPSS 17.0 . Differences between two groups were analyzed by independent samples χ2 test showed no significant differences, indicating equal efficacies of the two drugs for assisting SR reversion . Therefore, we proved that both WK and sotalol could successfully maintain SR.Data in the second investigation were analyzed by two methods. First, at the end of twelve-month follow-up, recurrent PAF happened in 1/54 (1.852%), 2/54 (3.704%), and 9/55 (16.364%) cases in WK, sotalol, or control groups, respectively. We found no differences of thyroid hormones at any follow-up time-point among the groups . Howeverl groups . Second,l groups . Log-RanSince there is always an inherent bitter taste in Chinese medicine, some patients would unavoidably complain about the gastrointestinal discomfort or related symptoms after taking WK. Altogether, there were 10/91 cases (10.989%) in the first investigation and 6/54 (11.111%) in the second investigation who reported various degrees of nausea and dizziness after taking WK. However, all patients showed endurance and continued with the medication. For sotalol groups, the gastrointestinal discomfort was far less frequent; there were only 3/89 cases (3.371%) in the first investigation and 2/54 (3.704%) in the second investigation who reported mild stomach discomfort. However, after taking sotalol, 2/89 cases (2.247%) in the first investigation developed symptomatic bradycardia, whose PAF disappeared though. The problems completely dissolved after dose reduction from 80 mg bid to 40 mg bid for one patient and from 80 mg bid to 40 mg qd for the other patient. These two patients' heart rhythm maintained SR during the rest of the study. WK showed no bradycardia side effect. No other unwanted incidences were recorded.131I or ATD is associated with a reversion to SR in a majority of patients [β-blockers can provide rapid symptom relief by reducing the ventricular rate, but these agents are unlikely to convert PAF to SR. Pharmacotherapy of atrial fibrillation has an advantage over electrical cardioversion and the catheter ablation methods, because it can be used on an outpatient basis [The risk of developing atrial fibrillation in patients with hyperthyroidism is approximately 6-fold of the euthyroidism population, which aggravates the overall conditions of such patients . Successpatients , 21, 22.patients . Selectint basis . HoweverWK is identified as a novel drug against atrial fibrillation. Its mechanism has been elucidated recently. Burashnikov and colleagues  have impContrary to the relatively new discovery of the MK mechanisms, traditional Chinese medicines were first documented about 2500 years ago by Confucian scholars and are now still being used by tens of millions in China as well as around the world , 24, 25.131I was much more effective for hyperthyroidism management and thereafter to gain better SR reversion. We believed that this was largely due to better therapeutic results of 131I to control thyroid hormones (Tables In the current investigation, we provided the first clinical evidence of WK as well as sotalol on the management of hyperthyroidism-induced PAF in two aspects. First, the drugs could assist SR reversion from PAF caused by hyperthyroidism. Second, the drugs could maintain SR afterwards. The second application seemed more important, since the first application was very dependent on the degree of thyroid hormone reduction. We showed that there were nearly the same efficacies of both WK and sotalol to assist SR restoration. However, s Tables  and 5. Is Tables . Our stuAlthough WK's effect on hyperthyroidism-related atrial fibrillation has never been reported before, WK's anti-atrial-fibrillation ability is not new discovery. All of WK's clinical studies are published in Chinese language so far; however, after considering their relevancy to the current study, further comments are deserved. Chen and colleagues  compiled131I was better to control thyroid hormone and to gain SR reversion. We also showed that both WK and sotalol could maintain SR with equal abilities in those PAF hyperthyroidism patients who had already gained SR after treatments. Therefore, WK is a useful drug that should be advocated in the initial treatment of PAF caused by hyperthyroidism, as well as in the follow-up management strategy.In conclusion, we demonstrated the same efficacies of WK and sotalol to assist SR reversion from hyperthyroidism-related PAF.


Asthma and COPD (chronic obstructive pulmonary disease) lead to persistent airway inflammation and are associated with lung cancer. The objective of the study was to assess the relationship between inhaled (ICS) and oral corticosteroid (OCS) use, and risk of lung squamous cell carcinoma (SqCC).This study was a nested case–control study. Patients with newly diagnosed asthma or COPD between 2003 and 2010 were identified from the National Health Insurance Database. Cases were defined as patients diagnosed with SqCC after enrollment. For each case, four control individuals who were randomly matched for sex and age and date diagnosis of asthma or COPD were selected.From the 1,672,455 eligible participants, 793 patients with SqCC were matched with 3,172 controls. The odds ratios (ORs) of SqCC in men who received high and low-dose ICS were 2.18  and 1.77 (1.22–2.57), respectively. Similarly, the ORs were 1.46  and 1.55  for men who were placed on low and high dose OCS. However, there was no significant association between cumulative ICS and/or OCS and risk of SqCC in women. Recent dose increase in corticosteriod was significantly associated with risk of SqCC. Specifically, among men, the ORs for SqCC were 8.08  for high-dose ICS + OCS, 4.49  for high-dose ICS, and 3.54  for high-dose OCS treatments, respectively. The OR for SqCC in women who received high-dose OCS was 6.72 .Corticosteroid use did not decrease SqCC in patients with asthma or COPD. Recent dose increase in corticosteroids was associated with SqCC. Squamous cell carcinoma (SqCC) accounts for approximately 20 % of all lung cancers in the United States . It is tOral (OCS) and inhaled corticosteroid (ICS) have reduced local and systemic inflammation among patients with asthma or COPD , 12. HowData used in this study were obtained from 2003 to 2010 using the National Health Insurance Research Database (NHIRD). Taiwan’s National Health Insurance covers more than 99 % of the 23 million residents and contains enrollment files, claims data, catastrophic illness files, and registry for treatments. The database is one of the largest datasets described in most epidemiological studies –15. ThisA nested case–control study was conducted to overcome the time-varying nature of corticosteroid treatment. Analytic data included subjects aged ≥20 years with newly diagnosed asthma or COPD from 2003 to 2010. The date of the first diagnosis of asthma or COPD was defined as the initiation date. Excluded were individuals with incomplete information on sex and registry data. Also excluded were individuals diagnosed with lung cancer before 2002, initiation date, or 2 years after the initiation date.The study began in 2003. Cases with newly diagnosed lung cancer were followed until death, loss to follow-up, or the study end in 2010. Lung cancer was identified using the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code 162. The index date was defined as the date of first assignment of the above ICD codes.The TCRD was used to confirm cell types of lung cancer. All major cancer care hospitals in Taiwan are obligated to submit cancer type, initial tumor stages and histology to Taiwan Cancer Registry funded by the Ministry of Health and Welfare . Lung caFor each case, up to four control individuals, who were randomly matched for sex, age diagnosed with asthma or COPD, and initiation date were selected without replication from individuals without lung cancer.The NDRD, NHIRD, and TCRD were used to assess the age at cancer onset, person-year follow-up, death and survival time, and potentially unconfirmed cases diagnosed with cancer.We identified patients who were prescribed OCS and ICS between the initiation and index date. Data were collected on prescription dates, prescribed daily dose, and the number of days supplied. In accordance with the Anatomic Therapeutic Chemical classification of ICS drugs, beclomethasone, budesonide, fluticasone, and ciclesonide were selected as the major drugs of interest, whether dispensed alone or in combination with an inhaled β2 agonist. The defined daily dose (DDD) recommended by the World Health Organization is a unit for measuring a prescribed amount of drug to standardize the comparison of drug usage between different drugs . All ICSAll OCS prescriptions were converted to hydrocortisone equivalents (4 mg of hydrocortisone = 1 mg of prednisolone = 5 mg of cortisone = 0.8 mg methylprednisolone = 0.8 mg of triamcinolone = 0.4 mg of paramethasone = 0.15 mg of betamethasone = 0.15 mg of dexamethasone) .An average quarter dose was calculated by dividing the total number of milligrams of OCS or cDDDs of ICS by the number of quarter prescribed during the assessment period.The diagnoses of pulmonary diseases and comorbidities were confirmed by either 2 outpatient visits or one hospitalization in one year. Baseline pulmonary diseases and other comorbidities are listed as follows: asthma (ICD-9-CM: 493), COPD , pulmonary tuberculosis , pneumonia (ICD-9-CM: 486), chronic renal disease (ICD-9-CM: 585 and 586), hyperlipidemia (ICD-9-CM: 272), and smoking-related cancers . Type 2 DM  is characterized by hyperinsulinemia in the context of insulin resistance and an increased level of circulating insulin-like growth factor 1 which are associated with an increased risk of cancer . IndividP value of less than 0.05 was considered statistical significance.Data analyses were made using SAS 9.3 software . Chi-square and t tests were used to compare baseline sociodemographic characteristics and comorbidities between cases and controls. Conditional logistic regression was used to assess the association between corticosteroid use and SqCC. Low or high-dose corticosteroid was defined by the median of corticosteroid dose per quarter. Adjusted odd ratios (ORs) were presented with 95 % confidence intervals (CIs) and a n = 559,589), incomplete data on sex  and registration , type 1 DM (n = 123), diagnosed with lung cancer before 2002 or initiation date, and 2 years after the initiation date . The final enrolment included 1,672,455 individuals. A total of 4032 patients were identified with lung cancer, 793 of whom were patients with SqCC.We identified 2,384,046 individuals with first-time diagnosis of asthma or COPD from 2003 to 2010. The total number of individuals excluded were as follows; <20 years of age (The baseline characteristics of patients with SqCC (793) and their controls 3172) are summarized in Table 72 are suCompared with non-ICS users (Model 1), the ORs for SqCC in low and high-dose ICS were 2.09  and 1.88 , respectively (Table P = 0.234). The interactions between sex and corticosteroids were also not significant (P = 0.764).In Table Over the past decade, some studies have documented a possible link between chronic inflammatory lung diseases and lung cancer , 6. CortOur results demonstrated that patients with SqCC had significantly higher pneumonia, pulmonary tuberculosis, and smoking-related cancers, but lower hyperlipidemia. Because national databases do not contain detailed information regarding smoking history, occupational exposures, and other risk factors of lung cancer, other diseases (excluding COPD and asthma) may affect the risk of lung SqCC. In a cohort study with 17,859,318 Taiwanese residents, Jian et al. evaluated gender disparities in pulmonary diseases, comorbidities, and effects on SqCC . These rWith effective treatment and control of allergens and irritants, majority of patients with asthma or COPD have a controlled disease though some patients can still be exposed to frequent exacerbations , 23. ICSFew studies on the relationship between corticosteroids and lung cancer have yielded conflicting results. Gundisch et al. found that glucocorticoids promoted tumor cell proliferation in a pre-clinical mouse model of lung carcinoma . BudesonHowever, none of these trials has evaluated the relationship between corticosteroids and specific types of lung cancer. Asthma and COPD have been closely associated with SqCC . In thisOur study results indicated no association between cumulative dose of corticosteroids and SqCC in women. In Taiwan, smoking is almost ten times more prevalent in men (45.7 %) than women (4.8 %) . This miThis study has several strengths. First, the sample size is large with a long follow-up. It was based on nationwide databases, hence selection bias was possibly minimized. Second, to enhance the reliability of temporal relationship between corticosteroid use and SqCC, we excluded individuals who were diagnosed with lung cancer before 2002 and initiation date, or 2 years after initiation date. Nevertheless, this study had some limitations. First, corticosteroid exposure was assessed solely by refills recorded in the NHIRD, not by whether the subjects actually used their medication. Second, NHIRD, NDRD, and TCRD do not contain detailed information regarding smoking history, radon exposure, occupational exposures, diet preference, and family history, all of which may be risk factors for lung cancer.The use of corticosteroids in patients with asthma and COPD was associated with lung SqCC, especially in men. Recent dose-increase in corticosteroids was associated with SqCC. Lung cancer screening is necessary when treatment goals for asthma or COPD are not being met or when patients are not responding to current therapy.


Castleman’s disease (CD) is a rare lymphoproliferative disorder with clinical features and prognostic factors that are incompletely characterized. This retrospective single-center study reviewed the largest HIV-negative CD patient cohort (n = 145) to date. By clinical classification, we identified 69 patients (47.6%) as unicentric CD (UCD) and 76 patients (52.4%) as multicentric CD (MCD). Pathological classification identified 74 patients (51.0%) with the hyaline-vascular variant, 51 patients (35.2%) with the plasma-cell variant, and 20 patients (13.8%) with a mixed variant. After a median follow-up duration of 58 months , the 1-year and 5-year survival rates were 95.1% and 91.0%, respectively. UCD patients exhibited significantly better survival  compared with MCD patients . By univariate and multivariate Cox regression analyses, the estimated glomerular filtration rate < 60 ml/min  was clinically significant and represented an independent predictor for death in MCD patients. In summary, this large-scale study suggests that UCD patients enjoy better survival than MCD patients and that renal function is an important prognostic factor for MCD patients. Castleman’s disease  is a rare lymphoproliferative disorder that was first described in 1956 by Benjamin Castleman and colleagues456735This retrospective single-center study screened 151 patients who were diagnosed as CD at Peking Union Medical College Hospital (PUMCH) from January 2000 to June 2015. Pathological specimens were retrieved from the PUMCH pathology database and reviewed by two experienced pathologists. The diagnosis of CD was corrected in three patients and confirmed in 148 patients. Because patients with an underlying systemic inflammatory disease or malignancies that might have a CD etiology of that differed from that observed in most CD patients5This study was performed in accordance with relevant guidelines and regulations and was approved by the PUMCH Ethics Committee. Informed consent was obtained from our patients.Patient follow-up was conducted via interviews at an outpatient clinic, telephone contacts, letters, and analyses of information documented in the PUMCH databases. Patients were followed until May 31, 2015 and the survival status for each patient was documented. Survival time was defined as the period from diagnosis to either death or the last follow-up.t-tests for continuous covariates; p < 0.05 was used as a threshold for statistically significant differences. Univariate Cox regression was used to estimate hazard ratios (HRs) and to test survival differences between groups. Variables with p < 0.10 by univariate analysis were further analyzed by multivariate Cox regression. All clinically significant parameters (p < 0.05) were considered to represent independent predictors of survival.Statistical analyses were performed with SPSS statistical software . The Kaplan–Meier method was used to depict survival curves and calculate survival rates at 1 and 5 years after the diagnosis of CD. The log-rank test was used to compare survival curves for UCD and MCD patients. Because MCD was considered to be a disease that was very distinct from UCD, both in its clinical course and underlying pathogenesis2Appendix 1 see  shows thThere were seven patients (4.8%) who received no treatment other than biopsy. All UCD patients (n = 69) and 11 MCD patients  received surgery. A total of 13 patients (9.0%) received steroids with or without intravenous immunoglobulin and immunosuppressive agents. Among 54 patients (37.2%) who received combination chemotherapy, 47 patients received CHOP  or CHOP-like chemotherapy (eight had Rituximab-CHOP), and seven patients were administered high-dose dexamethasone based therapy (see Appendix 1). Additionally, eight patients (5.5%) received autologous stem cell transplantation (ASCT), which was a recommended first-line treatment for young POEMS patients with normal organ functionThe median follow-up duration was 58 months (1–180 months), and one patient  was lost to follow up. In the remaining 144 patients, 15 (10.4%) had died, 13 had MCD, and two had UCD. The 1-year and 5-year survival rates for all CD patients were 95.1% and 91.0%, respectively . For UCDIn total, 76 MCD patients were eligible for further analysis. The clinical features of MCD patients are listed in Appendix 2 see , includiBased on univariate logistic regression , three rAfter incorporating variables with P < 0.10 in univariate analysis, multivariate Cox regression  revealedBefore 2011, knowledge about CD was mainly based on case reports475First, they enrolled patients since the 1940s or 1970s, respectively, which might introduce considerable error, especially for retrospective studies. Moreover, HIV is an important factor in the pathogenesis of CDSecond, a recent study from Chinaet al.et al.Third, HIV status is vital for CD studies as it is both related to the pathogenesis of CDUCD is a localized disease that is sometimes associated with systemic symptoms. In contrast, MCD is a systemic disease that is often associated with generalized peripheral lymphadenopathy, hepatosplenomegaly, fevers, and night sweats. Unlike UCD, MCD is strongly associated with immunosuppression  and human herpesvirus 8 (HHV-8) infection92The identification of prognostic factors for CD patients  is important for making clinical decisions. However, because of the low incidence of CD, few studies have investigated this issue. Indeed, most studies only used univariate analysis because of the limited number of patients457Notably, despite the fact that this current study is of clinical importance, it also carries considerable limitations. First, as a retrospective study, it has an implicit bias for data collection and interpretation. Second, although the patient HIV status was known in this study, the status of another important virus (HHV-8) was unknown. HHV-8 has been recognized to play an important role in the pathogenesis of MCDThis largest study carried out to date was based on recently available data. Our findings indicate that UCD patients enjoy better survival than MCD patients and that renal function is an important prognostic factor in MCD patients.How to cite this article: Zhang, L. et al. Clinical spectrum and survival analysis of 145 cases of HIV-negative Castleman’s disease: renal function is an important prognostic factor. Sci. Rep.6, 23831; doi: 10.1038/srep23831 (2016).


HPV persistence is a key determinant of cervical carcinogenesis. The influence of postpartum on HPV clearance has been debated. This study aimed to assess HPV clearance in later pregnancy and postpartum among HIV-positive and negative women.We conducted a follow-up study with 151 HPV-positive women coinfected with HIV, in 2007–2010. After baseline assessment, all women were retested for HPV infection using PCR in later pregnancy and after delivery. Multivariable logistic regressions assessed the putative association of covariates with HPV status in between each one of the successive visits.Seventy-one women (47%) have eliminated HPV between the baseline visit and their second or third visits. HIV-positive women took a significantly longer time (7.0 ± 3.8 months) to clear HPV, compared to those not infected by HIV (5.9 ± 3.0 months). HPV clearance was significantly more likely to take place after delivery than during pregnancy (84.5% x 15.5%).Both HIV-positive and negative women presented a significant reduction in HPV infection during the postpartum period. HIV-positive status was found to be associated with a longer period of time to clear HPV infection in pregnant women. Human papillomavirus (HPV) is highly prevalent in non-vaccinated sexually active women worldwide, even among those presenting normal cytology . Most HPHPV clearance is more frequent in the first six months after prime infection, with rates of 50-70% per follow-up year ,13. AdeqHIV-positive women have a higher risk of acquiring new HPV infections and a lower chance of clearing them compared to women not infected by HIV -21. MarkChanges in immunity and other biological parameters (e.g. changes in the levels of different hormones) during pregnancy and postpartum may modulate the natural history of HPV infection. Some recent studies did not observe differences in HPV status over time, during pregnancy -25. MostThe dynamics of HPV infection during pregnancy is not well established and information remains scarce and controversial. Few studies have studied HPV clearance and persistence during and after pregnancy, and just one of them has prospectively evaluated HPV infection in HIV-positive women. In this study, we aimed to evaluate HPV clearance in the postpartum period of both HIV-positive and negative women.A prospective follow-up study was carried out between January 2007 and January 2010, in Ribeirão Preto, São Paulo state, Brazil. Pregnant women were selected consecutively from the Prenatal Outpatient Clinic of the Infectious Diseases Unit, Obstetrics and Gynecology Department of the University Hospital, Medical School of Ribeirão Preto, University of São Paulo. This is a tertiary service, which follows-up with women previously diagnosed with HIV or HPV infection at primary units, selected from a large catchment population mainly composed of women from middle and low socioeconomic strata. Women were evaluated according to their HIV status and/or HPV-related lesion. HPV infection type was classified as clinical (in case of warts), subclinical (in case that colposcopic lesions were made evident), or HPV-positive (if only PCR-positive).Inclusion criteria were as follows: 1) be in the 1st half of pregnancy; 2) be positive for HPV, as defined by a PCR exam; 3) be an adult ; and 4) have read and signed the informed consent form.Cervicovaginal samples  were collected from all women and tested for HPV by Polymerase Chain Reaction (PCR). Women who tested positive for HPV were re-evaluated at two subsequent visits: at the 2nd half of pregnancy and after delivery.The study protocol was approved by the Ethics Review Board of the Ribeirao Preto’s Medical School Hospital – Sao Paulo University, and all women voluntarily signed informed consent before enrolment. All patients followed antenatal care routine of the service and no procedure was altered for those women enrolled in the study, except for the three PCR exams performed in each one of the visits .Cervicovaginal samples were stored at -80°C until DNA extraction, which was performed by QIAamp® DNA Mini Kit . Consensus primers GP5+ / GP6+ were used to identify HPV positivity, as previously described . After tSample size was calculated with the help of Graphpad StatMate® . Assuming a 15% differential clearance over time (from early pregnancy up to postpartum), it would be necessary to include at least 99 patients, considering an 80% power and an alpha of 5%.HPV clearance was defined as having at least one negative result after a confirmed positive one. A negative test detected in the 2nd visit (followed by a negative one in the 3rd visit) was defined as “clearance”, taking place between the 1st and the 2nd visit (i.e. clearance during pregnancy). Women with positive tests in the first and second visits, followed by a negative test in the 3rd visit, were defined as “clearance” occurring between the 2nd and the 3rd visits (i.e. clearance during the postpartum period).Persistent infection was defined for the sake of our study as “the consecutive detection of at least two positive HPV tests between the second and the third visit” . When a negative result appeared in between two positive tests (i.e. between the baseline and the third visit), the intermediate result was defined as a false negative, in agreement with previous studies and their respective criteria ,35. DuraStatistical analysis was carried out with the help of EpiInfo 3.5.3  and SAS 9.0 . Paired t-tests  and chi-square test (or Fisher Exact test for table cells with less than 5 events) were used for univariate analysis. Multiple log-binomial regression models were used, as the outcome was binary; procedure PROC GENMOD was applied for model adjustment. Multivariable analyses were controlled for participants’ age, smoking/non-smoking, mode of delivery, HIV status, nature of HPV infection , and HPV type (dichotomous variable: high vs. low risk).One hundred and fifty-one HPV-positive women were enrolled and followed-up with, of which 54 were HIV-positive and 97 were HIV-negative Figure . Mean geOverall genotype prevalences were as follows (in descending order of frequency): 37.1% (HPV 6 / 11), 23.8% (HPV 18), 13.2% (HPV 16), 4.0% (HPV 31), and 4.0% (HPV 33). None of these genotypes were identified in 43 samples (28.5%). The socio-demographic characteristics, as well as clinical and laboratory data did not significantly differ between these women and those form whom HPV infection was typed.3 and 44% of them present viral loads above 10,000 copies/mL.More than half of the HIV-positive women had their condition diagnosed less than a year ago (28/54). More than 60% of these women had CD4+ counts above 350 cells/mmOverall, clearance rate was 47% (71/151). The groups among whom HPV had persisted or cleared did not differ in median age , nor in gestational age at their 1st visit . HIV-positive and negative women did not significantly differ regarding HPV clearance rates ; but HIV-positive women took a significantly longer time (7.0 ± 3.8 months) to clear HPV, compared to those not infected by HIV (5.9 ± 3.0 months).Univariate analyses are summarised in Table Clearance rate was slightly higher in women with LR-HPV than those with HR-HPV infection, but such difference was not found to be statistically significant (20/39 (51.3%) vs. 22/56 (39.3%); p = 0.25).3 cleared HPV during follow-up (20/26) and only 4 women with less than 1,000 copies/mL had a persistent HPV infection over the follow-up period (4/11).Almost half of the women living with HIV infection cleared HPV infection during follow-up (26/54). Lower HIV viral load and higher CD4+ cell counts were found to be associated with HPV clearance. More than 75% of HIV-women with CD4+ count over 350 cells/mmIn multivariable analysis, non-smoker women had a two-fold higher chance to clear HPV  compared with smokers. Additional data are presented in Table HPV clearance was made evident between the 2nd and the 3rd (postpartum) visits in 84.5% of women who cleared HPV infection (60/71), compared to 15.5% during the 1st and 2nd visits  (11/71). Such difference was statistically significant for women, irrespectively of their HIV status, as well as when analyses were stratified by HIV status . More than 90% of our patients have used this therapy during pregnancy, which might influence our results. A recent study has demonstrated that highly active antiretroviral therapy may increase the clearance of some oncogenic HPV types . These fOurs is the first study to assess HPV clearance among HIV-positive women during pregnancy and postpartum. Minkoff et al.  compared3. A South-African study found that low CD4+ count was associated with a higher HPV infection incidence, but no association with clearance was made evident by it. Clearance was found to be exclusively associated with HIV viral load [In the HER Study (HIV Epidemiology Research), which included more than 500 HIV-positive women, higher CD4+ cell counts were found to be associated with a higher HPV clearance . Rowhaniral load . Furtherral load . In the We further analysed the database in order to assess a putative linear relationship between successive categories of CD4+ counts  and HPV clearance (data not shown). Unfortunately, we did not observe any gradient, either because it does not exist or because of our small sample size (beta error).The study assessed HPV types using a modest set of primers and HPV type-persistence was not assessed. Further studies, profiting from a comprehensive variety of HPV primers and a follow-up of HPV-types incidence/clearance over time is sorely needed in order to complement and validate our findings.In longitudinal studies, clearance rates and median infection duration might be underestimated due to left-censoring of infections present at the beginning of the study and right-censoring of infections that have not been cleared until the end of follow-up. The current study had a relatively short follow-up and intervals between visits, which were limited to pregnancy and post-partum. Such a short period of time  might compromise our ability to assess HPV evaluation. Women might have cleared their infection between visits, and it is not possible to specify the precise moment during which clearance may take place. The association between HPV and HIV coinfection might be underestimated in which case a longer follow-up period would be necessary to properly assess it over time.Chlamydia trachomatis. Therefore, some unmeasured confounding may have affected the adjusted risk ratios.Our findings should not be generalised to other groups of pregnant women, due to its small sample size, short follow-up time, and the fact that our study enrolled women from a tertiary referral care unit. Other sexually transmitted infections were not investigated such as HPV pathophysiology is especially complex due to the impossibility to fully differentiate persistent infections from those which might have occurred during the interval of two consecutive visits. Besides such caveat, a negative HPV test may not represent the full elimination of the virus, since latent infection may remain elusive from the perspective of both clinical and laboratory assessments. According to Schiffman et al. , HPV reaThe current study aimed to better assess some controversial aspects of HPV’s natural history. Postpartum period appears to be a period of significant decline of HPV infection, among both HIV-negative and positive women. The mode of delivery does not seem to influence this decline. A better understanding of HPV dynamics may help to identify women at higher risk of developing HPV-related lesions during antenatal care, contributing to prevention of cervical cancer, especially among HIV-positive women, among whom such cancers tend to be especially aggressive and harmful.HPV: Human papillomavirus; HIV: Human immunodeficiency virus; PCR: Polymerase chain reaction; LR-HPV: Low-risk HPV; HR-HPV: High-risk HPV.The authors declare that they have no competing interests.EMJ and SMQ conceived and designed the project, and analysed data. PPSM participated in data collection. GD and FIB provided guidance on this project. RTS and AYY carried out the PCR analysis. RAAM contributed to the data analysis and in the review process. All authors have been involved in drafting or revising the manuscript and have approved its final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/564/prepub


Functional MRI and neuropsychological assessments were carried out at baseline and after 3 months of supplementation. We analyzed resting state functional connectivity with the FSL fMRI analysis package. There were no differences in neuropsychological scores between the groups at baseline. After 3 months of supplementation, the carnosine/anserine group had better verbal episodic memory performance and decreased connectivity in the default mode network, the posterior cingulate cortex and the right fronto parietal network, as compared with the placebo group. Furthermore, there was a correlation between the extents of cognitive and neuroimaging changes. These results suggest that daily carnosine/anserine supplementation can impact cognitive function and that network connectivity changes are associated with its effects.Carnosine and anserine are strong antioxidants, previously demonstrated to reduce cognitive decline in animal studies. We aimed to investigate their cognitive and neurophysiological effects, using functional MRI, on humans. Thirty-one healthy participants  were recruited to a double-blind placebo-controlled study. Participants were assigned to twice-daily doses of imidazole dipeptide formula ( We live in a world with an aging population. The number of people aged 60 and over is projected to double as a proportion of the global population, from 7% in 2000 to 16% in 2050, taking the number of people aged 60 years and over to 2 billion Nations, .Aging is a risk factor for most common neurodegenerative disease, for example Alzheimer's disease  is acquired by performing fMRI on participants asked to lie quietly and think of nothing in particular and assesses brain activity in the absence of specific task demands  is an important resting state network, which typically shows high activity during rest and is deactivated during task performance  and Left Fronto-Parietal Network (LFPN) are two strongly lateralized maps and have broad involvement in many cognitive functions, including language and memory  were recruited from June 2012 to November 2013 from the Tokyo metropolitan area. Each participant gave written informed consent to participate in the study. The study was approved by the Ethics Committee of the University of Tokyo and by the Ethics Committee of the National Center of Neurology and Psychiatry. Exclusion criteria were:Those who had a pre-existing neuropsychiatric disorder or head injury.Having focal lesions, such as a brain tumor or cerebral infarction as detected on baseline MRI scan.Having contraindications to MRI.Subjects with Mini-mental state examination (MMSE) score below 24 , 500 mg in total (carnosine/anserine=1/3) derived from chicken meat , or an identical placebo (n = 17) in a double-blinded study. The groups were matched according to age and gender , Wechsler Memory Scale Logical Memory 1&2 (WMS-LM1&2), and Beck Depression Inventory (BDI).All used tests were translated into Japanese. The WMS-LM1&2 were used to assess logical and episodic memory for immediate recall and delayed recall respectively. The ADAS-J cog  is the Japanese version of cognitive subscale of ADAS, and was used to evaluate changes in cognitive function over time , located at the National Center of Neurology and Psychiatry Facility . All data were acquired using a 32-channel phased array head coil. MRI data of volunteers were collected at two time points: pre-supplementation (baseline) and the post-supplementation (3 month follow up). In the scanner, headgear and ear plugs were used to limit head motion and reduce scanner noise. rsfMRI scans were acquired using gradient-echo echo-planar sequence with repetition time TR = 3000 ms; echo time TE = 30 ms; flip angle = 80°; with 48 axial slices; slice thickness being 3.3 mm and no gap; each slice consisted of 64 × 64 voxels, resulting in 3.30 × 3.31 × 3.31 voxel dimension. Whole brain high resolution T1-weighted anatomical scan was acquired for registration purposes using magnetization prepared rapid gradient echo (MP-RAGE) sequence with following parameters: TR = 1900 ms, TE = 2.52 ms, TI = 900 ms, flip angle = 9°, field of view = 250 × 250 mm, acquisition matrix = 192 × 256, 256 sagittal slices, slice thickness = 1.0 mm, slice gap = 0 mm, axial slice number = 192, voxel dimension = 1.00 × 0.98 × 0.98. All subjects underwent 7 min (140 images) of scanning and were instructed to remain awake and think of nothing particular with their eyes open.Structural data preprocessing consisted of removal of non-brain tissue and celebro spinal fluid by using Statistical Parametric Mapping software package (SPM12).www.fmrib.ox.ac.uk/fsl). Registration to high resolution structural and/or standard space images was carried out using FLIRT  Version 6.00, part of FSL  and resampled to 4 × 4 × 4 mm resolution. Afterwards, data were temporally concatenated across all subjects to create a single 4-dimensional dataset and inputed to group-ICA analysis.The full workflow is presented in Figure We assessed head motion translation and rotation separately using the formula . Detailed information on motion is presented in Table Spike-like movements and other movement or physiological artifacts were removed using FSL-FIX software.Groups spatial ICA was conducted on all 31 subjects and both timepoints combined together to ensure that the resulting components had similar resting state fluctuations in all the participants at all timepoints of the study. Dimension estimation was done automatically, resulting in 33 spatial components. The components were identified by LL and JR according to Smith et al. .p < 0.05  = 0.005.Components of interest  were identified visually and additionally PCC and HC ROIs were constructed Figure . The HC http://www.r-project.org/). The voxel (4 × 4 × 4 mm) with the highest significance value on the family-wise error corrected comparison maps was identified (Table 4). These were used to extract a connectivity value from the spatial maps produced from the second stage of the dual regression procedure of each participant at both time points (baseline and 3 months). The change in connectivity within each person then was calculated as the difference between the connectivity values at the two different timepoints.Spearman's correlation was performed to investigate the correlation between functional connectivity changes as assessed by fMRI and cognitive score changes using packages hmisc (for full correlation) and ppcor  in R . There were no differences between the carnosine/anserine and placebo groups in BDI and ADAS-Cog score. The cognitive results are summarized in Table There were no significant differences between the carnosine/anserine and placebo groups at the baseline in WMS-LM2, BDI, and ADAS-Cog. After 3 months supplementation, the decline in verbal episodic memory score as measured with WMS-LM2 was significantly less in the carnosine/anserine group (−0.29±3.45) than in the placebo group (−3.06±3.86) (There was no any significant differences between the carnosine/anserine and placebo groups at the baseline Table .p < 0.05. If correction for multiple maps and contrasts is used (p = 0.005) only two masks survive significance threshold: PCC and RFPN.The group which had taken carnosine/anserine showed decreased connectivity for DMN, RFPN, and PCC masks in comparison with the placebo group at p < 0.05; Figure The DMN map showed decreased connectivity in the carnosine/anserine group, as compared to the placebo group with bilateral thalami, hippocampal areas, putamen and pallidium, precuneus cortex, right lateral occipital cortex and PCC .The PCC mask showed decreased connectivity with carnosine/anserine group in the areas similar to DMN Figure , but witp < 0.005).The carnosine/anserine group also showed decreased connectivity between the RFPN Figure  and bilaThere was no significant difference in hippocampal or LFPN connectivity when comparing the carnosine/anserine and placebo groups.All results are listed in Table x = −18, y = −34, z = −8) represented the area where the change in connectivity between the two groups was most significant . That is, a greater decrease in connectivity was associated with less decline in WMS-LM2 score , indicating that older age correlated with greater decline in WMS-LM2 score .The left posterior hippocampus , from the Ministry of Agriculture, Forestry, and Fishery (MAFF), the Government of Japan.Guarantors of integrity of entire study: TH; study concepts and study design: TH, HM; data acquisition: EI, TH, JK, HM; data analysis: JR, LL; literature research: LL, JR; statistical analysis: JR, LL; manuscript preparation: JR, LL; data visualization: JR; manuscript final version approval: all authors.NH foods Ltd., company provided us the supplements free of charge but provided no other financial support. 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.