Rational design of large flat nitrogen-doped graphene oxide quantum dots with green-luminescence suitable for biomedical applications.

Rational synthesis and simple methodology for the purification of large (35-45 nm in lateral size) and flat (1.0-1.5 nm of height) nitrogen-doped graphene oxide quantum dots (GOQDs) are presented. The methodology allows robust metal-free and acid-free preparation of N-GOQDs with a yield of about 100% and includes hydrothermal treatment of graphene oxide with hydrogen peroxide and ammonia. It was demonstrated that macroscopic impurities can be separated from N-GOQD suspension by their coagulation with 0.9% NaCl solution. Redispersible in water and saline solutions, particles of N-GOQDs were characterized using tip-enhanced Raman spectroscopy (TERS), photoluminescent, XPS, and UV-VIS spectroscopies. The size and morphology of N-GOQDs were studied by dynamic light scattering, AFM, SEM, and TEM. The procedure proposed allows nitrogen-doped GOQDs to be obtained, having 60-51% of carbon, 34-45% of oxygen, and up to 7.2% of nitrogen. The N-GOQD particles obtained in two hours of synthesis contain only pyrrolic defects of the graphene core. The fraction of pyridine moieties grows with the time of synthesis, while the fraction of quaternary nitrogen declines. Application of TERS allows demonstration that the N-GOQDs consist of a graphene core with an average crystallite size of 9 nm and an average distance between nearest defects smaller than 3 nm. The cytotoxicity tests reveal high viability of the monkey epithelial kidney cells Vero in the presence of N-GOQDs in a concentration below 60 mg L-1. The N-GOQDs demonstrate green luminescence with an emission maximum at 505 nm and sedimentation stability in the cell culture medium.

Low lineage diversity and increased virulence of group C Streptococcus dysgalactiae subsp. equisimilis.

Introduction. In some species, the population structure of pathogenic bacteria is clonal. However, the mechanisms that determine the predominance and persistence of specific bacterial lineages of group C Streptococcus remain poorly understood. In Brazil, a previous study revealed the predominance of two main lineages of Streptococcus dysgalactiae subsp. equisimilis (SDSE).Aim. The aim of this study was to assess the virulence and fitness advantages that might explain the predominance of these SDSE lineages for a long period of time.Methodology. emm typing was determined by DNA sequencing. Adhesion and invasion tests were performed using human bronchial epithelial cells (16HBE14o-). Biofilm formation was tested on glass surfaces and the presence of virulence genes was assessed by PCR. Additionally, virulence was studied using Caenorhabditis elegans models and competitive fitness was analysed in murine models.Results. The predominant lineages A and B were mostly typed as emm stC839 and stC6979, respectively. Notably, these lineages exhibited a superior ability to adhere and invade airway cells. Furthermore, the dominant lineages were more prone to induce aversive olfactory learning and more likely to kill C. elegans. In the competitive fitness assays, they also showed increased adaptability. Consistent with the increased virulence observed in the ex vivo and in vivo models, the predominant lineages A and B showed a higher number of virulence-associated genes and a superior ability to accumulate biofilm.Conclusion. These results suggest strongly that this predominance did not occur randomly but rather was due to adaptive mechanisms that culminated in increased colonization and other bacterial properties that might confer increased bacteria-host adaptability to cause disease.

Liquid biopsy of HPV DNA in cervical cancer.

BACKGROUND: A blood test to serve as a tumor marker for cervical cancer would be useful to clinicians to guide treatment and provide an early signal for recurrence. The development of droplet digital PCR has enabled the detection of HPV DNA in patient serum, providing a potential marker for cervical cancer. OBJECTIVES: To report on a blood-based test for HPV-specific E7 and L1 genes, which may serve as a tumor marker to guide treatment and detect early recurrence in cervical cancer. STUDY DESIGN: Pre-treatment plasma samples were investigated from 138 Hong Kong Chinese women with primary invasive squamous cell carcinoma and adenocarcinoma of the cervix with tumor samples expressing HPV16 or HPV18. Two genes specific to the human papillomavirus, E7 and L1, were measured in cell free DNA (cfDNA) extracted from plasma using droplet digital PCR. Analysis of detectable E7 and L1 levels was performed to investigate the potential of liquid biopsy of E7 and L1 as a clinically useful molecular biomarker. RESULTS: The majority of patients had HPV16 (71.7%), squamous cell carcinoma (78.3%) and stage IB-II disease (82.6%). HPV E7 and L1 sequences were detected in plasma cfDNA from 61.6% (85/138) of patients. Patients with high viral load (defined as ≥20 E7 or L1 copies per 20 µL reaction volume) had increased risk of recurrence and death at 5 years on univariate analysis but not multivariate analysis. CONCLUSIONS: HPV DNA can be quantitatively detected with the use of cfDNA. This has the potential to provide a clinically useful tumor marker for patients with cervical cancer that can aid in post-treatment surveillance and estimating the risk of disease relapse.

Stability of different influenza subtypes: How can high hydrostatic pressure be a useful tool for vaccine development?

BACKGROUND: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. METHODS: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. RESULTS: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. CONCLUSIONS: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice.

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.

To Study Endothelial Dysfunction in Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with an increased morbidity and mortality. This excess mortality attributable to cardiovascular events. Endothelial dysfunction represents the earliest stage of atherosclerosis. It can be measured noninvasively by peripheral tests of function, such as pulse wave analysis. OBJECTIVES: To evaluate the influence of chronic inflammatory state on endothelial function in patients with RA free of cardiovascular disease or risk factors by measuring endothelial reactivity. METHOD: A total number of 62 patients of RA and 18 normal healthy controls participated in the study. The pulse wave velocity (PWV), reflection index (RI) and augmentation or stiffness Index (SI) were measured at baseline and vasodilatory response was measured. Waveform analyzer and Micromedical Pulse Trace Analyser were used. RESULTS: Heart brachial PWV and brachial ankle PWV were not statistically significant in healthy and RA patient groups. RI was higher in RA patients than controls. SI in RA patient group (7.94 ± 1.20) was statistically significant (p < .0001) as compared to healthy controls (6.75 ± 0.65). RA patients with low SI had active disease indicated by DAS28 (5.03 ± 1.20) increased ESR and CRP levels as compared to the controls. CONCLUSIONS: RA with high disease activity, free from cardiovascular risk factors and overt cardiovascular disease had premature aging (increased vascular stiffness). Inflammatory process associated with RA was responsible for findings. It is suggested that the increased arterial stiffness contributes to the observed increased cardiovascular mortality and morbidity in subjects with RA.

Full inactivation of human influenza virus by high hydrostatic pressure preserves virus structure and membrane fusion while conferring protection to mice against infection.

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.

Detection of avian paramyxoviruses in migratory and resident birds in the state of Rio de Janeiro, Brazil.

Paramyxoviruses and avian influenza viruses are present worldwide, and wild birds are known natural reservoirs of these viruses. This study monitored the circulation of these viruses in migratory and resident coastal birds captured in the state of Rio de Janeiro, Brazil. In total, 494 birds were trapped, and their fecal samples were collected and inoculated into embryonated chicken eggs. The allantoic fluids were evaluated using a hemagglutination test and PCR amplification of the genes of the M and L proteins of influenza A virus and paramyxovirus, respectively. Avian paramyxovirus was detected in 5 (1.01%) of the birds. The majority of these viruses were isolated from migratory birds classified into the order Charadriiformes (families Scolopacidae and Charadriidae). Four samples were characterized as avian paramyxovirus serotype-2 (APMV-2) by a hemagglutination inhibition test. These results reinforce the importance of continuous surveillance of wild species in Brazil.

Flavonoids in the therapy and prophylaxis of flu: a patent review.

INTRODUCTION: Influenza viruses are common agents of flu outbreaks, epidemics, and pandemics that have occurred through the centuries. Prevention and control of flu are of great clinical importance, since they cause serious damage to health, with a consequent impact on quality of life and economy of a country. Resistance against the current drugs justifies the development of new anti-influenza molecules. Flavonoids exhibit significant activity against flu through their anti-inflammatory and antiviral properties. The profile of these molecules makes them particularly promising as therapeutic agents against flu. AREAS COVERED: This review focus on the activity of flavonoids on different influenza virus targets as well as their use in patented pharmaceutical formulations. Twenty-one patents of these compounds for prophylaxis and treatment of influenza infection are discussed. EXPERT OPINION: The H1N1 influenza pandemic in 2009 resulted in a significant increase in the number of patents claiming pharmaceutical formulations for prophylaxis and treatment of flu. The research advances on flavonoids showing anti-influenza activity and the efforts made by researchers and industries consolidate the interest on new alternatives for the therapy of an infectious disease that represents a serious public health problem throughout the world.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast.

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.

CHD5 Downregulation Associated with Poor Prognosis in Epithelial Ovarian Cancer.

BACKGROUND: The CHD5 gene located on 1p36 encodes a protein-chromodomain helicase DNA-binding protein 5. CHD5 has been shown to be a tumor suppressor gene candidate. This study investigated the involvement of CHD5 in ovarian cancer and its clinicopathological significance. METHODS: CHD5 expression in ovarian cancer and its counterpart were determined by quantitative RT-PCR. The correlation of CHD5 expression to clinicopathological features of the tumor was analyzed. RESULTS: CHD5 expression was downregulated by at least twofold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 controls in Hong Kong Chinese women. CHD5 downregulation was correlated to clinical status (p < 0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (p > 0.05). Survival analysis showed that patients with CHD5 downregulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 downregulation (p < 0.05). Cox proportional-hazards regression analysis indicated that downregulation of CHD5 is an independent adverse prognostic factor in ovarian cancer. CONCLUSION: This study shows that CHD5 is downregulated in a certain number of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival.

Clinical use of laparoscopic ultrasonography in detecting nodal metastasis in advanced-stage cervical carcinoma.

OBJECTIVE: To assess the clinical use of a laparoscopic ultrasound scan (LUS) to identify pelvic and para-aortic node metastasis in patients with advanced-stage cervical cancer. METHODS: After examination under general anesthesia and cystoscopy, LUS was used to examine the pelvic nodes of patients with advanced-stage cervical cancer. Abnormal nodes were excised before definitive treatment to confirm the nodal status. Patients without abnormal para-aortic nodes on preoperative computer tomography/magnetic resonance imaging in the past 3 years were surgically staged via laparoscopic extraperitoneal aortic node sampling, and the findings were correlated with LUS findings. The predictive values of abnormal pelvic nodes on LUS for pelvic and aortic node metastasis were determined. RESULTS: A total of 119 advanced-stage cervical cancer patients underwent LUS of pelvic nodes. Abnormal pelvic nodes were found in 62 (52.1%) patients, and metastasis was confirmed by histology in 38 (31.9%) patients. Three patients had micro-metastasis in para-aortic nodes, and all of these patients had abnormal pelvic lymph nodes on LUS. CONCLUSION: Abnormal pelvic nodes are commonly found on LUS in patients with advanced-stage cervical cancer, and selective excision biopsy is needed to confirm pelvic node metastasis. Surgical staging of aortic nodes might be considered for patients with abnormal pelvic nodes on LUS.

Do we need new surgical techniques to repair vesico-vaginal fistulas?

INTRODUCTION AND HYPOTHESIS: The urogenital fistula is a devastating condition for women. Despite advances in medical care, the vesicovaginal fistula continues to be a distressful problem. Complex vesicovaginal fistulae repair may need tissue interposition. It can be achieved by vaginal or abdominal approach and depends on the surgeon's experience and local factors like size, location, and previous radiotherapy. The aim of this study was to demonstrate that using traditional approaches is possible and reasonable to treat any sort of vesicovaginal fistula. METHODS: Between January 2004 and August 2007, we treated 23 patients with complex urogenital fistulae. Of those with concomitant ureteral fistula requiring re-implantation or bladder augmentation, the vaginal approach was the first choice in 17 and abdominal approach in six. Patients were clinically evaluated at 1, 4, and 12 weeks postoperatively, then every 3 months in the first year. RESULTS: Seventeen women were treated by vaginal approach and six patients were treated by abdominal approach. Hysterectomy was the major etiology (73.9%). Ten patients (43.5%) had at least one previous abdominal surgery for fistulae repair without success before. In those patients with abdominal approach, the hospitalization was longer than vaginal approach (80.5+/-6 h versus 48+/-3 h). In both, there were no major intraoperative or postoperative complications; 13% developed urgency and 4% developed stress urinary incontinence. No patients have recurrence of fistulae (success rate 100%). CONCLUSIONS: Complex vesicovaginal fistulas are a big challenge for the urologist, and there is no gold standard surgical approach. The majority of complex vaginal fistula can be successfully managed by vaginal repair. As the vaginal approach is a minimally invasive procedure with low costs, easy learning curve, and high cure rates, new approaches must be carefully evaluated before being suggested as an alternative.

Dysregulated microRNAs and their predicted targets associated with endometrioid endometrial adenocarcinoma in Hong Kong women.

The objective of this study, a parallel study to global gene expression profiling, was to identify dysregulated microRNAs (miRNAs) associated with endometrioid endometrial adenocarcinoma (EEC), examine their correlation with clinico-pathological characteristics and identify predicted target genes of the dysregulated miRNAs. Using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), profiling of miRNA expression was performed in 30 EECs and 22 normal counterparts in which genome-wide gene expression had been previously profiled and reported. Clustering analysis identified 30 miRNAs which were significantly dysregulated in EEC. The expression of a sub-group of miRNAs was significantly correlated with clinico-pathological characteristics including stage, myometrial invasion, recurrence and lymph node involvement. By searching for predicted miRNA targets that were linked to the dysregulated genes previously identified, 68 genes were predicted as candidate targets of these 30 dysregulated miRNAs. miR-205 was significantly overexpressed in EECs compared with normal controls. After transfection of a miR-205 inhibitor, the expression of miR-205 in endometrial cancer cell line RL95-2 cells decreased whereas its predicted target gene, JPH4, showed increased protein expression. JPH4 seems to be a real miR-205 target in vitro and in vivo, and a candidate tumor suppressor gene in EEC. Based on this study in EEC, miRNAs predicted to be involved in tumorigenesis and tumor progression have been identified and placed in the context of the transcriptome of EEC. This work provides a framework on which further research into novel diagnosis and treatment of EEC can be focused.

Risk association between human leukocyte antigen-A allele and high-risk human papillomavirus infection for cervical neoplasia in Chinese women.

To examine the association between human leukocyte antigen-A (HLA-A) allele polymorphism, human papillomavirus (HPV) infection, and risk for cervical neoplasia in Chinese women, 263 patients (155 with cervical intraepithelial neoplasia [CIN] II/III and 108 with invasive cervical cancer [ICC]) were compared with 572 controls. Overall, regardless of HPV status, a decreased risk for ICC was observed for patients with A*0207/0215N or A*2402, and an increased risk was observed for patients with A*1104. The protective association of A*0207/0215N was reproduced in HPV-16-positive patients with ICC, but not in subgroups infected with other HPV types. The risk association between A*1104 and both HPV-16 and HPV-18 was reproduced in the subgroups with CIN III/ICC. The protective association between A*2402 and HPV-16, HPV-18, HPV-52, and HPV-58 was consistently observed in all subgroups with CIN III/ICC, suggesting a linkage with a general antioncogenic genetic factor. The results of the present study indicate that HLA-A polymorphism is one of the host genetic factors that alter the risk for the development of cervical cancer in Chinese women.

Diclofenac-induced embryotoxicity is associated with increased embryonic 8-isoprostaglandin F2alpha level in rat whole embryo culture.

Diclofenac is embryotoxic in animals but the mechanism of its embryotoxicity is not fully understood. We postulated that diclofenac-induced embryotoxicity might be related to free oxygen radical damage. Rat embryos were explanted at 9.5 days gestation and cultured in medium containing various concentrations of diclofenac. The direct effect of diclofenac on embryonic free oxygen radical content was evaluated by measuring the 8-isoprostaglandin F2alpha level. We found that embryos exposed to high concentrations of diclofenac (7.5 and 15.0 micro g/ml) had significantly higher levels of 8-isoprostaglandin F2alpha (2.85 and 3.50 pg/mm embryonic crown-rump length, respectively) than the control group (1.73 pg/mm, P<0.05), while the 8-isoprostaglandin F2alpha level in embryos exposed to low concentration of diclofenac (1.5 micro g/ml) was 2.54 pg/mm, which was not significantly different from the control group. Median embryo morphologic score was also lower in the high concentration group compared to the control group (39.0 versus 44.0, P<0.05). These results suggest that diclofenac-induced embryopathy may be related to free oxygen radical damage.

Changes in soft tissue profile of African-Americans following extraction treatment.

The purpose of this investigation was to determine changes in soft tissue profile of African-Americans following orthodontic treatment involving extraction of four premolars. The sample consisted of pretreatment and postreatment lateral cephalometric radiographs of 30 males and 30 females of African-American descent exhibiting bimaxillary protrusion. The age of the patients ranged between 10 years 4 months and 17 years 6 months at the start of treatment. Average time between pretreatment and postreatment radiographs was 2 years 11 months in the male group and 3 years 3 months in the female group. Changes in the dentofacial complex and facial soft tissue as a result of treatment and growth were evaluated with cephalometric analysis. Student's t-tests were performed to compare differences. Nasolabial angle increased 9.1 degrees in males and 7.1 degrees in females. Upper lip procumbency relative to SnPg' decreased 1.5 mm in males and 1.7 mm in females. Lower lip retraction relative to SnPg' was 2.7 mm in males and 2.5 mm in females.

The Broadbent-Bolton Growth Study--past, present, and future.

A computerized database of 14 textual elements has been created to describe the 4309 subjects enrolled in the Broadbent-Bolton Growth Study. The database can be searched by using Boolean operators to select subsets of subjects matched on the 14 data elements. The structure of the database, along with representative tables and charts of the data, is presented to aid researchers contemplating use of the collection for scientific investigation.