SENP1 induced protein deSUMO modification increased the chemotherapy sensitivity of endometrial cancer side population cells / 中华肿瘤杂志

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.

Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli / 生物工程学报

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.

Flavonoid glycosides and their putative human metabolites as potential inhibitors of the SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp)

BACKGROUND Since the World Health Organization (WHO) declared Coronavirus disease 2019 (COVID-19) to be a pandemic infection, important severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural proteins (nsp) have been analysed as promising targets in virtual screening approaches. Among these proteins, 3-chymotrypsin-like cysteine protease (3CLpro), also named main protease, and the RNA-dependent RNA polymerase (RdRp), have been identified as fundamental targets due to its importance in the viral replication stages. OBJECTIVES To investigate, in silico, two of the most abundant flavonoid glycosides from Dysphania ambrosioides; a medicinal plant found in many regions of the world, along with some of the putative derivatives of these flavonoid glycosides in the human organism as potential inhibitors of the SARS-CoV-2 3CLpro and RdRp. METHODS Using a molecular docking approach, the interactions and the binding affinity with SARS-CoV-2 3CLpro and RdRp were predicted for quercetin-3-O-rutinoside (rutin), kaempferol-3-O-rutinoside (nicotiflorin) and some of their glucuronide and sulfate derivatives. FINDINGS Docking analysis, based on the crystal structure of 3CLpro and RdRp, indicated rutin, nicotiflorin, and their glucuronide and sulfate derivatives as potential inhibitors for both proteins. Also, the importance of the hydrogen bond and π-based interactions was evidenced for the presumed active sites. MAIN CONCLUSIONS Overall, these results suggest that both flavonoid glycosides and their putative human metabolites can play a key role as inhibitors of the SARS-CoV-2 3CLpro and RdRp. Obviously, further researches, mainly in vitro and in vivo experiments, are necessary to certify the docking results reported here, as well as the adequate application of these substances. Furthermore, it is necessary to investigate the risks of D. ambrosioides as a phytomedicine for use against COVID-19.

Coagulation modifiers targeting SARS-CoV-2 main protease Mpro for COVID-19 treatment: an in silico approach

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on viral polyprotein processing, catalysed by the main proteinase (Mpro). The solution of the SARS-CoV-2 Mpro structure allowed the investigation of potential inhibitors. This work aims to provide first evidences of the applicability of commercially approved drugs to treat coronavirus disease-19 (COVID-19). We screened 4,334 compounds to found potential inhibitors of SARS-CoV-2 replication using an in silico approach. Our results evidenced the potential use of coagulation modifiers in COVID-19 treatment due to the structural similarity of SARS-CoV-2 Mpro and human coagulation factors thrombin and Factor Xa. Further in vitro and in vivo analysis are needed to corroborate these results.

Active constituents and mechanisms of Respiratory Detox Shot, a traditional Chinese medicine prescription, for COVID-19 control and prevention: Network-molecular docking-LC-MS analysis / 中西医结合学报

OBJECTIVE@#Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.@*METHODS@#To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.@*RESULTS@#For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.@*CONCLUSION@#RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.

Ribosomal Protein S9 Expression in Multiple Myeloma and Its Effect on Cell Biological Function / 中国医学科学院学报

Objective To identify the expression of ribosomal protein S9(RPS9)in multiple myeloma(MM)and explore its effect on the biological characteristics of myeloma cells and the corresponding mechanisms. Methods Bone marrow mononuclear cells were harvested in 10 healthy volunteers(CON group)and bone marrow CD138 +cells from 30 MM patients(CD138+group).Quantitative polymerase chain reaction(qPCR)was performed to detect RPS9 expression at mRNA level.In three cases from CON group and 11 cases from CD138+group,Western blot was performed to detect RPS9 at protein level.GSE19784 dataset was employed to detect the relationships of RPS9 expression with the overall survival rate,nuclear factor-κB(NF-κB),small ubiquitin-like modifier(SUMO),and ubiquitin pathway.After the RPS9 knock-down vector was constructed,flow cytometry was performed to detect the infection efficiency and qPCR and Western blot to detect the knock-down efficiency.RPMI8226 was divided into CON group and RPS9-short hairpin RNA(shRNA)group,in which annexin V allophycocyanin/propidium iodide(PI)double staining was performed to detect the change of apoptosis,CCK8 to detect the proliferation change,and PI staining to detect cell cycle change.After sentrin-specific protease 1(SENP1)overexpression vector was constructed,Western blot was performed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)from NF-κB pathway in CON,RPS9-shRNA,and RPS9-shRNA-SENP1 cells;in addition,annexin V/PI double staining was also performed to detect the apoptosis in these three cells. Results The relative expression of RPS9 in CON group and CD138+group was(1.00±0.12)and(5.45±0.71),respectively(t=4.291,P=0.0036).Western blot showed RPS9 expression was high in most myeloma CD138+cells.The high expression of RPS9 was associated with both extramedullary invasion and overall survival in GSE19784 dataset.After RPMI8226 was infected with CON or RPS9-shRNA lentivirus for 48 hours,flow cytometry confirmed that the infection efficiencies were above 90% in both groups.qPCR and Western blot confirmed that RPS9 expression was inhibited at both mRNA and protein levels.After RPMI8226 CON and RPS9-shRNA infected with virus for 48 hours,the proportion of annexin V-positive cells in CON and RPS9-shRNA cells was(3.47±0.37)% and(18.60±64.00)%(t=9.015,P=0.0008).The proliferation index significantly differed between CON group and RPS9-shRNA group at 72 hours(t=6.846,P=0.0024).When CON and RPS9-shRNA were infected with virus for 48 hours,the proportion of G2 phase cells was(29.28±3.42)% and(10.43±1.43)%,respectively(t=9.329,P=0.0007).The RPS9 expression was positively correlated with SENP1 in GSE19784 dataset and negatively correlated with IκBα coding gene NFKBIA.Western blot further confirmed that RPS9 knockdown inhibited the expression of SENP1,inhibited the phosphorylation of NF-κB subunit P65 and inhibitor IκBα,and promoted the expression of IκBα.Overexpression of SENP1 not only impeded this effect but also reduced RPS9-induced apoptosis. Conclusions RPS9 is highly expressed in MM CD138+cells and is associated with overall survival and extramedullary infiltration.Inhibition of RPS9 can promote apoptosis,cell cycle arrest,and proliferation of myeloma cells.RPS9 can affect the activation of NF-κB pathway and cell apoptosis through SENP1,suggesting that SENP1 may be a key factor in the biological effect of RPS9.

Experimental research on Arginine-gingipain A gene vaccine from Porphyromonas gingivalis that prevents peri-implantitis in Beagle dogs / 华西口腔医学杂志

OBJECTIVE@#This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development.@*METHODS@#Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants.@*RESULTS@#Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants.@*CONCLUSIONS@#IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.

Sodium valprovate suppresses autophagy in SH-SY5Y cells activating miR-34c-5p/ATG4B signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.@*METHODS@#Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.@*RESULTS@#VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.@*CONCLUSIONS@#VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

Successful application of virtual screening and molecular dynamics simulations against antimalarial molecular targets

The main challenge in the control of malaria has been the emergence of drug-resistant parasites. The presence of drug-resistant Plasmodium sp. has raised the need for new antimalarial drugs. Molecular modelling techniques have been used as tools to develop new drugs. In this study, we employed virtual screening of a pyrazol derivative (Tx001) against four malaria targets: plasmepsin-IV, plasmepsin-II, falcipain-II, and PfATP6. The receiver operating characteristic curves and area under the curve (AUC) were established for each molecular target. The AUC values obtained for plasmepsin-IV, plasmepsin-II, and falcipain-II were 0.64, 0.92, and 0.94, respectively. All docking simulations were carried out using AutoDock Vina software. The ligand Tx001 exhibited a better interaction with PfATP6 than with the reference compound (-12.2 versus -6.8 Kcal/mol). The Tx001-PfATP6 complex was submitted to molecular dynamics simulations in vacuum implemented on an NAMD program. The ligand Tx001 docked at the same binding site as thapsigargin, which is a natural inhibitor of PfATP6. Compound TX001 was evaluated in vitro with a P. falciparum strain (W2) and a human cell line (WI-26VA4). Tx001 was discovered to be active against P. falciparum (IC50 = 8.2 µM) and inactive against WI-26VA4 (IC50 > 200 µM). Further ligand optimisation cycles generated new prospects for docking and biological assays.

Association Analysis of Proteasome Subunits and Transporter Associated with Antigen Processing on Chinese Patients with Parkinson's Disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Proteasome subunits (PSMB) and transporter associated with antigen processing (TAP) loci are located in the human leukocyte antigen (HLA) Class II region play important roles in immune response and protein degradation in neurodegenerative diseases. This study aimed to explore the association between single nucleotide polymorphisms (SNPs) of PSMB and TAP and Parkinson's disease (PD).</p><p><b>METHODS</b>A case-control study was conducted by genotyping SNPs in PSMB8, PSMB9, TAP1, and TAP2 genes in the Chinese population. Subjects included 542 sporadic patients with PD and 674 healthy controls. Nine identified SNPs in PSMB8, PSMB9, TAP1, and TAP2 were genotyped through SNaPshot testing.</p><p><b>RESULTS</b>The stratified analysis of rs17587 was specially performed on gender. Data revealed that female patients carry a higher frequency of rs17587-G/G versus (A/A + G/A) compared with controls. But there was no significant difference with respect to the genotypic frequencies of the SNPs in PSMB8, TAP1, and TAP2 loci in PD patients.</p><p><b>CONCLUSION</b>Chinese females carrying the rs17587-G/G genotype in PSMB9 may increase a higher risk for PD, but no linkage was found between other SNPs in HLA Class II region and PD.</p>

SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

Resultados de dos encuestas dirigidas a inmigrantes y nativos del sureste español: salud, uso de servicios y necesidad de asistencia médica / Results of two surveys of immigrants and natives in Southeast Spain: health, use of services, and need for medical assistance

Objetivo. Comparar la salud, uso de servicios sanitarios y necesidad insatisfecha de atención médica (NIAM) entre inmigrantes y nativos del sureste español. Material y métodos. Estudio transversal de dos muestras representativas de población: inmigrante (n=1150) y nativa (n=1303; Encuesta Nacional de Salud). Se creó una única base de datos con ponderación específica para cada muestra y se estimaron razones de prevalencia (RP) mediante regresión multivariante. Resultados. Marroquíes, ecuatorianos y europeos del este (EE) declararon peor salud que los nativos (RPs [IC95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] y 1.44 [1.08-1.93], respectivamente). Los inmigrantes hicieron mayor uso de las urgencias (excepto EE) y consumieron menos fármacos. Los marroquíes mostraron la mayor diferencia en la frecuencia de NIAM (RP [IC95%]: 12.20 [5.25-28.37]), principalmente por razones laborales (46%). Conclusiones. La salud y el uso de servicios sanitarios difirieron significativamente entre inmigrantes y nativos. Destaca la NIAM alta en marroquíes por causa laboral.

Objective. To compare the self-perceived health, use of health services and unmet need for health care (UNHC) among immigrants and native populations of Southeast Spain. Materials and methods. Cross-sectional study of two representative samples of 1150 immigrants, and 1303 native participants from the National Health Survey. A single database was created with specific weights for each sample, and prevalence ratios (PR) were estimated by multivariate regression. Results. Moroccans, Ecuadorians and Eastern Europeans (EE) reported poorer health than the native population (PRs [CI95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] and 1.44 [1.08-1.93], respectively). Immigrants made greater use of emergencies that natives (except for EE) and had lower use of medication. Moroccan showed the greatest difference in the frequency of UNHC (PR [CI95%]:12.20 [5.25 - 28.37]), mainly because of working limitations (46%). Conclusions. The health status and use of health services among immigrants differ significantly from those of natives. Results highlight the higher frequency of UNHC among immigrants, especially high in Moroccans.

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Educação permanente em alimentação e nutrição na Estratégia Saúde da Família: encontros e desencontros em municípios brasileiros de grande porte / Ongoing food and nutrition education within the family health strategy: points of agreement and disagreement in major Brazilian cities

O objetivo foi conhecer fatores facilitadores e dificultadores e estratégias utilizadas por 28 municípios brasileiros de grande porte na realização de Educação Permanente em alimentação e nutrição, na Estratégia Saúde da Família. Método qualitativo de múltiplos casos. A técnica de coleta de dados foi a entrevista com coordenadores municipais das ações de alimentação e nutrição, das cinco regiões do país. O estudo foi realizado entre 2009 e 2010. Utilizou-se o software NVivo e a análise de conteúdo foi orientada por categorias temáticas. Foram entrevistados 44 profissionais, sendo 19 nutricionistas. A maioria dos municípios era do Nordeste e Sudeste, 14 eram capitais, 7 metrópoles e 14 tinham Núcleo de Apoio à Saúde da Família. Os fatores facilitadores para Educação Permanente em nutrição mais citados foram as parcerias e a disponibilidade de recursos. Os dificultadores mais frequentes foram a indisponibilidade de agendas e a falta de profissionais na gestão das ações de nutrição. As estratégias mais utilizadas foram a realização de ações educativas no nível local, por grupos e o planejamento e programação. Concluiu-se que são necessários maiores investimentos para que a Educação Permanente em alimentação e nutrição se concretize.

This study sought to ascertain the facilitating and inhibiting factors and strategies used by 28 major Brazilian cities in conducting ongoing food and nutrition education within the Family Health Strategy. It involved a qualitative study of multiple cases. The data collection technique was conducted in interviews with municipal coordinators of food and nutrition campaigns from the five regions of the country. The study was conducted between 2009 and 2010. NVivo 10 software was used and content analysis was divided up into thematic categories. Forty-four professionals were interviewed, 19 of which were nutritionists. Most cities were from the Northeast and Southeast; 14 were capitals, 7 were metropolises and 14 had Family Health Suppor Units. The most frequently mentioned facilitating factors for Ongoing Education in Nutrition were partnerships and the availability of funds. The most frequent inhibiting factors were difficulty in scheduling and a lack of professionals in management of nutrition actions. The strategies most commonly used were conducting training at the local level, in groups and planning and programming. The conclusion drawn is that more investment is needed for Ongoing Education in Feeding and Nutrition to be effectively implemented.

Diagnosis, classification and treatment of gestational trophoblastic neoplasia / Diagnóstico, classificação e tratamento da neoplasia trofoblástica gestacional

Gestational trophoblastic neoplasia (GTN) is the term to describe a set of malignant placental diseases, including invasive mole, choriocarcinoma, placental site trophoblastic tumor and epithelioid trophoblastic tumor. Both invasive mole and choriocarcinoma respond well to chemotherapy, and cure rates are greater than 90%. Since the advent of chemotherapy, low-risk GTN has been treated with a single agent, usually methotrexate or actinomycin D. Cases of high-risk GTN, however, should be treated with multiagent chemotherapy, and the regimen usually selected is EMA-CO, which combines etoposide, methotrexate, actinomycin D, cyclophosphamide and vincristine. This study reviews the literature about GTN to discuss current knowledge about its diagnosis and treatment.

Neoplasia trofoblástica gestacional (NTG) é o termo que descreve o conjunto de anomalias malignas da placenta, incluindo a mola invasora, coriocarcinoma, tumor trofoblástico do sítio placentário e tumor trofoblástico epitelióide. Ambos a mola invasora e o coriocarcinoma respondem bem à quimioterapia, com taxas de cura superiores a 90%. Desde o advento da quimioterapia, NTG de baixo risco tem sido tratada com monoquimioterapia, pelo geral methotrexate ou actinomicina-D. Casos de NTG de alto risco, contudo, devem ser tratados com poliquimioterapia, e o regime usualmente escolhido é o EMA-CO que combina etoposide, methotrexate, actinomicina-D, ciclofosfamida e vincristina. Esse estudo revê a literatura sobre NTG a fim de discutir os conhecimentos atuais sobre seu diagnóstico e tratamento.

Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway.</p><p><b>METHODS</b>The nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay.</p><p><b>RESULTS</b>The recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1.</p><p><b>CONCLUSION</b>We successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.</p>

Structural basis for complementary and alternative medicine: Phytochemical interaction with non-structural protein 2 protease-a reverse engineering strategy / 中国结合医学杂志

<p><b>OBJECTIVE</b>To understand the druggability of the bioactive compounds from traditional herbal formulations "Nilavembu Kudineer" and "Swasthya Raksha Amruta Peya" to heal chikungunya virus (CHIKV) infection.</p><p><b>METHODS</b>The efficiency of twenty novel chemical entities from "Nilavembu Kudineer" and "Swasthya Raksha Amruta Peya" to inhibit CHIKV infection in silico were evaluated. Ligands were prepared using Ligprep module of Schrödinger. Active site was identified using SiteMap program. Grid box was generated using receptor grid generation wizard. Molecular docking was carried out using Grid Based Ligand Docking with Energetics (GLIDE) program.</p><p><b>RESULTS</b>Molecular docking studies showed that among twenty compounds, andrographoside, deoxyandrographoside, neoandrographolide, 14-deoxy-11-oxoandrographolide, butoxone and oleanolic acid showed GLIDE extra precision (XP) score of -9.10, -8.72, -8.25, -7.38, -7.28 and -7.01, respectively which were greater than or comparable with chloroquine (reference compound) XP score (-7.08) and were found to interact with the key residues GLU 1043, LYS 1045, GLY 1176, LEU 1203, HIS 1222 and LYS 1239 which were characteristic functional unit crucial for replication of CHIKV.</p><p><b>CONCLUSION</b>The binding affinity and the binding mode of chemical entities taken from herbal formulations with non-structural protein 2 protease were understood and our study provided a novel strategy in the development and design of drugs for CHIKV infection.</p>

Effect of formaldehyde inhalation on allergic rhinitis in mice / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe the effect of formaldehyde inhalation on the allergic rhinitis mice model.@*METHOD@#Forty-eight male BALB/C mice in six experimental group were exposure to (A) saline control; (B) Der p1; (C) formaldehyde (3.0 mg/m3); (D) Derp1 + formaldehyde (1.5 mg/m3); (E) Der p1 + formaldehyde (3.0 mg/M3); (F) Der p1+ formaldehyde (6.0 mg/m3). The concentrations of IL-4, IL-10 and IFN-γ in the peripheral serum were measured by enzyme-linked immunosorbent assay(ELISA). Nasal mucosal inflammation was evaluated by HE staining. Result: Formaldehyde exposure could increase the number of allergic rhinitis mice with sneezing and rubbing nose. The levels of IL-4 and IL-10 in group B, D, E and F were higher than that ingroup A (P < 0.05). Compared with the group C, the group D, E and F could effectively increase serum IL-4 and IL-10. The concentration of IL-4 in group E and F was higher than that of group B, while the group C was lower (P < 0.05). The concentration of IL-10 in group D, E and F was higher than that in group B (P < 0.05). The expression of IFN-γ in group B, D, E and F was lower than that in group A. While, the IFN-γ expression in group B was lower than that of group C and higher than that in group F (P < 0.05). Moreover, the concentration of IFN-γ in group D, E and F was lower compared with group C (P < 0.05). The nasal mucosa HE staining showed that the density of EOS increased simultaneously in formaldehyde exposure allergic rhinitis groups.@*CONCLUSION@#The study showed that formaldehyde exposure can promote Th2 cytokines and eosinophil infiltration and then aggravate the allergic rhinitis symptoms.