Recent advances in small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase / 药学学报

Influenza virus causes serious threat to human life and health. Due to the inherent high variability of influenza virus, clinically resistant mutant strains of currently approved anti-influenza virus drugs have emerged. Therefore, it is urgent to develop antiviral drugs with new targets or mechanisms of action. RNA-dependent RNA polymerase is directly responsible for viral RNA transcription and replication, and plays key roles in the viral life cycle, which is considered an important target of anti-influenza drug design. From the point of view of medicinal chemistry, this review summarizes current advances in diverse small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase, hoping to provide valuable reference for development of novel antiviral drugs.

Multi-level scoring approach to discover multi-targeting potency of medicinal plant phytochemicals against protein targets in SARS-CoV-2 and human ACE-2 receptor

SARS-CoV-2 pandemic has become a major threat to human healthcare and world economy. Due to the rapid spreading and deadly nature of infection, we are in a situation to develop quick therapeutics to combat SARS-CoV-2. In this study, we have adopted a multi-level scoring approach to identify multi-targeting potency of bioactive compounds in selected medicinal plants and compared its efficacy with two reference drugs, Nafamostat and Acalabrutinib which are under clinical trials to treat SARS-CoV-2. In particular, we employ molecular docking and implicit solvent free energy calculations (as implemented in the Molecular Mechanics -Generalized Born Surface Area approach) and QM fragmentation approach for validating the potency of bioactive compounds from the selected medicinal plants against four di?erent viral targets and one human receptor (Angiotensin-converting enzyme 2 -ACE-2) which facilitates the SARS-CoV-2entry into the cell. The protein targets considered for the study are viral 3CL main protease (3CLpro), papain-like protease (PLpro), RNA dependent RNA polymerase (RdRp), and viral spike protein-human hACE-2 complex (Spike:hACE2)including human protein target (hACE-2). Herein, thereliable multi-level scoring approach was used to validate the mechanism behind the multi-targeting potency of selected phytochemicals from medicinal plants. The present study evidenced that the phytochemicals Chebulagic acid, Stigmosterol, Repandusinic acid and Geranin exhibited efficient inhibitory activity against PLpro while Chebulagic acid was highly active against 3CLpro. Chebulagic acid andGeranin also showed excellent target specific activity against RdRp.Luteolin, Quercetin, Chrysoeriol and Repandusinic acid inhibited the interaction of viral spike protein with human ACE-2 receptor. Moreover Piperlonguminine and Piperine displayed significant inhibitory activity against human ACE-2 receptor. Therefore, the identified compounds namely Chebulagic acid, Geranin and Repandusinic acid can serve as potent multi-targeting phytomedicine for treating COVID-19

Las variantes de SARS-CoV-2 y la llamada resistencia a las vacunas / SARS-CoV-2 variants and the so-called resistance to vaccines

Resumen Los virus ARN, excepto los retrovirus, se replican por acción de una ARN polimerasa ARN-dependiente que carece de exonucleasa correctora y, en consecuencia, en cada replicación puede co meter errores. Así se originan mutantes que, según su menor o mayor fitness, se extinguen o bien prosperan y originan variantes que escapan al sistema inmune. Las mutaciones de SARS-CoV-2 más importantes son las que alteran la proteína viral S, porque ella tiene la llave de ingreso del virus a la célula humana. Cuanto más se replican los virus, más mutan, y se hace más probable que aparezcan variantes resistentes dominantes. En esos casos, se requerirá una aplicación más estricta de las medidas de protección de la comunidad. Las vacunas y los anticuerpos policlonales, que inducen una respuesta dirigida hacia toda la proteína S, mantendrían protec ción efectiva contra las variantes del SARS-CoV-2. Además, las vacunas inducirían una mayor respuesta de células T helper y citotóxicas, lo que puede ser un biomarcador de protección. En áreas densamente pobladas con escasas medidas de protección, el virus se difunde libremente y aumenta la probabilidad de mutaciones de escape. India y Manaos ejemplifican esa situación. La evolución natural selecciona las mutantes que se repro ducen con mayor eficiencia sin eliminar al huésped, lo que facilita la propagación. En cambio, la circulación de virus de alta virulencia y letalidad (Ebola, hantavirus), que eliminan al huésped, se circunscribe a determinadas áreas geográficas, sin mayor difusión. Por lo tanto, sería esperable que SARS-CoV-2 evolucione a variantes más infecciosas y menos virulentas.

Abstract RNA viruses (except retroviruses) replicate by the action of an RNA-dependent RNA polymerase, which lacks a proofreading exo nuclease and, consequently, errors may occur in each replication giving place to viral mutants. Depending on their fitness, these mutants either become extinct or thrive, spawning variants that escape the immune system. The most important SARS-CoV-2 mutations are those that alter the amino acid sequence in the viral S protein because this protein holds the key for the virus to enter the human cell. The more viruses replicate, the more they mutate, and the more likely it is that dominant resistant variants will appear. In such cases, more stringent measures for community protection will be required. Vaccines and polyclonal antibodies, which induce a response directed towards several sites along the S protein, would maintain effective protection against SARS-CoV-2 vari ants. Furthermore, vaccines appear to induce an increased helper and cytotoxic T-cell response, which may also be a biomarker of protection. In densely populated areas with insufficient protection measures, the virus spreads freely, thus increasing the likelihood of generating escape mutants. India and Manaus exemplify this situation. Natural evolution selects the mutants that multiply most efficiently without eliminating the host, thus facilitating their spread. Contrastingly, the circulation of viruses of high virulence and lethality (Ebola, hantavirus) that elimi nate the host remain limited to certain geographic areas, without further dissemination. Therefore, it would be expected that SARS-CoV-2 will evolve into more infectious and less virulent variants.

Exploration of bioflavonoids targeting dengue virus NS5 RNA-dependent RNA polymerase: In silico molecular docking approach

Dengue viral infection becomes highly epidemic and rashes the economic stability of most of the developing countriesdue to its wide prevalence with limited therapeutic ailments. Alarming demographic data urge the need for thedevelopment of new antiviral agents which are safe and efficacious. This study aimed to evaluate the antiviral potentialof bioflavonoids (apigenin, hesperidin, kaempferol, myricetin, and naringenin) against dengue virus nonstructural(NS)5 RNA-dependent RNA polymerase (RdRp) by AutoDock and tox prediction tools. The results of moleculardocking analysis strongly suggested that the lead phytocomponents such as apigenin, hesperidin, and kaempferolreveal potential RdRp inhibition as ascertained by its interaction with core active amino acid residues (710 SER, 729ARG, and 737 ARG) on the target. Apigenin exhibited the best binding affinity of −8.28kcal/mol with RdRp, followedby kaempferol (−7.00 kcal/mol), myricetin (−4.37 kcal/mol), naringenin (−4.35 kcal/mol), and hesperidin(−3.20 kcal/mol). The present research finding clearly advocates that plant-derived bioflavonoids possess excellent antiviralproperty against the selected target.

Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim:To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryoticMSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.Place and Duration of Study: School of Biotechnology, MaduraiKamaraj University, Madurai, India, between 2007-2013. Methodology:Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results:Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the templatebinding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9thposition from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regionsare highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, inthe eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6thposition are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present

Synthesis and antibacterial activity of dithiolopyrrolone derivatives / 国际药学研究杂志

Objective: To design and synthesize novel dithiolopyrrolone derivatives with antibacterial activity. Methods: Fourteen N-aryldithiolopyrrolone derivatives with 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-one skeleton were synthesized via amidation and nucleophilic substitution reactions. The in vitro antimicrobial activity against Staphy- lococcus aureus was assayed for all of the synthesized fourteen derivatives by the paper disk diffusion method. For select- ed compounds,the minimum inhibition concentration on S. aureus was determined and the inhibitory activity on the Esch- erichia coli RNA polymerase(RNAP)was further evaluated. In addition,a predictive investigation for the action mecha- nism of the most potent compound 2i was performed by the molecular docking study on RNAP. Results and Conclu- sion: The structures of the synthesized compounds were confirmed by 1H NMR,13C NMR and ESI-MS. The compound 2i showed a potent antibacterial activity and the molecular docking study revealed the interaction of 2i with the switch re- gion of the bacterial RNA polymerase. Compound 2i might serve as a new lead compound for further optimization into po- tential antibacterial agents.

Correlation between expression of Brf1 and prognosis in non-small cell lung cancer / 中华胸心血管外科杂志

Objective To explore the relationship between the expression of transcription factor ⅡB-related factor 1 (Brf1) and the prognosis of non-small cell lung cancer (NSCLC).Methods Collected 96 cases of NSCLC Surgical specimens and clinical data of patients from January 2013 to August 2015 in our hospital.First of all,we compared the expression of Brf1 in NSCLC tissues and adjacent lung tissues by Western blot and RT-qPCR.Then,Immunohistochemistry was used to detect the expression of Brf1 in NSCLC tissues,and analysis of the relationship between Brf1 expression level and clinical case characteristics.Survival curves were plotted using the Kaplan-Meier method and Log-rank test and multivariate Coxv regression analysis were performed.Results Western blot and RT-qPCR results showed that the expression of Brf1 in NSCLC tissues was significantly higher than that in adjacent lung tissues (P ＜0.01).The positive expression rate of Brf1 in 96 cases of NSCLC was 72.9％.The Brf1 expression level was higher in the poorly differentiated group than in the moderately-highly differentiated group(Mean Rank 62.33 ＞ 43.89,Z =-2.914,P =0.004),and the lymph node metastasis group was higher than the non-metastasis group(Mean Rank 60.34 ＞ 42.58,Z =-3.055,P =0.002),which was independent of patient gender,age,smoking status,tumor size,TNM stage,and pathological type (P ＞0.05).Single-factor survival analysis by Log-rank test showed that the survival rate of Brf1 positive expression group was lower than that of the negative group (x2 =7.560,P ＜0.01).Multivariate analysis of Cox regression model found that Brf1 positive expression (HR =2.043,95％ CI:1.082-3.860) was an independent observational index that affects the prognosis of patients with NSCLC.Conclusion Brf1 is overexpressed in NSCLC tissues,and Brf1 negative expression has a good clinical prognosis,suggesting that Brf1 may be one of the indicators of malignant degree and prognosis of NSCLC.

Patterns of rpoC Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea / 결핵및호흡기질환

BACKGROUND: Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea. METHODS: Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence. RESULTS: In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L. CONCLUSION: Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.

Z1, a novel DENV2 NS5 RdRp small molecular inhibitor, inhibits DENV2 replication and infection / 中国药理学通报

Aim To screen novel NS5 inhibitors a-gainst dengue virus ( DENV) replication. Methods His-tagged DENV2 NS5 RNA-dependent polymerase ( NS5 RdRp ) was expressed and purified in BL21 cells. The binding ability of the small molecules to NS5 polymerase was determined by SPR assay. The activity of dengue inhibition by Z1 was determined by CPE, LDH and plaque assay. RNA synthesis was as-sessed by Real-time PCR. The dsRNA synthesis and viral proteins were detected by immunofluorescence as-say. The level of viral proteins was examined by West-ern blot. The stage of DENV life cycle was evaluated by time of drug-addition assay. Results A small mo-lecular Z1 was discovered, which could bind to NS5 RdRp. Z1 inhibited DENV2 RNA replication, synthe-sis of dsRNA and protein synthesis in post-entry stage of dengue life-cycle. Cell based assay confirmed that Z1 inhibited DENV-induced cell death with EC50 val-ues of 4. 75μmol·L-1 . Conclusions The novel NS5 inhibitor Z1, inhibits DENV2 RNA replication, protein synthesis and release of progeny virus, which may be severed as an anti-DENV2 antiviral drug for further de-velopment.

Regulation of gene expression via control of transcription elongation during immune responses / 中国免疫学杂志

Transcription is a multi-stage process that coordinates several steps during the transcription cycle including pre-initiation,initiation and elongation.Recent advances by genome-wide study suggest that control of transcription elongation is a critical step for precise regulation of gene expression across species from Drosophila to mammals.Here we review the molecular mechanisms of how transcription elongation of RNA polymeraseⅡ(PolⅡ) is modulated by the major pausing factors-NELF,DSIF and the positive elongation factor P-TEFb,which is the key player in pause release.We also discuss the potential implications of regulation of transcription elongation in pathogenesis of cancer,inflammation and virus infection.

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Research advances in poly (ADP-ribose) polymerase inhibitors in combination with radiotherapy / 中华放射肿瘤学杂志

As a DNA damage sensor,poly (ADP-ribose) polymerase (PARP) is involved in a wide variety of cellular activities,such as DNA damage repair.PARP inhibitors regulate a series of cellular activities by inhibiting PARP function,which have become a focus of current research.Recently,several in vivo and in vitro studies showed that PARP inhibitors combined with radiotherapy effectively enhanced the efficacy of radiotherapy.This paper reviews the research advances in the mechanisms of action of PARP inhibitors and their combination with radiotherapy.

Construction and application of N-terminal Strep-tagged protein expression vector / 中国药理学通报

Aims To construct the N-terminal Strep-tagged ( NS-tagged) fusion protein expression vector, and to apply the vector to express NS-tagged fusion proteins of Chlamydia RNA polymerase subunit. Meth-ods By using PCR method, NS fusion protein tag and a new multiple cloning sites (MCS) were inserted into pET21c-DH plasmid by primers to replace the original T7 protein tag and MCS. The newly introduced Not I cutting site was chosen for self-ligation of PCR prod-uct. Then, the cyclized PCR product was transformed into DH-5α competent cells. The positive clones were selected by PCR and sequencing. To get NS-tagged fu-sion proteins of chlamydial RNA polymerase subunits, the α, β and β′ subunits were inserted between BamH I and Sal I cutting sites of the newly constructed ex-pression vector. Then, the NS-α, NS-β and NS-β′ ex-pression vectors were transformed into Arctic Express expression cells. The fusion protein expression statuses of transformed cells were identified by Commassie blue staining and Western blot. Results The NS-tagged fusion protein expression vector pET21c-NS-MCS was successfully constructed, and NS-α, NS-β and NS-β′fusion proteins were obtained by using this newly con-structed expression vector. Conclusions In this pro-ject, we constructed an NS-tagged fusion protein ex-pression vector and applied it to express NS-α, NS-βand NS-β′ fusion proteins. Our study can lay a solid foundation for the study of transcriptional regulation of Chlamydia genes.

Autoanticorpos em esclerose sistêmica e sua correlação com as manifestações clínicas da doença em pacientes do Centro-Oeste do Brasil / Autoantibodies in systemic sclerosis and their clinical correlation in patients from a Midwestern region of Brazil

Introdução: a esclerose sistêmica (ES) é uma enfermidade do tecido conjuntivo de caráter autoimune caracterizada pela tríade de injúria vascular, autoimunidade (celular e humoral) e fibrose tecidual. Os autoanticorpos não parecem ser simplesmente epifenômenos, mas sim estarem envolvidos na patogênese da doença. Acredita-se que os autoanticorpos específicos da ES são responsáveis tanto pela amplificação da resposta imune quanto por alvejar os tipos celulares que são relevantes na fisiopatologia da ES. Objetivos: correlacionar o perfil de autoanticorpos específicos (anti-SCL70, ACA, anti-POL3) com as manifestações clínicas e laboratoriais observadas em 46 pacientes com ES da região Centro-Oeste do Brasil. Métodos: pesquisou-se a ocorrência de autoanticorpos específicos em 46 pacientes com diagnóstico de ES e correlacionou-se o tipo de autoanticorpo com as manifestações clínicas e laboratoriais encontradas. Resultados: dentre todos os pacientes avaliados, encontrou-se predomínio feminino (97,8%), idade média de 50,21 anos, cor branca (50%), forma limitada da doença (47,8%), tempo de diagnóstico entre cinco e 10 anos (50%) e tempo de evolução da doença de 9,38 anos. De acordo com o autoanticorpo específico, 24 pacientes apresentavam ACA positivo (52,2%), 15 apresentavam positividade para anti-SCL70 (32,6%) e sete apresentavam anti-POL3 positivo (15,2%). O autoanticorpo anti-SCL70 se correlacionou com a forma difusa da doença, com maior gravidade e atividade da doença, com pior qualidade de vida medida pelo índice HAQ, com maior prevalência de fenômeno de Raynaud objetivo e microcicatrizes de polpas digitais. O ACA se correlacionou com a forma limitada da doença, com o início mais precoce da enfermidade, bem como com maior prevalência de telangiectasias nos pacientes. Já o anti-POL3 se correlacionou com a forma difusa da doença, com maior ocorrência de fenômeno de Raynaud subjetivo e de atrofia muscular. Para as demais variáveis relacionadas ...

Introduction: Systemic sclerosis (SSc) is a connective tissue disease of autoimmune nature characterized by the triad of vascular injury, autoimmunity (cellular and humoral) and tissue fibrosis. Autoantibodies do not seem to be simply epiphenomena, but are involved in disease pathogenesis. It is believed that the SSc-specific autoantibodies are responsible both for amplifying immune response and targeting cell types that are relevant in the pathophysiology of SSc. Objectives: To correlate the profile of the following specific autoantibodies: anti-centromere (ACA), anti-topoisomerase I (topo I) and anti-RNA polymerase III (RNAP III) with clinical and laboratory manifestations were observed in 46 patients with SSc in the Midwest region of Brazil. Methods: The occurrence of specific autoantibodies in 46 patients with SSc was investigated, correlating the type of autoantibody with clinical and laboratory manifestations found. Results: Among all patients evaluated, we found a predominance of females (97.8%), mean age 50.21 years old, Caucasian (50%), limited cutaneous SSc (47.8%), time of diagnosis between 5 and 10 years (50%), and disease duration of 9.38 years. According to the specific autoantibody profile, 24 patients were ACA-positive (52.2%), 15 were positive for anti-topo I (32.6%), and 7 showed positive anti-RNAP III (15.2%). The anti-topo I autoantibody correlated with diffuse scleroderma, with greater disease severity and activity, with worse quality of life measured by the SHAQ index, with a higher prevalence of objective Raynaud's phenomenon and digital pitting scars of fingertips. The ACA correlated with limited scleroderma, with earlier onset of disease, as well as higher prevalence of telangiectasias. The anti-RNAP III correlated with diffuse scleroderma, with a higher occurrence of subjective Raynaud's phenomenon and muscle atrophy. There was no association between the positivity for anti-topo I, ACA and anti-RNAP III antibodies ...

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: A study from Eastern India.

Background: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very effi cient opportunity for the diagnosis of drug resistance by in vitro method. Aim: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. Methods: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplifi ed by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI – BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. Results: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp – 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specifi c for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). Limitations: The major limitations of multipleprimer PCR amplifi cation refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. Conclusion: The study indicates the existence of rifampicin drug resistance in Eastern India.

Noble strain of Sparassis latifolia produces high content of β-glucan

Objective: To classify four new Sparassis strains (CLM1, CKM1, CKM2, and KJM1) using the internal transcribed spacer sequence and to elucidate their β-glucan content and mycelial growth. Methods: Two different microbiological media were used to determine growth rate. The β-glucan contents were analyzed using the Megazyme Mushroom and Yeast Beta-Glucan kit. To determine the genetic relationships, phylogenetic trees were constructed using ClustalX. Multiple sequence alignments were printed and shaded with the BOXSHADE 3.21 program. Results: In this study, four new Sparassis strains were isolated from the southern region of the Korea Peninsula. They were all classified into the Sparassis latifolia clade as a monophyletic group based on the internal transcribed spacer sequence. Mycelial growth rate of the CLM1 strain was highest in potato dextrose agar and potato dextrose agar larch. The β-glucan content of the CLM1 strain was highest at 29.5% (w/w). A high degree of sequence divergence was detected in the RNA polymerase second largest subunit II gene (RPB2) within Sparassis spp. tested. The putative amino acid sequences of the RPB2 had a distinct sequence. The nucleotide sequences of the RPB2's intron were also divergent among Sparassis spp., even though their nucleotide length was well conserved within Sparassis latifolia. Conclusions: These results indicate that the nucleotide sequences and the amino acid sequences of RPB2 can be used to identify individual Sparassis sp. The Sparassis strain CLM1 may be best for developing a remedy to prevent or treat cancer and other chronic diseases.

Integrator complex and its research progress / 军事医学

The integrator complex is multifunctional and contains at least 12 evolutionarily conserved subunits in hu-mans.It interacts with the C-terminal tail of the largest subunit of RNA-polymeraseⅡ ( RNAPⅡ) to promote 3′-end pro-cessing of small nuclear RNA (snRNA) U1/U2.It also interacts with RNAPⅡ, NELF and Spt5 to regulate NELF-mediated RNAPⅡpause/release and processivity at coding genes .Recently, the integrator complex is also reported to be involved in DNA damage response , dynein recruitment to the nuclear envelope , integrity of Cajal bodies , adipose differentiation , hem-atopoiesis , ciliogenesis , tumorigenesis and generation of viral microRNAs .This review discusses related research progress in the integrator complex .

A New Record of Penicillium antarcticum from Marine Environments in Korea

During a survey of marine fungi from the waters surrounding Jeju Island, Korea, several Penicillium strains were isolated from seawater and marine sponges. Based on morphological characteristics and phylogenetic analyses of the internal transcribed spacer and RNA polymerase subunit II, four strains were identified as Penicillium antarcticum, a fungus that, to the best of our knowledge, had not been previously reported in Korea. Here, we provide detailed descriptions of the morphological characteristics and extracellular enzyme activities of the four strains.