RESUMO
Protein-protein interactions (PPIs) lead the formation of protein complexes that perform biochemical reactions that maintain the living state of the living cell. Although therapeutic drugs should influence the formation of protein complexes in addition to PPI network, the methodology analyzing such influences remain to be developed. Here, we demonstrate that a new approach combining HPLC (high performance liquid chromatography) for separating protein complexes, and the SILAC (stable isotope labeling using amino acids in cell culture) method for relative protein quantification, enable us to identify the protein complexes influenced by a drug. We applied this approach to the analysis of thalidomide action on HepG2 cells, assessed the identified proteins by clustering data analyses, and assigned 135 novel protein complexes affected by the drug. We propose that this approach is applicable to elucidating the mechanisms of actions of other therapeutic drugs on the PPI network, and the formation of protein complexes.
Assuntos
Aminoácidos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismo , Proteômica , Células Hep G2 , Humanos , Marcação por Isótopo , Talidomida/farmacologiaRESUMO
The U1 small nuclear ribonucleoprotein (snRNP) plays pivotal roles in pre-mRNA splicing and in regulating mRNA length and isoform expression; however, the mechanism of U1 snRNA quality control remains undetermined. Here, we describe a novel surveillance pathway for U1 snRNP biogenesis. Mass spectrometry-based RNA analysis showed that a small population of SMN complexes contains truncated forms of U1 snRNA (U1-tfs) lacking the Sm-binding site and stem loop 4 but containing a 7-monomethylguanosine 5' cap and a methylated first adenosine base. U1-tfs form a unique SMN complex, are shunted to processing bodies and have a turnover rate faster than that of mature U1 snRNA. U1-tfs are formed partly from the transcripts of U1 genes and partly from those lacking the 3' box elements or having defective SL4 coding regions. We propose that U1 snRNP biogenesis is under strict quality control: U1 transcripts are surveyed at the 3'-terminal region and U1-tfs are diverted from the normal U1 snRNP biogenesis pathway.
Assuntos
RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Adenosina/metabolismo , Citoplasma/metabolismo , Guanosina/análogos & derivados , Guanosina/análise , Espectrometria de Massas , Metilação , Estabilidade de RNA , RNA Nuclear Pequeno/genética , Proteínas do Complexo SMN/metabolismoRESUMO
BACKGROUND: Kenya introduced a monovalent rotavirus vaccine administered orally at 6 and 10 weeks of age into her National Immunization Program in July 2014. The study evaluated the long-term impact of the vaccine on hospitalization for all-cause and rotavirus-specific acute gastroenteritis (AGE) and strain epidemiology in Kenya. METHODS: Data on all-cause and rotavirus-specific AGE and strain distribution were derived from an eleven-year hospital-based surveillance of AGE among children aged <5 years at Kiambu County Teaching and Referral Hospital (KCTRH) in Central Kenya between 2009 and 2020. Fecal samples were screened for group A rotavirus using ELISA and genotyped using multiplex semi-nested RT-PCR. Trends in all-cause and rotavirus-related AGE and strain distribution were compared between the pre-vaccine (July 2009-June 2014), early post-vaccine (July 2014-June 2016) and late post-vaccine (February 2019-October 2020) periods. RESULTS: Rotavirus-specific AGE was detected at 27.5% (429/1546, 95% CI: 25.5-30.1%) in the pre-vaccine period; 13.8% (91/658, 95% CI: 11.3-16.6%) in the early post-vaccine period (July 2014-June 2016); and 12.0% (229/1916, 95% CI: 10.6-13.5%) in the late post-vaccine period (February 2019-October 2020). This amounted to a decline of 49.8% (95% CI: 34.6%-63.7%) in rotavirus-specific AGE in the early post-vaccine period and 53.4% (95% CI: 41.5-70.3%) in the late post-vaccine period when compared to the pre-vaccine period. All-cause AGE hospitalizations declined by 40.2% (95% CI: 30.8%-50.2%) and 75.3% (95% CI: 65.9-83.1%) in the early post-vaccine and late post-vaccine periods, respectively, when compared to the pre-vaccine period. G3P [8] was the predominant strain in the late post-vaccine period, replacing G1P[8] which had predominated in the pre-vaccine and early post-vaccine periods. Additionally, we detected considerable proportions of uncommon strains G3P[6] (4.8%) and G12P[6] (3.5%) in the post-vaccine era. CONCLUSION: Rotavirus vaccination has resulted in a significant decline in all-cause and rotavirus-specific AGE, and thus, provides strong evidence for public health policy makers in Kenya to support the sustained use of the rotavirus vaccine in routine immunization. However, the shift in strain dominance and age distribution of rotavirus AGE in the post-vaccine era underscores the need for continued surveillance to assess any possible vaccine-induced selective pressure that could diminish the vaccine effectiveness over time.
Assuntos
Gastroenterite , Programas de Imunização , Análise de Séries Temporais Interrompida , Infecções por Rotavirus , Vacinas contra Rotavirus , Rotavirus , Vacinação , Humanos , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/imunologia , Infecções por Rotavirus/prevenção & controle , Infecções por Rotavirus/epidemiologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Gastroenterite/prevenção & controle , Quênia/epidemiologia , Pré-Escolar , Rotavirus/imunologia , Rotavirus/genética , Lactente , Vacinação/estatística & dados numéricos , Hospitalização/estatística & dados numéricos , Feminino , Fezes/virologia , Masculino , Genótipo , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/administração & dosagemRESUMO
BACKGROUND: Virus-like particles (VLPs) are unable to replicate in the recipient but stimulate the immune system through recognition of repetitive subunits. Parenterally delivered rotavirus-VLP (Ro-VLP) vaccine could have the potential to overcome the weaknesses of licensed oral live-attenuated rotavirus vaccines, namely, low efficacy in low-income and high mortality settings and a potential risk of intussusception. METHODS: A monovalent Ro-VLP composed of viral protein (VP) 7, VP6 and VP2 of G1 genotype specificity was produced in Nicotiana benthamiana using Agrobacterium tumefaciens infiltration-based transient recombinant expression system. Plants expressing recombinant G1 Ro-VLP were harvested, then the resultant biomass was processed through a series of clarification and purification steps including standard extraction, filtration, ultrafiltration and chromatography. The purified G1 Ro-VLP was subsequently examined for its immunogenicity and toxicological profile using animal models. RESULTS: G1 Ro-VLP had a purity of ≥90% and was structurally similar to triple-layered rotavirus particles as determined by cryogenic transmission electron microscopy. Two doses of aluminum hydroxide-adjuvanted G1 Ro-VLP (1 µg, 5 µg or 30 µg), administered intramuscularly, elicited a robust homotypic neutralizing antibody response in rats. Also, rabbits administered G1 Ro-VLP (10 µg or 30 µg) four times intramuscularly with aluminum hydroxide adjuvant did not show any significant toxicity. CONCLUSIONS: Plant-derived Ro-VLP composed of VP7, VP6 and VP2 structural proteins would be a plausible alternative to live-attenuated oral rotavirus vaccines currently distributed worldwide.
Assuntos
Infecções por Rotavirus , Vacinas contra Rotavirus , Rotavirus , Vacinas de Partículas Semelhantes a Vírus , Animais , Anticorpos Antivirais , Coelhos , Ratos , Rotavirus/genética , Infecções por Rotavirus/prevenção & controle , Vacinas Atenuadas , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
BACKGROUND: This study is the first clinical trial for a parenteral non-replicating rotavirus vaccine developed using virus-like particle (VLP) technology. METHODS: This open-labeled, randomized, placebo-controlled trial was conducted in two parts: Part A (a first-in-human study in Australian adults) and Part B (ascending dose and descending age in South African adults, toddlers and infants). In Part A, two cohorts of 10 adults were assigned to receive a single intramuscular injection of 1 of 2 escalating dose levels of the rotavirus VLP (Ro-VLP) vaccine (7 µg or 21 µg) or placebo. In Part B, one cohort of 10 adults was assigned to receive a single injection of the Ro-VLP vaccine (21 µg) or placebo, two cohorts of 10 toddlers were assigned to receive 2 injections of 1 of 2 escalating dose levels of the Ro-VLP vaccine (7 µg or 21 µg) or placebo 28 days apart, and three cohorts of 20 infants were assigned to receive 3 injections of 1 of 3 escalating dose levels of the Ro-VLP vaccine (2.5 µg, 7 µg or 21 µg) or placebo or 2 doses of oral Rotarix 28 days apart. Safety, reactogenicity and immunogenicity were assessed. RESULTS: There were no safety or tolerability concerns after administration of the Ro-VLP vaccine. The Ro-VLP vaccine induced an anti-G1P[8] IgG response in infants 4 weeks after the second and third doses. Neutralizing antibody responses against homologous G1P[8] rotavirus were higher in all Ro-VLP infant groups than in the placebo group 4 weeks after the third dose. No heterotypic immunity was elicited by the Ro-VLP vaccine. CONCLUSIONS: The Ro-VLP vaccine was well tolerated and induced a homotypic immune response in infants, suggesting that this technology platform is a favorable approach for a parenteral non-replicating rotavirus vaccine. CLINICAL TRIAL REGISTRATION: NCT03507738.
Assuntos
Vacinas contra Rotavirus , Rotavirus , Vacinas de Partículas Semelhantes a Vírus , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , Austrália , Pré-Escolar , Método Duplo-Cego , Humanos , Imunogenicidade da Vacina , Lactente , Vacinas contra Rotavirus/efeitos adversosRESUMO
Human rotavirus strains having the unconventional G4P[6] genotype have been sporadically identified in diarrheic patients in different parts of the world. However, the whole genome of only one human G4P[6] strain from Africa (central Africa) has been sequenced and analyzed, and thus the exact origin and evolutionary pattern of African G4P[6] strains remain to be elucidated. In this study, we characterized the full genome of an African G4P[6] strain (RVA/Human-wt/KEN/KCH148/2019/G4P[6]) identified in a stool specimen from a diarrheic child in Kenya. Full genome analysis of strain KCH148 revealed a unique Wa-like genogroup constellation: G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1. NSP3 genotype T7 is commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis showed that 10 of the 11 genes of strain KCH148 (VP7, VP4, VP6, VP1-VP3, NSP1, and NSP3-NSP5) appeared to be of porcine origin, the remaining NSP2 gene appearing to be of human origin. Therefore, strain KCH148 was found to have a porcine rotavirus backbone and thus is likely to be of porcine origin. Furthermore, strain KCH148 is assumed to have been derived through interspecies transmission and reassortment events involving porcine and human rotavirus strains. To our knowledge, this is the first report on full genome-based characterization of a human G4P[6] strain from east Africa. Our observations demonstrated the diversity of human G4P[6] strains in Africa, and provide important insights into the origin and evolutionary pattern of zoonotic G4P[6] strains on the African continent.