RESUMO
The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.
Assuntos
Evolução Molecular , Imunoterapia , Neoplasias Pulmonares , Platina , Carcinoma de Pequenas Células do Pulmão , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Genes myc/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Platina/farmacologia , Platina/uso terapêutico , Recidiva , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapiaRESUMO
The human prefrontal cortex (PFC) constitutes the structural basis underlying flexible cognitive control, where mixed-selective neural populations encode multiple task features to guide subsequent behavior. The mechanisms by which the brain simultaneously encodes multiple task-relevant variables while minimizing interference from task-irrelevant features remain unknown. Leveraging intracranial recordings from the human PFC, we first demonstrate that competition between coexisting representations of past and present task variables incurs a behavioral switch cost. Our results reveal that this interference between past and present states in the PFC is resolved through coding partitioning into distinct low-dimensional neural states; thereby strongly attenuating behavioral switch costs. In sum, these findings uncover a fundamental coding mechanism that constitutes a central building block of flexible cognitive control.
Assuntos
Cognição , Córtex Pré-Frontal , HumanosRESUMO
BACKGROUND: Patients with congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD) and dementia are underrepresented in specialist palliative home care (SPHC). However, the complexity of their conditions requires collaboration between general practitioners (GPs) and SPHC teams and timely integration into SPHC to effectively meet their needs. OBJECTIVE: To facilitate joint palliative care planning and the timely transfer of patients with advanced chronic non-malignant conditions to SPHC. METHODS: A two-arm, unblinded, cluster-randomised controlled trial. 49 GP practices in northern Germany were randomised using web-based block randomisation. We included patients with advanced CHF, COPD and/or dementia. The KOPAL intervention consisted of a SPHC nurse-patient consultation followed by an interprofessional telephone case conference between SPHC team and GP. The primary outcome was the number of hospital admissions 48 weeks after baseline. Secondary analyses examined the effects on health-related quality of life and self-rated health status, as measured by the EuroQol 5D scale. RESULTS: A total of 172 patients were included in the analyses. 80.4% of GP practices had worked with SHPC before, most of them exclusively for cancer patients. At baseline, patients reported a mean EQ-VAS of 48.4, a mean quality of life index (EQ-5D-5L) of 0.63 and an average of 0.80 hospital admissions in the previous year. The intervention did not significantly reduce hospital admissions (incidence rate ratio = 0.79, 95%CI: [0.49, 1.26], P = 0.31) or the number of days spent in hospital (incidence rate ratio = 0.65, 95%CI: [0.28, 1.49], P = 0.29). There was also no significant effect on quality of life (∆ = -0.02, 95%CI: [-0.09, 0.05], P = 0.53) or self-rated health (∆ = -2.48, 95%CI: [-9.95, 4.99], P = 0.51). CONCLUSIONS: The study did not show the hypothesised effect on hospitalisations and health-related quality of life. Future research should focus on refining this approach, with particular emphasis on optimising the timing of case conferences and implementing discussed changes to treatment plans, to improve collaboration between GPs and SPHC teams.
Assuntos
Insuficiência Cardíaca , Cuidados Paliativos , Atenção Primária à Saúde , Doença Pulmonar Obstrutiva Crônica , Qualidade de Vida , Humanos , Cuidados Paliativos/métodos , Masculino , Feminino , Idoso , Alemanha , Doença Pulmonar Obstrutiva Crônica/terapia , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Idoso de 80 Anos ou mais , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/diagnóstico , Demência/terapia , Doença Crônica , Serviços de Assistência Domiciliar , Equipe de Assistência ao Paciente , Fatores de Tempo , Comunicação Interdisciplinar , Prestação Integrada de Cuidados de Saúde/organização & administraçãoRESUMO
Highly sulfated malto-oligomers, similar to heparin and heparan-sulfate, have good antiviral, antimetastatic, anti-inflammatory and cell growth inhibitory effects. Due to their broad biological activities and simple structure, sulfated malto-oligomer derivatives have a great therapeutic potential, therefore, the development of efficient synthesis methods for their production is of utmost importance. In this work, preparation of α-(1â4)-linked oligoglucosides containing a sulfonatomethyl moiety at position C-6 of each glucose unit was studied by different approaches. Malto-oligomeric sulfonic acid derivatives up to dodecasaccharides were prepared by polymerization using different protecting groups, and the composition of the product mixtures was analyzed by MALDI-MS methods and size-exclusion chromatography. Synthesis of lower oligomers was also accomplished by stepwise and block synthetic methods, and then the oligosaccharide products were persulfated. The antiviral, anti-inflammatory and cell growth inhibitory activity of the fully sulfated malto-oligosaccharide sulfonic acids were determined by in vitro tests. Four tested di- and trisaccharide sulfonic acids effectively inhibited the activation of the TNF-α-mediated inflammatory pathway without showing cytotoxicity.
Assuntos
Oligossacarídeos , Sulfatos , Polimerização , Oligossacarídeos/farmacologia , Ácidos Sulfônicos , Anti-Inflamatórios/farmacologia , Antivirais/farmacologiaRESUMO
This review article deals with the pathways of cellular and global molybdate distribution in plants, especially with a full overview for the model plant Arabidopsis thaliana. In its oxidized state as bioavailable molybdate, molybdenum can be absorbed from the environment. Especially in higher plants, molybdenum is indispensable as part of the molybdenum cofactor (Moco), which is responsible for functionality as a prosthetic group in a variety of essential enzymes like nitrate reductase and sulfite oxidase. Therefore, plants need mechanisms for molybdate import and transport within the organism, which are accomplished via high-affinity molybdate transporter (MOT) localized in different cells and membranes. Two different MOT families were identified. Legumes like Glycine max or Medicago truncatula have an especially increased number of MOT1 family members for supplying their symbionts with molybdate for nitrogenase activity. In Arabidopsis thaliana especially, the complete pathway followed by molybdate through the plant is traceable. Not only the uptake from soil by MOT1.1 and its distribution to leaves, flowers, and seeds by MOT2-family members was identified, but also that inside the cell. the transport trough the cytoplasm and the vacuolar storage mechanisms depending on glutathione were described. Finally, supplying the Moco biosynthesis complex by MOT1.2 and MOT2.1 was demonstrated.
Assuntos
Arabidopsis , Molibdênio , Humanos , Homeostase , Transporte BiológicoRESUMO
d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.
Assuntos
COVID-19 , Nucleosídeos de Pirimidina , Tioaçúcares , Humanos , Camundongos , Animais , Arabinonucleosídeos/química , Arabinonucleosídeos/farmacologia , Nucleosídeos/farmacologia , Nucleosídeos/química , Antivirais/farmacologia , Acetais , Compostos de Sulfidrila/química , Purinas , Relação Estrutura-AtividadeRESUMO
Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.
Assuntos
COVID-19 , Vírus da Encefalite Transmitidos por Carrapatos , Humanos , Pandemias , RNA Polimerase Dependente de RNA , SARS-CoV-2RESUMO
Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein-protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.
Assuntos
Arabidopsis , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Molibdênio/metabolismo , Cofatores de MolibdênioRESUMO
The brain's ability to act as an input filter and to suppress actions is crucial to navigate everyday life and impairments in these abilities affect quality of life substantially. Although Parkinson's disease (PD) is primarily known as a movement disorder, recent research has redefined it as a multisystem disorder affecting cognition, in particular inhibitory control and attentional resource allocation. Analysing the neural mechanisms underlying this cognitive deficit provides a better understanding of brain changes observed in patients affected by PD. Therefore, this study aimed to identify resource allocation to relevant and irrelevant stimuli in patients affected by PD. Besides neuropsychological tests, we employed electroencephalographic recordings during an auditory oddball paradigm in 13 patients suffering from idiopathic PD and 11 healthy controls (HC). Participants were instructed to ignore the standard stimulus and to respond as fast as possible to the rarely presented target tone. Event-related potentials (ERP) and time-frequency representations (TFR) were analyzed. Patients affected by PD showed faster response latencies to the task-irrelevant standard tones, but slower response latencies to target tones compared to HC. This observation was prominent at frontal sites during later P3-like processing stages. Reaction time, however, was prolonged in patients with PD, suggesting inefficient resource allocation. Additionally, TFR revealed reduced parietal alpha activity, which is associated with distractor suppression and functional inhibition in patients with PD compared to healthy controls. Thus, our results point towards inefficient resource allocation in patients with PD possibly driven by less functional inhibition through parietal alpha activity.
Assuntos
Doença de Parkinson , Eletroencefalografia , Humanos , Lobo Parietal , Qualidade de Vida , Tempo de Reação , Alocação de RecursosRESUMO
BACKGROUND: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. RESULTS: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. CONCLUSIONS: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.
Assuntos
Trifosfato de Adenosina/análise , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Frutose/análise , Ácido Pirúvico/análise , Proteínas Recombinantes/biossíntese , Trifosfato de Adenosina/metabolismo , Carbono/metabolismo , Frutose/química , Frutose/metabolismo , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Viral infections cause life-threatening diseases in millions of people worldwide every year and there is an urgent need for new, effective antiviral drugs. Hybridization of two chemically diverse compounds into a new bioactive effector product is a successful concept to improve the properties of a hybrid drug relative to the parent compounds. In this study, (iso)quinoline-artemisinin hybrids, obtained through copper-catalyzed azide-alkyne cycloaddition or metal-free click reactions (in organic solvents or in the presence of water), were analyzed in vitro, for the first time, for their inhibitory activity against human cytomegalovirus (HCMV), relative to their parent compounds and the reference drug ganciclovir. EC50 (HCMV) values were obtained in a range 0.22-1.20â µm, which indicated highly potent antiviral properties in the absence of cytotoxic effects on normal cells (CC50 >100â µm). The most active hybrid, 1 (EC50 =0.22â µm), is 25 times more potent than its parent compound artesunic acid (EC50 =5.41â µm) and 12 times more efficient than the standard drug ganciclovir (EC50 =2.6â µm). Interestingly, hybrid 1 also shows inhibitory activity against hepatitisâ B virus in vitro (EC50 (HBeAg)=2.57â µm).
Assuntos
Vírus , Antivirais/farmacologia , Artemisininas/farmacologia , Química Click , Citomegalovirus , Humanos , Quinolinas/farmacologiaRESUMO
BACKGROUND: Addiction is associated with health-related and social risks. The interministerial harmonized Lower Saxon concept on drug prevention was most recently revised in 2008 and is predominantly implemented by drug counselling services, drug prevention specialists, the police, self-help groups, corporate addiction prevention, child welfare, and local prevention committees. OBJECTIVE: The study aimed at providing the necessary information for an intended enhancement of the Lower Saxon concept on drug prevention. METHOD: Descriptive analysis of the ratings of a sample of 459 stakeholders involved in preventive activities who completed an online questionnaire on the current performance of Lower Saxon drug prevention. RESULTS AND DISCUSSION: Moderate levels of success have been rated in terms of the achievement of selected preventive goals as well as certain quality standards. An additional need for drug prevention was described for several target groups, including children living in families with a drug-addicted family member, youth aged between 13 and 17, refugees, children with poor educational background, people suffering from mental illness, trainees and university students, as well as the unemployed. In comparison to other institutions, German employment offices, job centers, and churches were nominated to a lesser extent as being responsible for executing drug prevention. Participants saw great potential in a more consistent implementation of current regulations on advertisement, access, and consumption as well as for additional situation-based-prevention. In addition, stakeholders felt restricted in their preventive work due to financial constraints and a lack of recourse in terms of evaluation and documentation. Disregarding their various institutional backgrounds participants had a common agreement when evaluating the questions. Differences led back to their deferring public service obligations.
Assuntos
Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Família , Alemanha/epidemiologia , Humanos , Transtornos Mentais , Inquéritos e QuestionáriosRESUMO
Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (â¼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.
Assuntos
Antivirais , Materiais Biomiméticos , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 2/metabolismo , Nanopartículas , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/farmacologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologiaRESUMO
The increasing demand of space flights requires a profound knowledge of the chronologic reactions of the human body to extreme conditions. Prior studies already have shown the adverse effects of long-term isolation on psycho-physiological well-being. The chronology of the effects and whether short-term isolation periods already lead to similar effects has not been investigated. Therefore, the aim of the current study was to investigate the effects of short-term isolation (30 days) on mood, cognition, cortisol, neurotrophic factors, and brain activity. 16 participants were isolated in the Human Exploration Research Analog at NASA for 30 days. 17 non-isolated control participants were tested simultaneously. On mission days - 5, 7, 14, 28, and + 5, multiple tests including the Positive and Negative Affect Schedule-X and cognitive tests were conducted, and a 5-min resting electroencephalography was recorded. A fasted morning blood drawing was also done. Increased stress was observed via augmented cortisol levels during the isolation period. Activity within the parietal cortex was reduced over time, probably representing a neural adaptation to less external stimuli. Cognitive performance was not affected, but rather enhanced in both groups. No further significant changes in neurotrophic factors BDNF/IGF-1 and mood could be detected. These results suggest that 30 days of isolation do not have a significant impact on brain activity, neurotrophic factors, cognition, or mood, even though stress levels were significantly increased during isolation. Further studies need to address the question as to what extent increased levels of stress do not affect mental functions during isolation periods.
Assuntos
Afeto/fisiologia , Fator Neurotrófico Derivado do Encéfalo/sangue , Cognição/fisiologia , Hidrocortisona/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Lobo Parietal/fisiologia , Isolamento Social , Estresse Psicológico/metabolismo , Adulto , Astronautas , Eletroencefalografia , Feminino , Humanos , Masculino , Fatores de TempoRESUMO
The silent information regulator 2/3/4 (Sir2/3/4) complex is required for gene silencing at the silent mating-type loci and at telomeres in Saccharomyces cerevisiae. Sir3 is closely related to the origin recognition complex 1 subunit and consists of an N-terminal bromo-adjacent homology (BAH) domain and a C-terminal AAA(+) ATPase-like domain. Here, through a combination of structure biology and exhaustive mutagenesis, we identified unusual, silencing-specific features of the AAA(+) domain of Sir3. Structural analysis of the putative nucleotide-binding pocket in this domain reveals a shallow groove that would preclude nucleotide binding. Mutation of this site has little effect on Sir3 function in vivo. In contrast, several surface regions are shown to be necessary for the Sir3 silencing function. Interestingly, the Sir3 AAA(+) domain is shown here to bind chromatin in vitro in a manner sensitive to histone H3K79 methylation. Moreover, an exposed loop on the surface of this Sir3 domain is found to interact with Sir4. In summary, the unique folding of this conserved Sir3 AAA(+) domain generates novel surface regions that mediate Sir3-Sir4 and Sir3-nucleosome interactions, both being required for the proper assembly of heterochromatin in living cells.
Assuntos
Inativação Gênica , Histonas/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Alelos , Cromatina/metabolismo , Metilação de DNA , Histonas/química , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genéticaRESUMO
Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S. cerevisiae, which lies within the essential N-terminal domain (END) of the extended Cse4 N-terminus. Significantly, we identified histone H4-K5, 8, 12R to cause a temperature-sensitive growth defect with mutations in Cse4-S33 and sensitivity to nocodazole and hydroxyurea. Furthermore, the absence of Cse4-S33ph reduced the levels of Cse4 at centromeric sequences, suggesting that Cse4 deposition is defective in the absence of S33 phosphorylation. We furthermore identified synthetic genetic interactions with histone H2A-E57A and H2A-L66A, which both cause a reduced interaction with the histone chaperone FACT and reduced H2A/H2B levels in chromatin, again supporting the notion that a combined defect of H2A/H2B and Cse4 deposition causes centromeric defects. Altogether, our data highlight the importance of correct histone deposition in building a functional centromeric nucleosome and suggests a role for Cse4-S33ph in this process.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Centrômero/genética , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Espectrometria de Massas , Mutação , Fosforilação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Progression rates from initial HIV-1 infection to advanced AIDS vary significantly among infected individuals. A distinct subgroup of HIV-1-infected individuals-termed viremic non-progressors (VNP) or controllers-do not seem to progress to AIDS, maintaining high CD4+ T cell counts despite high levels of viremia for many years. Several studies have evaluated multiple host factors, including immune activation, trying to elucidate the atypical HIV-1 disease progression in these patients; however, limited work has been done to characterize viral factors in viremic controllers. METHODS: We analyzed HIV-1 isolates from three VNP individuals and compared the replicative fitness, near full-length HIV-1 genomes and intra-patient HIV-1 genetic diversity with viruses from three typical (TP) and one rapid (RP) progressor individuals. RESULTS: Viremic non-progressors and typical patients were infected for >10 years (range 10-17 years), with a mean CD4+ T-cell count of 472 cells/mm3 (442-529) and 400 cells/mm3 (126-789), respectively. VNP individuals had a less marked decline in CD4+ cells (mean -0.56, range -0.4 to -0.7 CD4+/month) than TP patients (mean -10.3, -8.2 to -13.1 CD4+/month). Interestingly, VNP individuals carried viruses with impaired replicative fitness, compared to HIV-1 isolates from the TP and RP patients (p < 0.05, 95% CI). Although analyses of the near full-length HIV-1 genomes showed no clear patterns of single-nucleotide polymorphisms (SNP) that could explain the decrease in replicative fitness, both the number of SNPs and HIV-1 population diversity correlated inversely with the replication capacity of the viruses (r = -0.956 and r = -0.878, p < 0.01, respectively). CONCLUSION: It is likely that complex multifactorial parameters govern HIV-1 disease progression in each individual, starting with the infecting virus (phenotype, load, and quasispecies diversity) and the intrinsic ability of the host to respond to the infection. Here we analyzed a subset of viremic controller patients and demonstrated that similar to the phenomenon observed in patients with a discordant response to antiretroviral therapy (i.e., high CD4+ cell counts with detectable plasma HIV-1 RNA load), reduced viral replicative fitness seems to be linked to slow disease progression in these antiretroviral-naïve individuals.
Assuntos
Aptidão Genética , Infecções por HIV/virologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/isolamento & purificação , HIV-1/fisiologia , Replicação Viral , Adulto , Estudos de Coortes , Variação Genética , Genoma Viral , HIV-1/classificação , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
A series of benzaldehyde and salicylaldehyde-S-benzylisothiosemicarbazones was synthesized and tested against 12 different strains of mycobacteria, Gram-positive and Gram-negative bacteria, and the significant selectivity toward mycobacteria was proved. Twenty-eight derivatives were evaluated for the inhibition of isocitrate lyase, which is a key enzyme of the glyoxylate cycle necessary for latent tuberculosis infection, and their iron-chelating properties were investigated. Two derivatives, 5-bromosalicylaldehyde-S-(4-fluorobenzyl)-isothiosemicarbazone and salicylaldehyde-S-(4-bromobenzyl)-isothiosemicarbazone, influenced the isocitrate lyase activity and caused a better inhibition at 10 µmol/L than 3-nitropropionic acid, a standard inhibitor. The compounds were also found to act as exogenous chelators of iron, which is an obligate cofactor for many mycobacterial enzymes. Due to their low cytotoxicity, together with the activity against isocitrate lyase and the ability to sequester iron ions, the compounds belong to potential antibiotics with the main effect on mycobacteria.
Assuntos
Antibacterianos/farmacologia , Antituberculosos/farmacologia , Mycobacterium/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antituberculosos/síntese química , Antituberculosos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Isocitrato Liase/antagonistas & inibidores , Relação Estrutura-Atividade , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/químicaRESUMO
BACKGROUND: Healthcare-acquired infections by pathogenic microorganisms including viruses represent significant health concern worldwide. Next to direct transmission from person-to-person also indirect transmission from contaminated surfaces is well documented and important route of infections. Here, we tested antiviral properties of hybrid coating containing silver, copper and zinc cations that was previously shown to be effective against pathogenic bacteria including methicillin-resistant Staphylococcus aureus. Hybrid coatings containing silver, copper and zinc cations were prepared through radical polymerization via sol-gel method and applied on glass slides or into the wells of polymethylmethacrylate plates. A 10 µl droplet of several viruses such as human immunodeficiency virus type 1 (HIV-1), influenza, dengue virus, herpes simplex virus, and coxsackievirus was added to coated and uncoated slides or plates, incubated usually from 5 to 240 min and followed by titer determination of recovered virus. RESULTS: Scanning electron microscopy analysis showed better adhesion of coatings on glass surfaces, which resulted in 99.5-100 % HIV-1 titer reduction (3.1 ± 0.8 log10TCID50, n = 3) already after 20 min of exposure to coatings, than on coated polymethylmethacrylate plates with 75-100 % (1.7 ± 1.1 log10TCID50, n = 3) and 98-100 % (2.3 ± 0.5 log10TCID50, n = 3) HIV-1 titer reduction after 20 and 120 min of exposure, respectively. Slower virucidal kinetics was observed with other enveloped viruses, where 240 min exposure to coated slides lead to 97 % (dengue), 100 % (herpes simplex) and 77 % (influenza) reduction in virus titers. Interestingly, only marginal reduction in viral titer after 240 min of exposure was noticed for non-enveloped coxsackie B3 virus. CONCLUSIONS: Our hybrid coatings showed virucidal activity against HIV and other enveloped viruses thus providing further findings towards development of broad-spectrum antimicrobial coating suitable for surfaces in healthcare settings.
Assuntos
Antivirais/farmacologia , Cobre/farmacologia , Infecção Hospitalar/prevenção & controle , HIV-1/efeitos dos fármacos , Prata/farmacologia , Vírus/efeitos dos fármacos , Zinco/farmacologia , Antivirais/química , Cátions/química , Cátions/farmacologia , Cobre/química , Infecção Hospitalar/virologia , Contaminação de Equipamentos/prevenção & controle , Equipamentos e Provisões Hospitalares/virologia , Infecções por HIV , Humanos , Prata/química , Zinco/químicaRESUMO
We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.