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Representing the probability and uncertainty of outcomes facilitates adaptive behavior by allowing organisms to prepare in advance and devote attention to relevant events. Probability and uncertainty are often studied only for valenced (appetitive or aversive) outcomes, raising the question of whether the identified neural machinery also processes the probability and uncertainty of motivationally neutral outcomes. Here, we aimed to dissociate valenced from valence-independent (i.e., generic) probability (p; maximum at pâ =â 1) and uncertainty (maximum at p = 0.5) signals using human neuroimaging. In a Pavlovian task (n = 41; 19 females), different cues predicted appetitive, aversive, or neutral liquids with different probabilities (p = 0, p = 0.5, p = 1). Cue-elicited motor responses accelerated, and pupil sizes increased primarily for cues that predicted valenced liquids with higher probability. For neutral liquids, uncertainty rather than probability tended to accelerate cue-induced responding and decrease pupil size. At the neural level, generic uncertainty signals were limited to the occipital cortex, while generic probability also activated the anterior ventromedial prefrontal cortex. These generic probability and uncertainty signals contrasted with cue-induced responses that only encoded the probability and uncertainty of valenced liquids in medial prefrontal, insular, and occipital cortices. Our findings show a behavioral and neural dissociation of generic and valenced signals. Thus, some parts of the brain keep track of motivational charge while others do not, highlighting the need and usefulness of characterizing the exact nature of learned representations.
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Sinais (Psicologia) , Humanos , Feminino , Masculino , Incerteza , Adulto , Adulto Jovem , Probabilidade , Imageamento por Ressonância Magnética , Pupila/fisiologia , Condicionamento Clássico/fisiologia , Motivação/fisiologia , Mapeamento Encefálico , Córtex Pré-Frontal/fisiologia , Córtex Pré-Frontal/diagnóstico por imagemRESUMO
PURPOSE: Customizing a Siemens 32-channel coil for use in a Philips 3T MRI system with incorporated magnetic field probes for collecting high-quality MRI and magnetic-field monitoring data concurrently. METHODS: The development process of the custom coil involved several (iterative) phases. Standard temporal SNR and B1 + data were collected with the 32-channel Siemens and for reference the 32-channel/8-channel Philips head coils before and after the custom coil was made compatible with the 3T Philips Achieva system, and magnetic field probes were installed into it along with ancillary electronics around it. Quality assurance tests were conducted in each of the build phases to ensure that the modifications did not affect MRI or field-monitoring data negatively. To test the finished custom coil, we collected high angular resolution diffusion imaging (HARDI) datasets on a spherical silicon oil phantom both with and without concurrent field monitoring and a 32-channel Philips coil without concurrent field monitoring, where the latter two served as reference scans to assess the improved performance of the custom coil with field monitoring. Similar HARDI-MRI data were also collected in vivo with the finished custom coil together with field monitoring data. RESULTS: The custom coil provided excellent temporal SNR especially at the edges where cortical gray matter is expected. When using concurrent field monitoring in HARDI acquisitions, the custom coil alleviated ghosting artifacts in phantom data and provided in vivo images with 1.4-mm isotropic resolution. CONCLUSION: The custom MRI coil with integrated magnetic-field monitoring probes provided improved imaging performance.
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PURPOSE: Inherited retinal diseases (IRDs) are a group of monogenic conditions that can lead to progressive blindness. Their missing heritability is still considerable, due in part to the presence of disease genes that await molecular identification. The purpose of this work was to identify novel genetic associations with IRDs. METHODS: Patients underwent a comprehensive ophthalmological evaluation using standard-of-care tests, such as detailed retinal imaging (macular optical coherence tomography and short-wavelength fundus autofluorescence) and electrophysiological testing. Exome and genome sequencing, as well as computer-assisted data analysis were used for genotyping and detection of DNA variants. A minigene-driven splicing assay was performed to validate the deleterious effects of 1 of such variants. RESULTS: We identified 8 unrelated families from Hungary, the United States, Israel, and The Netherlands with members presenting with a form of autosomal recessive and nonsyndromic retinal degeneration, predominantly described as rod-cone dystrophy but also including cases of cone/cone-rod dystrophy. Age of disease onset was very variable, with some patients experiencing first symptoms during their fourth decade of life or later. Myopia greater than 5 diopters was present in 5 of 7 cases with available refractive data, and retinal detachment was reported in 2 cases. All ascertained patients carried biallelic loss-of-function variants in UBAP1L (HGNC: 40028), a gene with unknown function and with homologies to UBAP1, encoding a protein involved in ubiquitin metabolism. One of these pathogenic variants, the intronic NM_001163692.2:c.910-7G>A substitution, was identified in 5 unrelated families. Minigene-driven splicing assays in HEK293T cells confirmed that this DNA change is responsible for the creation of a new acceptor splice site, resulting in aberrant splicing. CONCLUSION: We identified UBAP1L as a novel IRD gene. Although its function is currently unknown, UBAP1L is almost exclusively expressed in photoreceptors and the retinal pigment epithelium, hence possibly explaining the link between pathogenic variants in this gene and an ocular phenotype.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linhagem , Degeneração Retiniana , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Distrofias de Cones e Bastonetes/genética , Genes Recessivos , Predisposição Genética para Doença , Hungria , Mutação com Perda de Função , Degeneração Retiniana/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: There exist several fMRI quality assurance measures to assess scanner stability. Because they have practical and/or theoretical limitations, a different and more practical measure for instability would be desirable. PURPOSE: To develop and test a sensitive, reliable and widely applicable temporal instability measure (TIM) for fMRI quality assurance. STUDY TYPE: Technical development. PHANTOM: Spherical gel phantom. POPULATION: A total of 120 datasets from a local Philips scanner with two different receive-only head coils (32ch and 8ch, 60 datasets per coil) were collected as well as 29 additional datasets with three different receive-only head coils (20ch, 32ch, and 64ch) from two additional sites with GE (seven runs with 32ch) and Siemens scanners (seven runs with 32ch and Multiband imaging, five runs with 20ch, 32ch, and 64ch) were borrowed. FIELD STRENGTH/SEQUENCE: 2D Echo-planar-imaging (EPI). ASSESSMENT: A new TIM was proposed that is based on the eigenratio of the correlation coefficient matrix, where each entry of the matrix is a correlation coefficient between two time-points of the time-series. STATISTICAL TESTS: Nonparametric bootstrap resampling was used twice to estimate confidence intervals (CI) of the TIM values and to assess the improved sensitivity of this measure. Differences in coil performance were assessed via a nonparametric bootstrap two-sample t-test. P-values <0.05 were considered significant. RESULTS: The TIM values ranged between 60 parts-per-million and 10,780 parts-per-million across all 149 experiments. The mean CI was 2.96% and 2.16% for the 120 and 29 fMRI datasets, respectively (the repeated bootstrap analysis gave 2.9% and 2.19%, respectively). The 32ch coils of the local Philips data provided more stable measurements than the 8ch coil (observed two-sample t-values = 26.36, -0.2 and -6.2 for TIM, tSNR, and RDC, respectively. PtSNR = 0.58). DATA CONCLUSION: The proposed TIM is particularly useful for multichannel coils with spatially nonuniform receive sensitivity and overcomes several limitations of other measures. As such, it provides a reliable test for ascertaining scanner stability for fMRI experiments. EVIDENCE LEVEL: 5. TECHNICAL EFFICACY: Stage 1.
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Imagem Ecoplanar , Imageamento por Ressonância Magnética , Humanos , Imageamento por Ressonância Magnética/métodos , Imagem Ecoplanar/métodos , Imagens de Fantasmas , Reprodutibilidade dos TestesRESUMO
There is emerging evidence that sampling the blood-oxygen-level-dependent (BOLD) response with high temporal resolution opens up new avenues to study the in vivo functioning of the human brain with functional magnetic resonance imaging. Because the speed of sampling and the signal level are intrinsically connected in magnetic resonance imaging via the T1 relaxation time, optimization efforts usually must make a trade-off to increase the temporal sampling rate at the cost of the signal level. We present a method, which combines a sparse event-related stimulus paradigm with subsequent data reshuffling to achieve high temporal resolution while maintaining high signal levels (HiHi). The proof-of-principle is presented by separately measuring the single-voxel time course of the BOLD response in both the primary visual and primary motor cortices with 100-ms temporal resolution.
Assuntos
Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Humanos , Imageamento por Ressonância Magnética/métodos , Processamento de Imagem Assistida por Computador/métodos , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico/métodos , Hemodinâmica/fisiologia , OxigênioRESUMO
OBJECTIVE: Despite the prevalent use of the general linear model (GLM) in fMRI data analysis, assuming a pre-defined hemodynamic response function (HRF) for all voxels can lead to reduced reliability and may distort the inferences derived from it. To overcome the necessity of presuming a specific model for the hemodynamic response, we introduce a semi-supervised automatic detection (SAD) method. MATERIALS AND METHODS: The proposed SAD method employs a Bi-LSTM neural network to classify high temporal resolution fMRI data. Network training utilized an fMRI dataset with 75-ms temporal resolution in an iterative scheme. Classification performance was evaluated on a second fMRI dataset from the same participant, collected on a different day. Comparative analysis with the standard GLM approach was conducted to evaluate the cooperative effectiveness of the SAD method. RESULTS: The SAD method performed well based on the classification scores: true-positive rate = 0.961, area under the receiver operating curve = 0.998, true-negative rate = 0.99, F1-score = 0.979, False-negative rate = 0.038, false-discovery rate = 0.002, false-positive rate = 0.002 at 75-ms temporal resolution. CONCLUSION: SAD can detect hemodynamic responses at 75-ms temporal resolution without relying on a specific shape of an HRF. Future work could expand the use cases to include more participants and different fMRI paradigms.
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In this work dynamic models of the continuous crystallization, filtration, deliquoring, washing, and drying steps are introduced, which are developed in the open-source pharmaceutical modeling tool PharmaPy. These models enable the simulation and digital design of an integrated continuous two-stage crystallization and filtration-drying carousel system. The carousel offers an intensified process that can manufacture products with tailored properties through optimal design and control. Results show that improved crystallization design enhances overall process efficiency by improving critical material attributes of the crystal slurry for downstream filtration and drying operations. The digital design of the integrated process achieves enhanced productivity while satisfying multiple design and product quality constraints. Additionally, the impact of model uncertainty on the optimal operating conditions is investigated. The findings demonstrate the systematic process development potential of PharmaPy, providing improved process understanding, design space identification, and optimized robust operation.
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In October 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown 2-year-old common sage (Salvia officinalis L. 'Icterina') in two ornamental nurseries in Somogy and Zala counties in Hungary. The disease occurred at a frequency of 15-20% (100 to 150 symptomatic plants in each nursery). A P. cryptogea-like species was isolated consistently from necrotic root collars of many plants on carrot (CA) PARPB agar. Six isolates from the nursery in Zala county and three isolates from the nursery in Somogy county were deposited in the culture collection of Plant Protection Institute (Budapest, Hungary). All developed slightly petaloid colonies on CA agar. Chlamydospores and gametangia were not present in single and dual culture combinations of isolates. Radial colony growth was the fastest at 25°C (6.8 to 7.4 mm/day) and no growth occurred above 34°C. On mycelial discs floating in nonsterile stream water, persistent, nonpapillate, mostly ovoid to obpyriform sporangia (37.4±3.5 to 47.8±4.6 µm long and 22.3±2.6 to 29.2±3.7 µm wide) and hyphal swellings were produced abundantly. Pathogenicity of one selected isolate from each nursery was tested on 3-month-old seedlings of S. officinalis 'Icterina' in 2010. Isolates were grown for 4 weeks at 20°C on autoclaved millet grains moistened with CA broth. Infested and uninfested grains were mixed with autoclaved soil (30 cm3 grain/liter), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth room (20-25°C, 16/8 h dark/light). Pots were flooded for 24 hours on the 1st day and every 2 weeks. All and only treated plants showed symptoms of wilt associated with basal stem and root necrosis within three weeks. The trial was repeated with the same result. The pathogen could be reisolated only from the treated plants. Identity of isolates from nurseries and inoculated plants was confirmed recently by amplification and sequence analysis of the rDNA internal transcribed spacers (ITS) and gene regions of cytochrome c oxidase subunit I (coxI) and ß-tubulin (tub) according to Jung et al. (2017). BLASTn searches showed 100% identity and only 97.3-99.0% similarity to the corresponding sequences of authenthic P. pseudocryptogea and P. cryptogea strains, respectively (e.g., GenBank accession nos. KP288336-KP288342, KP288370-KP288372, KP288386-KP288392, MN872725, MN872776). Sequences of the 9 field isolates were deposited in GenBank under accession nos. OR771701-OR771709 (ITS), OR787508-OR787516 (coxI) and OR787517-OR787525 (tub). P. pseudocryptogea was delineated from P. cryptogea sensu lato (Safaiefarahani et al. 2015), which has been reported from S. officinalis in the United States (Koike 1997), and S. leucantha (Cacciola et al. 2002) and S. officinalis (Garibaldi et al. 2015) in Italy. The known natural hosts of P. pseudocryptogea includes plant species in families other than Lamiaceae (cf. Aloi et al. 2023), but it was pathogenic on the lamiaceous Plectranthus scutellarioides in artificial inoculations (Christova 2020). The pathogen is present in European nurseries (Antonelli et al. 2023). This is the first report of P. pseudocryptogea on S. officinalis in Hungary. The causal agent threatens the production of sages and other ornamentals, and its spread in Hungary should be prevented by proper disease management and phytosanitary actions.
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We investigated the association between the SDF-1-3' (c801G > A) variant and the development of diabetic macular edema (DME) or proliferative diabetic retinopathy (PDR) in a Hungarian cohort. SDF-1-3' (c801G > A) was genotyped in 103 patients with diabetic retinopathy and 31 age- and sex-matched non-diabetic controls. Central retinal and choroidal thickness was measured by swept-source optical coherence tomography. The distribution of heterozygous and homozygous SDF-1-3' (c801G > A) genotypes was similar in diabetic and control subjects. The SDF-3'(c801AA) genotype was associated with DME (n = 94 eyes, allele distribution p = 0.006, genotype distribution p = 0.01 OR: 2.48, 95% CL: 1.21-5.08) in both univariable and multivariable modelling, independent of duration and type of diabetes, HbA1C, hypertension and microalbuminuria (p = 0.03). DME occurred earlier in patients carrying the SDF-1 (c801A) allele (Kaplan-Meier analysis, log-rank test p = 0.02). A marginally significant association was found between the presence of the SDF-1 (c801A) allele and the development of PDR (n = 89 eyes, p = 0.06). The SDF-1-3' (c801A) allele also showed a correlation with central retinal (p = 0.006) and choroidal (p = 0.08) thickness. SDF-1-3' (c801G > A) is involved in the development of macular complications in DM independent of critical clinical factors, suggesting that SDF-1 may be a future therapeutic target for high-risk patients, especially those carrying the SDF-1 (c801A) allele.
Assuntos
Quimiocina CXCL12 , Retinopatia Diabética , Humanos , Quimiocina CXCL12/genética , Retinopatia Diabética/genética , Feminino , Masculino , Hungria , Pessoa de Meia-Idade , Idoso , Alelos , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Genótipo , Estudos de Casos e Controles , Tomografia de Coerência Óptica , Edema Macular/genéticaRESUMO
Cystinosis is a low-prevalence lysosomal storage disease. The pathomechanism involves abnormal functioning of the cystinosine lysosomal cystine transporter (CTNS), causing intraliposomal accumulation of the amino acid cysteine disulfide, which crystallizes and deposits in several parts of the body. The most common ophthalmic complication of cystinosis is the deposition of "gold dust" cystine crystals on the cornea, which already occurs in infancy and leads to severe photosensitivity and dry eyes as it gradually progresses with age. In the specific treatment of cystinosis, preparations containing cysteamine (CYA) are used. The availability of commercialized eyedrops for the targeted treatment is scarce, and only Cystadrops® are commercially available with strong limitations. Thus, magistral CYA-containing compounded eyedrops (CYA-CED) could have a key role in patient care; however, a rationally designed comprehensive study on the commercialized and magistral products is still missing. This work aims to build up a comprehensive study about commercialized and magistral CYA eye drops, involving pharmacokinetic and physicochemical characterization (applying mucoadhesivity, rheology test, investigation of drug release, and parallel artificial membrane permeability assays), as well as ex vivo tests, well supported by statistical analysis.
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Cistinose , Humanos , Cistinose/metabolismo , Cisteamina/uso terapêutico , Cisteamina/metabolismo , Cistina/metabolismo , Soluções Oftálmicas/uso terapêutico , Córnea/metabolismoRESUMO
EZH2 (Enhancer of zeste homolog 2) promotes tumor growth and survival through numerous mechanisms and is a promising target for novel therapeutic approaches. We aimed to characterize the expression of EZH2 in the tumors of young head-and-neck squamous cell cancer (HNSCC) patients in comparison with the general HNSCC patient population. We used formalin-fixed, paraffin-embedded tissue blocks from 68 random young HNSCC patients (≤39 years, median age: 36 years; diagnosed between 2000 and 2018), which were compared with the samples of 58 age- and gender-matched general HNSCC subjects (median age: 62 years; all diagnosed in the year 2014). EZH2 and p53 expression of the tumors was detected using immunohistochemical staining. Lower EZH2 expression was found to be characteristic of the tumors of young HNSCC patients as opposed to the general population (median EZH2 staining intensity: 1 vs. 1.5 respectively, p < 0.001; median fraction of EZH2 positive tumor cells: 40% vs. 60%, respectively, p = 0.003, Mann-Whitney). Cox analysis identified a more advanced T status (T3-4 vs. T1-2), a positive nodal status, and alcohol consumption, but neither intratumoral EZH2 nor p53 were identified as predictors of mortality in the young patient group. The lower EZH2 expression of young HNSCC patients' tumors discourages speculations of a more malignant phenotype of early-onset tumors and suggests the dominant role of patient characteristics. Furthermore, our results might indicate the possibility of an altered efficacy of the novel anti-EZH2 therapies in this patient subgroup.
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Biomarcadores Tumorais , Proteína Potenciadora do Homólogo 2 de Zeste , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Background and purpose:
Pain intensity is the most frequently assessed health domain in clinical studies among patients with low-back pain. Visual analogue scale (VAS) and Numeric rating scale (NRS) have been the mostly used measurement tools for pain intensity. We proposed to correlate these instruments to a generic health-related quality of life measurement tool in order to show the scale with superior clinical relevance.
. Methods:We used cross-sectional, convenience sampling. 120 patients with chronic low-back pain administered the 29-item Patient Reported Outcomes Measurement Information System Profile with NRS included, and the VAS scale in the National Institute of Mental Health, Neurology and Neurosurgery. We determined the correlation between PROMIS domain T-scores and VAS and NRS scores.
. Results:We performed Spearman rank correlation test to calculate the correlation coefficient. We found VAS scales measuring pain had weak to moderate correlations with all PROMIS health domains (r = 0.24–0.55). Therefore, we compared correlation of PROMIS domain scores with PROMIS pain intensity numeric rating scale and VAS scales. PROMIS domains had moderate to strong correlations with pain intensity scale (r = 0.45–0.71). PROMIS physical function short form [r = –0.65, 95% CI (–0.75) – (–0.55)] and PROMIS pain interference short form (r = 0.71, 95% CI 0.63 – 0.79) had the strongest correlation with pain intensity item.
. Conclusion:NRS has showed greater correlation with PROMIS domain T-scores than VAS scale. This may prove that NRS has greater connection to another health domains, thus it correlated more to health-related quality of life than visual scale. We recommend NRS to use in further clinical studies conducted among patients with low-back pain.
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Dor nas Costas , Qualidade de Vida , Humanos , Medição da Dor , Estudos Transversais , Escala Visual AnalógicaRESUMO
Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.
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Proteínas de Escherichia coli/química , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Proteínas Periplásmicas/química , Sítios de Ligação , Lectina de Ligação a Manose da Via do Complemento , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Periplásmicas/metabolismo , Subunidades ProteicasRESUMO
Horizontal transfer (HT) of genes between multicellular animals, once thought to be extremely rare, is being more commonly detected, but its global geographic trend and transfer mechanism have not been investigated. We discovered a unique HT pattern of Bovine-B (BovB) LINE retrotransposons in vertebrates, with a bizarre transfer direction from predators (snakes) to their prey (frogs). At least 54 instances of BovB HT were detected, which we estimate to have occurred across time between 85 and 1.3â Ma. Using comprehensive transcontinental sampling, our study demonstrates that BovB HT is highly prevalent in one geographical region, Madagascar, suggesting important regional differences in the occurrence of HTs. We discovered parasite vectors that may plausibly transmit BovB and found that the proportion of BovB-positive parasites is also high in Madagascar where BovB thus might be physically transported by parasites to diverse vertebrates, potentially including humans. Remarkably, in two frog lineages, BovB HT occurred after migration from a non-HT area (Africa) to the HT hotspot (Madagascar). These results provide a novel perspective on how the prevalence of parasites influences the occurrence of HT in a region, similar to pathogens and their vectors in some endemic diseases.
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Transferência Genética Horizontal , Parasitos , Animais , Bovinos , Geografia , Parasitos/genética , Filogenia , Comportamento Predatório , Retroelementos , Vertebrados/genéticaRESUMO
Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.
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Quimases , Quimotripsina , Inibidores de Proteases , Animais , Bovinos , Humanos , Quimases/antagonistas & inibidores , Quimases/química , Quimotripsina/química , Pancreatite/prevenção & controle , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Especificidade por Substrato , Tripsinogênio , Biblioteca de PeptídeosRESUMO
BACKGROUND: FA patients are hypersensitive to preconditioning of bone marrow transplantation. OBJECTIVE: Assessment of the power of mitomycin C (MMC) test to assign FA patients. METHODS: We analysed 195 patients with hematological disorders using spontaneous and two types of chromosomal breakage tests (MMC and bleomycin). In case of presumed Ataxia telangiectasia (AT), patients' blood was irradiated in vitro to determine the radiosensitivity of the patients. RESULTS: Seven patients were diagnosed as having FA. The number of spontaneous chromosomal aberrations was significantly higher in FA patients than in aplastic anemia (AA) patients including chromatid breaks, exchanges, total aberrations, aberrant cells. MMC-induced ≥10 break/cell was 83.9 ± 11.4% in FA patients and 1.94 ± 0.41% in AA patients (p < .0001). The difference in bleomycin-induced breaks/cell was also significant: 2.01 ± 0.25 (FA) versus 1.30 ± 0.10 (AA) (p = .019). Seven patients showed increased radiation sensitivity. Both dicentric + ring, and total aberrations were significantly higher at 3 and 6 Gy compared to controls. CONCLUSIONS: MMC and Bleomycin tests together proved to be more informative than MMC test alone for the diagnostic classification of AA patients, while in vitro irradiation tests could help detect radiosensitive-as such, individuals with AT.
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Anemia Aplástica , Anemia de Fanconi , Humanos , Anemia Aplástica/etiologia , Anemia Aplástica/genética , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Quebra Cromossômica , Diagnóstico Diferencial , Mitomicina , BleomicinaRESUMO
PURPOSE: To detect immunoglobulins in aqueous humour of AMD patients after repeated administration of intravitreal aflibercept. PATIENTS AND METHODS: Twenty-one patients (age: 77.85 ± 9.21 years) previously treated with intravitreal aflibercept due to wet type age-related macular degeneration (AMD group) and 18 age-matched control subjects (age: 69.75 ± 12.67 years) were included in this study. Patients in the AMD group received a mean of 5 intravitreal injections (min: 1 max: 17) prior to the cataract surgery. Samples of aqueous humour (50 µl) were obtained by anterior chamber paracentesis as the first step of routine cataract surgery. The IgG content of the samples was analysed by an in-house developed ELISA system. RESULTS: A significant increase in nonspecific IgG levels in the AMD group was detected compared to the control group (13.37 ± 6.65 vs. 9.44 ± 6.55 µg/ml; p = 0.03). In 11 patients, intraocular anti-aflibercept immunoglobulins could be detected (0.05 ± 0.01 µg/ml) which was significantly higher than the limit of detection for anti-aflibercept (0.04 µg/ml; p = 0.001). No correlation was found between the number of injections or the type of CNV and the aqueous level of anti-aflibercept (r = 0.02; p = 0.95). CONCLUSION: According to our results, penetration of non-specific systemic antibodies through the impaired blood-retinal barrier is higher in patients with neovascular AMD than in subjects with an intact structural barrier. Evaluation of neutralizing antibodies to anti-VEGF agents in the aqueous humour can lead us to understanding tachyphylaxis and changes in intraocular immune mechanisms due to AMD.
Assuntos
Catarata , Degeneração Macular Exsudativa , Humanos , Idoso , Idoso de 80 Anos ou mais , Pessoa de Meia-Idade , Inibidores da Angiogênese/uso terapêutico , Ranibizumab/uso terapêutico , Degeneração Macular Exsudativa/tratamento farmacológico , Degeneração Macular Exsudativa/diagnóstico , Fator A de Crescimento do Endotélio Vascular , Anticorpos Neutralizantes/uso terapêutico , Acuidade Visual , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Injeções Intravítreas , Catarata/tratamento farmacológico , Imunoglobulina G , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
BACKGROUND: Anti-vascular endothelial growth factor (VEGF) therapy is currently the most effective therapy of exudative age-related macular degeneration (AMD). The aim of this study was to assess long-term benefits of intensive aflibercept and ranibizumab anti-VEGF therapy in patients with exudative AMD. METHODS: Two clinical trial sites recruited their original subjects for a re-evaluation 7 years after the baseline visit of the phase-3 Vascular Endothelial Growth Factor (VEGF) Trap-Eye: Investigation of Efficacy and Safety in Wet Age-Related Macular Degeneration (VIEW 2) trial. Forty-seven eyes of 47 patients with AMD originally treated with ranibizumab (14 eyes) or aflibercept (33 eyes) were included. RESULTS: Mean number of injections was 17.8 ± 3.0 during participation in the VIEW 2 trial. Fourteen of 47 (30%) eyes were given additional injections with a mean number of 5.7 ± 4.5 after the trial. At a mean follow-up time of 82 ± 5 months best corrected visual acuity (BCVA) remained stable or improved (≤ 10 letters lost) in 55% of patients in the entire study population, in 43% in the ranibizumab group and in 60% in the aflibercept group. In both groups combined mean BCVA was 54 ± 13 letters at baseline, 65 ± 17 letters at the end of the intensive phase and 45 ± 25 letters at the end of follow-up. There was no statistically significant difference in BCVA between the two groups at baseline (p = 0.88) and at the end of follow-up (p = 0.40). Macular atrophy was observed in 96% of eyes, average area was 7.22 ± 6.31 mm2 with no statistically significant difference between groups (p = 0.47). Correlation between BCVA at end-of-follow-up and the area of atrophy was significant (p < 0.001). At the end of follow-up, fluid was detected in 7 of 47 eyes (15%) indicating disease activity. CONCLUSION: Long-term efficacy of aflibercept and ranibizumab was largely consistent. Following a two-year intensive therapy with as-needed regimen, BCVA was maintained or improved in almost half of the patients and in the ranibizumab group and more than half of the patients in the aflibercept group with very few injections. In a remarkable proportion of eyes, BCVA declined severely which underlines the need for long-term follow-ups and may indicate a more prolonged intensive therapy. TRIAL REGISTRATIONS: VIEW 2 study: ClinicalTrials.gov ID: NCT00637377, date of registration: March 18, 2008. Long-term follow-up: IRB nr.: SE RKEB 168/2022, ClinicalTrials.gov ID: NCT05678517, date of registration: December 28, 2022, retrospectively registered.
Assuntos
Ranibizumab , Degeneração Macular Exsudativa , Humanos , Ranibizumab/uso terapêutico , Inibidores da Angiogênese , Fatores de Crescimento Endotelial/uso terapêutico , Resultado do Tratamento , Degeneração Macular Exsudativa/diagnóstico , Degeneração Macular Exsudativa/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Injeções Intravítreas , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Fast and reliable model development frameworks are required to support current trends in modernization of pharmaceutical processing, promoting the use of digital platforms to assist process design and operation. In this work, we use a parameter estimation framework built into the PharmaPy library to determine rate parameters and uncertainty regions of different mechanistic and semi-empirical kinetic expressions for the synthesis of the drug lomustine. The parameter estimation procedure was complemented by identifiability analysis, resulting in simplified reaction mechanisms. Comparison of parameters and their uncertainty in process design was demonstrated through design space analysis, showing important differences in model prediction and the extent of their corresponding design spaces. The results of this work can serve to analyze lomustine manufacturing processes that include separation and isolation steps, where parametric sensitivity is expected to propagate along the manufacturing line and impact process feasible operation, and attainment of critical quality attributes of the product.
RESUMO
Regular physical exercise is known to lower the incidence of age-related eye diseases. We aimed to assess the acute chorioretinal alterations in older adults following intense physical strain. Seventeen senior elite athletes were recruited who underwent an aerobic exercise on a cycle ergometer and macular scanning by optical coherence tomography. A significant thinning of the entire retina was observed 1 min after exercise, followed by a thickening at 5 min, after which the thickness returned to baseline. This trend was similar in almost every single retinal layer, although a significant change was observed only in the inner retina. Choroidal thickness changes were neither significant nor did they correlate with the thickness changes of intraretinal layers. The mechanism of how these immediate retinal changes chronically impact age-related sight-threatening pathologies that, in turn, result in a substantially reduced quality of life warrants further investigation on nontrained older adults as well.