4-Hydroxybenzoic acid rescues multisystemic disease and perinatal lethality in a mouse model of mitochondrial disease.

Coenzyme Q (CoQ) deficiency syndrome is conventionally treated with limited efficacy using exogenous CoQ10. Poor outcomes result from low absorption and bioavailability of CoQ10 and the clinical heterogenicity of the disease. Here, we demonstrate that supplementation with 4-hydroxybenzoic acid (4HB), the precursor of the benzoquinone ring in the CoQ biosynthetic pathway, completely rescues multisystemic disease and perinatal lethality in a mouse model of CoQ deficiency. 4HB stimulates endogenous CoQ biosynthesis in tissues of Coq2 mutant mice, normalizing mitochondrial function and rescuing cardiac insufficiency, edema, and neurodevelopmental delay. In contrast, exogenous CoQ10 supplementation falls short in fully restoring the phenotype. The treatment is translatable to human use, as proven by in vitro studies in skin fibroblasts from patients with pathogenic variants in COQ2. The therapeutic approach extends to other disorders characterized by deficiencies in the production of 4HB and early steps of CoQ biosynthesis and instances of secondary CoQ deficiency.

Membrane fission via transmembrane contact.

Division of intracellular organelles often correlates with additional membrane wrapping, e.g., by the endoplasmic reticulum or the outer mitochondrial membrane. Such wrapping plays a vital role in proteome and lipidome organization. However, how an extra membrane impacts the mechanics of the division has not been investigated. Here we combine fluorescence and cryo-electron microscopy experiments with self-consistent field theory to explore the stress-induced instabilities imposed by membrane wrapping in a simple double-membrane tubular system. We find that, at physiologically relevant conditions, the outer membrane facilitates an alternative pathway for the inner-tube fission through the formation of a transient contact (hemi-fusion) between both membranes. A detailed molecular theory of the fission pathways in the double membrane system reveals the topological complexity of the process, resulting both in leaky and leakless intermediates, with energies and topologies predicting physiological events.

Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif.

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.

Neuropharmacological Effects of the Dichloromethane Extract from the Stems of Argemone ochroleuca Sweet (Papaveraceae) and Its Active Compound Dihydrosanguinarine.

Argemone ochroleuca Sweet (Papaveraceae) is used in folk medicine as a sedative and hypnotic agent. This study aimed to evaluate the anxiolytic-like, sedative, antidepressant-like, and anticonvulsant activities of a dichloromethane extract of A. ochroleuca stems (AOE), chemically standardized using gas chromatography-mass spectrometry (GC-MS), and its active compound dihydrosanguinarine (DHS). The anxiolytic-like, sedative, antidepressant-like, and anticonvulsant activities of the AOE (0.1-50 mg/kg p.o.) and DHS (0.1-10 mg/kg p.o.) were evaluated using murine models. A possible mechanism for the neurological actions induced by the AOE or DHS was assessed using inhibitors of neurotransmission pathways and molecular docking. Effective dose 50 (ED50) values were calculated by a linear regression analysis. The AOE showed anxiolytic-like activity in the cylinder exploratory test (ED50 = 33 mg/kg), and antidepressant-like effects in the forced swimming test (ED50 = 3 mg/kg) and the tail suspension test (ED50 = 23 mg/kg), whereas DHS showed anxiolytic-like activity (ED50 = 2 mg/kg) in the hole board test. The AOE (1-50 mg/kg) showed no locomotive affectations or sedation in mice. A docking study revealed the affinity of DHS for α2-adrenoreceptors and GABAA receptors. The anxiolytic-like and anticonvulsant effects of the AOE are due to GABAergic participation, whereas the antidepressant-like effects of the AOE are due to the noradrenergic system. The noradrenergic and GABAergic systems are involved in the anxiolytic-like actions of DHS.

Allosteric control of dynamin-related protein 1-catalyzed mitochondrial fission through a conserved disordered C-terminal Short Linear Motif.

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial fission, but the regulatory mechanisms remain ambiguous. Here we found that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, leading to the fission of model membranes in vitro. In vivo, the availability of the native CT-SLiM is a requirement for productive mitochondrial fission, as both non-native extension and deletion of the CT-SLiM severely impair its progression. Thus, contrary to prevailing models, Drp1-catalyzed mitochondrial fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.

Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities.

Cell density is an important factor in all microbiome research, where interactions are of interest. It is also the most important parameter for the operation and control of most biotechnological processes. In the past, cell density determination was often performed offline and manually, resulting in a delay between sampling and immediate data processing, preventing quick action. While there are now some online methods for rapid and automated cell density determination, they are unable to distinguish between the different cell types in bacterial communities. To address this gap, an online automated flow cytometry procedure is proposed for real-time high-resolution analysis of bacterial communities. On the one hand, it allows for the online automated calculation of cell concentrations and, on the other, for the differentiation between different cell subsets of a bacterial community. To achieve this, the OC-300 automation device (onCyt Microbiology, Zürich, Switzerland) was coupled with the flow cytometer CytoFLEX (Beckman Coulter, Brea, USA). The OC-300 performs the automatic sampling, dilution, fixation and 4',6-diamidino-2-phenylindole (DAPI) staining of a bacterial sample before sending it to the CytoFLEX for measurement. It is demonstrated that this method can reproducibly measure both cell density and fingerprint-like patterns of bacterial communities, generating suitable data for powerful automated data analysis and interpretation pipelines. In particular, the automated, high-resolution partitioning of clustered data into cell subsets opens up the possibility of correlation analysis to identify the operational or abiotic/biotic causes of community disturbances or state changes, which can influence the interaction potential of organisms in microbiomes or even affect the performance of individual organisms.

In Vivo Neuropharmacological Effects of Neophytadiene.

Neophytadiene (NPT) is a diterpene found in the methanolic extracts of Crataeva nurvala and Blumea lacera, plants reported with anxiolytic-like activity, sedative properties, and antidepressant-like actions; however, the contribution of neophytadiene to these effects is unknown. This study determined the neuropharmacological (anxiolytic-like, antidepressant-like, anticonvulsant, and sedative) effects of neophytadiene (0.1-10 mg/kg p.o.) and determined the mechanisms of action involved in the neuropharmacological actions using inhibitors such as flumazenil and analyzing the possible interaction of neophytadiene with GABA receptors using a molecular docking study. The behavioral tests were evaluated using the light-dark box, elevated plus-maze, open field, hole-board, convulsion, tail suspension, pentobarbital-induced sleeping, and rotarod. The results showed that neophytadiene exhibited anxiolytic-like activity only to the high dose (10 mg/kg) in the elevated plus-maze and hole-board tests, and anticonvulsant actions in the 4-aminopyridine and pentylenetetrazole-induced seizures test. The anxiolytic-like and anticonvulsant effects of neophytadiene were abolished with the pre-treatment with 2 mg/kg flumazenil. In addition, neophytadiene showed low antidepressant effects (about 3-fold lower) compared to fluoxetine. On other hand, neophytadiene had no sedative or locomotor effects. In conclusion, neophytadiene exerts anxiolytic-like and anticonvulsant activities with the probable participation of the GABAergic system.

Neuropharmacological Activities of Ceiba aesculifolia (Kunth) Britten & Baker f (Malvaceae).

Ceiba aesculifolia (Kunth) Britten & Baker f (Malvaceae) is used for the folk treatment of mood disorders. C. aesculifolia bark was extracted in ethanol, and the extract (CAE) was chemically standardized using gas chromatography-mass spectrometry (GC-MS). This study evaluated the effects of CAE (10-100 mg/kg p.o.) on anxiolytic-like activity, sedation, locomotor activity, depression-like activity, and spatial working memory using in vivo rodent models. A possible mechanism for the anxiolytic-like and antidepressant-like actions induced by CAE was assessed using neurotransmission pathway inhibitors. Myristic acid was one of the compounds found in CAE using GC-MS. This study also evaluated the anxiolytic-like activity and the sedative actions of myristic acid and assessed a possible mechanism of action using neurotransmission pathway inhibitors and an in silico analysis. CAE elicited anxiolytic-like activity and antidepressant-like effects (ED50 = 57 mg/kg). CAE (10-100 mg/kg) did not affect locomotor coordination or induce sedation. The anxiolytic-like and antidepressant-like actions of CAE were reverted by prazosin, suggesting a possible participation of the noradrenergic system. The anxiolytic-like activity of myristic acid was reverted by the co-administration of prazosin and partially reverted by ketanserin. The docking study revealed that myristic acid can form favorable interactions within 5-HT2A and α1A-adrenoreceptor binding pockets.

Prevention, Diagnosis and Management of Post-Surgical Mediastinitis in Adults Consensus Guidelines of the Spanish Society of Cardiovascular Infections (SEICAV), the Spanish Society of Thoracic and Cardiovascular Surgery (SECTCV) and the Biomedical Research Centre Network for Respiratory Diseases (CIBERES).

This is a consensus document of the Spanish Society of Cardiovascular Infections (SEICAV), the Spanish Society of Thoracic and Cardiovascular Surgery (SECTCV) and the Biomedical Research Centre Network for Respiratory Diseases (CIBERES). These three entities have brought together a multidisciplinary group of experts that includes anaesthesiologists, cardiac and cardiothoracic surgeons, clinical microbiologists, infectious diseases and intensive care specialists, internal medicine doctors and radiologists. Despite the clinical and economic consequences of sternal wound infections, to date, there are no specific guidelines for the prevention, diagnosis and management of mediastinitis based on a multidisciplinary consensus. The purpose of the present document is to provide evidence-based guidance on the most effective diagnosis and management of patients who have experienced or are at risk of developing a post-surgical mediastinitis infection in order to optimise patient outcomes and the process of care. The intended users of the document are health care providers who help patients make decisions regarding their treatment, aiming to optimise the benefits and minimise any harm as well as the workload.

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.

KnowSeq R-Bioc package: The automatic smart gene expression tool for retrieving relevant biological knowledge.

KnowSeq R/Bioc package is designed as a powerful, scalable and modular software focused on automatizing and assembling renowned bioinformatic tools with new features and functionalities. It comprises a unified environment to perform complex gene expression analyses, covering all the needed processing steps to identify a gene signature for a specific disease to gather understandable knowledge. This process may be initiated from raw files either available at well-known platforms or provided by the users themselves, and in either case coming from different information sources and different Transcriptomic technologies. The pipeline makes use of a set of advanced algorithms, including the adaptation of a novel procedure for the selection of the most representative genes in a given multiclass problem. Similarly, an intelligent system able to classify new patients, providing the user the opportunity to choose one among a number of well-known and widespread classification and feature selection methods in Bioinformatics, is embedded. Furthermore, KnowSeq is engineered to automatically develop a complete and detailed HTML report of the whole process which is also modular and scalable. Biclass breast cancer and multiclass lung cancer study cases were addressed to rigorously assess the usability and efficiency of KnowSeq. The models built by using the Differential Expressed Genes achieved from both experiments reach high classification rates. Furthermore, biological knowledge was extracted in terms of Gene Ontologies, Pathways and related diseases with the aim of helping the expert in the decision-making process. KnowSeq is available at Bioconductor (https://bioconductor.org/packages/KnowSeq), GitHub (https://github.com/CasedUgr/KnowSeq) and Docker (https://hub.docker.com/r/casedugr/knowseq).

A new treehopper species of Cladonota (Stål) (Hemiptera: Membracidae:Membracinae: Hypsoprorini) from Mexico.

A new species of treehopper from Puebla, Mexico is described, Cladonota (Lecythifera) crucifixa n. sp., with illustrations and biological notes and compared with C. hoffmanni (Peláez, 1940).

Microfluidic chip with pillar arrays for controlled production and observation of lipid membrane nanotubes.

Lipid membrane nanotubes (NTs) are a widespread template for in vitro studies of cellular processes happening at high membrane curvature. Traditionally NTs are manufactured one by one, using sophisticated membrane micromanipulations, while simplified methods for controlled batch production of NTs are in growing demand. Here we propose a lab-on-a-chip (LOC) approach to the simultaneous formation of multiple NTs with length and radius controlled by the chip design. The NTs form upon rolling silica microbeads covered by lipid lamellas over the pillars of a polymer micropillar array. The array's design and surface chemistry set the geometry of the resulting free-standing NTs. The integration of the array inside a microfluidic chamber further enables fast and turbulence-free addition of components, such as proteins, to multiple preformed NTs. This LOC approach to NT production is compatible with the use of high power objectives of a fluorescence microscope, making real-time quantification of the different modes of the protein activity in a single experiment possible.

Prokaryotic Expression of the Immunoglobulin's Domains of CRTAM to Characterize a Monoclonal Antibody.

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.

Clinical and prognostic differences between methicillin-resistant and methicillin-susceptible Staphylococcus aureus infective endocarditis.

BACKGROUND: S. aureus (SA) infective endocarditis (IE) has a very high mortality, attributed to the age and comorbidities of patients, inadequate or delayed antibiotic treatment, and methicillin resistance, among other causes. The main study objective was to analyze epidemiological and clinical differences between IE by methicillin-resistant versus methicillin-susceptible SA (MRSA vs. MSSA) and to examine prognostic factors for SA endocarditis, including methicillin resistance and vancomycin minimum inhibitory concentration (MIC) values > 1 µg/mL to MRSA. METHODS: Patients with SA endocarditis were consecutively and prospectively recruited from the Andalusia endocarditis cohort between 1984 and January 2017. RESULTS: We studied 437 patients with SA endocarditis, which was MRSA in 13.5% of cases. A greater likelihood of history of COPD (OR 3.19; 95% CI 1.41-7.23), invasive procedures, or recognized infection focus in the 3 months before IE onset (OR 2.9; 95% CI 1.14-7.65) and of diagnostic delay (OR 3.94; 95% CI 1.64-9.5) was observed in patients with MRSA versus MSSA endocarditis. The one-year mortality rate due to SA endocarditis was 44.3% and associated with decade of endocarditis onset (1985-1999) (OR 8.391; 95% CI (2.82-24.9); 2000-2009 (OR 6.4; 95% CI 2.92-14.06); active neoplasm (OR 6.63; 95% CI 1.7-25.5) and sepsis (OR 2.28; 95% CI 1.053-4.9). Methicillin resistance was not associated with higher IE-related mortality (49.7 vs. 43.1%; p = 0.32). CONCLUSION: MRSA IE is associated with COPD, previous invasive procedure or recognized infection focus, and nosocomial or healthcare-related origin. Methicillin resistance does not appear to be a decisive prognostic factor for SA IE.

Mieloma múltiple en pacientes hospitalizados en hospital provincial pinareño / Multiple myeloma in patients admitted the provincial hospital of Pinar del Río

RESUMEN Introducción: el mieloma múltiple es un tumor derivado de las células plasmáticas, un tipo de células sanguíneas situadas en la médula ósea que se encargan de producir anticuerpos que sirven para combatir los gérmenes. Objetivo: describir el comportamiento clínico epidemiológico del mieloma múltiple, en pacientes hospitalizados en los servicios de Medicina Interna y Geriatría del Hospital Clínico Quirúrgico Docente Dr. León Cuervo Rubio de Pinar del Río, durante los años 2017 y 2018. Métodos: se realizó un estudio observacional, descriptivo, longitudinal y prospectivo. El universo estuvo conformado por 31 pacientes con diagnóstico de mieloma múltiple, la muestra se conformó por 25 pacientes mediante un muestreo simple aleatorio. Resultados: se observó predominio de la enfermedad en los pacientes masculinos y de los grupos de edades de 70-79 años, se obtuvo como principales comorbilidades y complicaciones la anemia y la insuficiencia renal. Conclusiones: es importante el conocimiento del comportamiento clínico epidemiológico del mieloma múltiple para un diagnóstico oportuno e integral y el mejoramiento de la calidad de vida del paciente.

ABSTRACT Introduction: multiple myeloma is a tumor derived from plasma cells, a type of blood cell in the bone marrow that produces antibodies to fight the germs. Objective: to describe the clinical epidemiological behavior of multiple myeloma in patients hospitalized in the Internal Medicine and Geriatrics services at Dr. León Cuervo Rubio Clinical Surgical Teaching Hospital during the years 2017 and 2018, in Pinar del Río. Methods: an observational, descriptive, longitudinal and prospective study was conducted. The target group included 31 patients diagnosed with multiple myeloma; the sample comprised 25 patients by simple random sample. Results: a prevalence of the disease was observed in male patients and in the age group from 70-79 years old. The main comorbidities and complications presented by patients with multiple myeloma were anemia in all of them, and kidney failure. Conclusions: knowledge of the clinical epidemiological behavior of multiple myeloma is important in order to achieve a timely and comprehensive diagnosis to improve the patient's quality of life.

Towards Improving Skin Cancer Diagnosis by Integrating Microarray and RNA-Seq Datasets.

Many clinical studies have revealed the high biological similarities existing among different skin pathological states. These similarities create difficulties in the efficient diagnosis of skin cancer, and encourage to study and design new intelligent clinical decision support systems. In this sense, gene expression analysis can help find differentially expressed genes (DEGs) simultaneously discerning multiple skin pathological states in a single test. The integration of multiple heterogeneous transcriptomic datasets requires different pipeline stages to be properly designed: from suitable batch merging and efficient biomarker selection to automated classification assessment. This article presents a novel approach addressing all these technical issues, with the intention of providing new sights about skin cancer diagnosis. Although new future efforts will have to be made in the search for better biomarkers recognizing specific skin pathological states, our study found a panel of 8 highly relevant multiclass DEGs for discerning up to 10 skin pathological states: 2 healthy skin conditions a priori, 2 cataloged precancerous skin diseases and 6 cancerous skin states. Their power of diagnosis over new samples was widely tested by previously well-trained classification models. Robust performance metrics such as overall and mean multiclass F1-score outperformed recognition rates of 94% and 80%, respectively. Clinicians should give special attention to highlighted multiclass DEGs that have high gene expression changes present among them, and understand their biological relationship to different skin pathological states.

Equality or disparity? Cardiovascular risk factors and their correlation within the metabolic syndrome. The secret of gods.

AIMS: The primary objective of our study is to determine the prevalence of the metabolic syndrome in the population. The secondary objective is to determine the prevalence of cardiovascular risk factors and their correlation with the metabolic syndrome. MATERIAL AND METHODS: The sample for the study was obtained by means of a consecutive population-based demonstration in 803 adults over 18 years of age belonging to the labor force of the company Grupo Delta SA. The study was carried out according to the guidelines of the Declaration of Helsinki. The individuals included in the study voluntarily participated, once informed of the purpose of the study, giving their prior verbal consent, to the company's human resources department, in the case of Delta Group workers. RESULTS: 23.8% of the population has metabolic syndrome. Each cardiovascular risk factor has different importance in the metabolic syndrome. CONCLUSION: The correlation of cardiovascular risk factors with the metabolic syndrome differs from one another.

Combining patch-clamping and fluorescence microscopy for quantitative reconstitution of cellular membrane processes with Giant Suspended Bilayers.

In vitro reconstitution and microscopic visualization of membrane processes is an indispensable source of information about a cellular function. Here we describe a conceptionally novel free-standing membrane template that facilitates such quantitative reconstitution of membrane remodelling at different scales. The Giant Suspended Bilayers (GSBs) spontaneously swell from lipid lamella reservoir deposited on microspheres. GSBs attached to the reservoir can be prepared from virtually any lipid composition following a fast procedure. Giant unilamellar vesicles can be further obtained by GSB detachment from the microspheres. The reservoir stabilizes GSB during deformations, mechanical micromanipulations, and fluorescence microscopy observations, while GSB-reservoir boundary enables the exchange of small solutes with GSB interior. These unique properties allow studying macro- and nano-scale membrane deformations, adding membrane-active compounds to both sides of GSB membrane and applying patch-clamp based approaches, thus making GSB a versatile tool for reconstitution and quantification of cellular membrane trafficking events.

Is TCR/pMHC Affinity a Good Estimate of the T-cell Response? An Answer Based on Predictions From 12 Phenotypic Models.

On the T-cell surface the TCR is the only molecule that senses antigen, and the engagement of TCR with its specific antigenic peptide (agonist)/MHC complex (pMHC) is determined by the biochemical parameters of the TCR-pMHC interaction. This interaction is the keystone of the adaptive immune response by triggering intracellular signaling pathways that induce the expression of genes required for T cell-mediated effector functions, such as T cell proliferation, cytokine secretion and cytotoxicity. To study the TCR-pMHC interaction one of its properties most extensively analyzed has been TCR-pMHC affinity. However, and despite of intensive experimental research, the results obtained are far from conclusive. Here, to determine if TCR-pMHC affinity is a reliable parameter to characterize T-cell responses, a systematic study has been performed based on the predictions of 12 phenotypic models. This approach has the advantage that allow us to study the response of a given system as a function of only those parameters in which we are interested while other system parameters remain constant. A little surprising, only the simple occupancy model predicts a direct relationship between affinity and response so that an increase in affinity always leads to larger responses. Conversely, in the others more elaborate models this clear situation does not occur, i.e., that a general positive correlation between affinity and immune response does not exist. This is mainly because affinity values are given by the quotient kon/koff where kon and koff are the rate constants of the binding process (i.e., affinity is in fact the quotient of two parameters), so that different sets of these rate constants can give the same value of affinity. However, except in the occupancy model, the predicted T-cell responses depend on the individual values of kon and koff rather than on their quotient kon/koff. This allows: a) that systems with the same affinity can show quite different responses; and b) that systems with low affinity may exhibit larger responses than systems with higher affinities. This would make affinity a poor estimate of T-cell responses and, as a result, data correlations between affinity and immune response should be interpreted and used with caution.