Therapeutic Targets of Monoclonal Antibodies Used in the Treatment of Cancer: Current and Emerging.

Cancer is one of the leading global causes of death and disease, and treatment options are constantly evolving. In this sense, the use of monoclonal antibodies (mAbs) in immunotherapy has been considered a fundamental aspect of modern cancer therapy. In order to avoid collateral damage, it is indispensable to identify specific molecular targets or biomarkers of therapy and/or diagnosis (theragnostic) when designing an appropriate immunotherapeutic regimen for any type of cancer. Furthermore, it is important to understand the currently employed mAbs in immunotherapy and their mechanisms of action in combating cancer. To achieve this, a comprehensive understanding of the biology of cancer cell antigens, domains, and functions is necessary, including both those presently utilized and those emerging as potential targets for the design of new mAbs in cancer treatment. This review aims to provide a description of the therapeutic targets utilized in cancer immunotherapy over the past 5 years, as well as emerging targets that hold promise as potential therapeutic options in the application of mAbs for immunotherapy. Additionally, the review explores the mechanisms of actin of the currently employed mAbs in immunotherapy.

Design of a Low-Cost Diffuse Optical Mammography System for Biomedical Image Processing in Breast Cancer Diagnosis.

Worldwide, breast cancer is the most common type of cancer that mainly affects women. Several diagnosis techniques based on optical instrumentation and image analysis have been developed, and these are commonly used in conjunction with conventional diagnostic devices such as mammographs, ultrasound, and magnetic resonance imaging of the breast. The cost of using these instruments is increasing, and developing countries, whose deaths indices due to breast cancer are high, cannot access conventional diagnostic methods and have even less access to newer techniques. Other studies, based on the analysis of images acquired by traditional methods, require high resolutions and knowledge of the origin of the captures in order to avoid errors. For this reason, the design of a low-cost diffuse optical mammography system for biomedical image processing in breast cancer diagnosis is presented. The system combines the acquisition of breast tissue photographs, diffuse optical reflectance (as a biophotonics technique), and the processing of digital images for the study and diagnosis of breast cancer. The system was developed in the form of a medical examination table with a 638 nm red-light source, using light-emitted diode technology (LED) and a low-cost web camera for the acquisition of breast tissue images. The system is automatic, and its control, through a graphical user interface (GUI), saves costs and allows for the subsequent analysis of images using a digital image-processing algorithm. The results obtained allow for the possibility of planning in vivo measurements. In addition, the acquisition of images every 30° around the breast tissue could be used in future research in order to perform a three-dimensional (3D) reconstruction and an analysis of the captures through deep learning techniques. These could be combined with virtual, augmented, or mixed reality environments to predict the position of tumors, increase the likelihood of a correct medical diagnosis, and develop a training system for specialists. Furthermore, the system allows for the possibility to develop analysis of optical characterization for new phantom studies in breast cancer diagnosis through bioimaging techniques.

Frecuencia de perforaciones radiculares en tratamientos endodónticos realizados por estudiantes de postgrados chilenos

Introducción: Este estudio evaluó la frecuencia de perforaciones radiculares provocadas por estudiantes de primer y segundo año de la especialidad de Endodoncia de la Facultad de Odontología de la Universidad Andrés Bello (UNAB), Santiago (Chile), entre 2019 y marzo de 2021. Materiales y Métodos: Se realizó un estudio observacional descriptivo a través de datos recopilados retrospectivamente en fichas clínicas de pacientes atendidos en el área de postgrado en Endodoncia de la universidad, durante 2019, 2020 y hasta marzo de 2021. Resultados: Un total de 569 pacientes fueron atendidos en el período, 118 fueron atendidos por estudiantes de primer año y 451 por los de segundo año. La frecuencia de perforaciones del primer año fue de un 0% y la del segundo año del 2 % (n = 9). Considerando el total general de pacientes, la frecuencia relativa de perforaciones fue de apenas 1,6 %. Con respecto a ubicación, 3 perforaciones fueron en el tercio cervical de la raíz, 3 en el tercio medio, 2 en el tercio apical y 1 en el piso cameral. Ninguna perforación resultó en la indicación inmediata de extracción y todas fueron selladas con materiales a base de silicato de calcio. Conclusiones: La frecuencia de perforaciones radiculares por los estudiantes de postgrado fue muy baja, lo que podría evidenciar la seguridad de los protocolos institucionales de tratamiento y enseñanza. La mayor ocurrencia de perforaciones fue con los estudiantes de segundo año, lo cual puede ser atribuido a que tratan casos de mayor complejidad.

Introduction: The present study evaluated the frequency of root perforations caused by first- and second-year students of the Endodontics specialty of the Faculty of Dentistry of the UNAB University in Santiago (Chile), between January 2019 and March 2021. Materials and Methods: A descriptive observational study was carried out through data collected retrospectively in clinical records of patients treated in the Postgraduate Endodontics Clinic of the Andrés Bello University (UNAB) during 2019, 2020 and until March 2021. Results: A total of 569 patients were treated during the period, 118 patients were seen by first-year students and 451 by second-year students. The frequency of perforations in the first year was 0% and in the second year it was 2% (n=9). Considering the overall total number of patients, the relative frequency of perforations was only 1.6%. Regarding location, 3 perforations were in the cervical third of the root, 3 in the middle third, 2 in the apical third and 1 in the pulp chamber floor. No perforation resulted in the immediate indication of extraction and all were sealed with calcium silicate-based materials. Conclusions: Te frequency of root perforations by postgraduate students was very low, which could evidence the safety of institutional treatment and teaching protocols. The greatest occurrence of perforations was with second-year students, which can be attributed to the fact that they treat cases of greater complexity.

Human adipose mesenchymal stromal cells growing into PCL-nHA electrospun scaffolds undergo hypoxia adaptive ultrastructural changes.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.

Norcantharidin toxicity profile: an in vivo murine study.

Norcantharidin (NCTD) is the demethylated analog of cantharidin, with allegedly reduced toxicity. However, there is still limited information regarding its posology and potential risk in its use in cancer treatment. Healthy BDF1 mice were intraperitoneally administered with norcantharidin (0, 3, 6, 12, and 25 mg/kg) every 24 h for 6 days. Survivor mice were euthanized, and the brain, lungs, kidneys, spleen, and liver were procured for enzymatic and histopathological analysis in the liver and kidney. DL50 were 8.86 mg/kg for females and 11.77 mg/kg for males. The treatments with 3.0 mg/kg and 6.0 mg/kg significantly modified the phosphorylase, alanine transaminase, and γ-glutamyl transferase activities; however, an organ-specific response was detected. A significant dose-dependent decrease was observed in the kidney for ROS, while the liver had the opposite effect. Histopathological analysis revealed a significant elevation in hepatocytes' nuclei average size and total area (3 mg/kg), as well as centrilobular vein and adjacent sinusoidal capillaries showed a significant difference. The portal triad presented a significant difference in veins and capillarity count in 6 mg/kg. Renal samples showed cortex convoluted tubules' average size significantly augmented in both doses' groups, and tubule count was found augmented in 6 mg/kg. These physiological effects of NCTD can be exploited as treatment strategies if able to operate in an established posology and proper testing.

Células madre en odontología: nuevas perspectivas / Stem cells in dentistry: new perspectives

En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)

In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)

When it is time to decide: factors associated to the choice of a medical specialty / Cuando es tiempo de decidir: factores asociados a la elección de especialidades médicas

Abstract After finishing their initial education, physicians can enter post-graduate training programs. In Chile, there are multiple medical specialties options. This review synthesizes the factors associated with the choice of specialty, according to international publications. Students coming from rural areas or small towns would tend to choose Family Medicine. Men prefer specialties such as Surgery. Having relatives in the medical profession, specializing in a specific field, also influence the choice. Concerning interests and preferences, these are linked to personal traits and some biological variables. The inclination towards clinical or surgical fields could be predicted. Academically, the curriculum, good learning experiences and teacher models favor choice, while the harassment suffered could contribute to avoidance. Besides, both prestige and economic prospects are influential factors in the choice and specialties such as Surgery are preferred. The choice of specialty must reach a balance between the students' interests and public health needs. Identifying these factors would allow a better understanding of the present shortfall (or surplus) in certain fields and contribute to the definition of policies and decision-making.

Concluida su formación, los médicos pueden acceder a programas de formación de postgrado; en Chile existen múltiples opciones de especialización médica. Esta revisión narrativa busca sintetizar los diversos factores asociados con la elección de especialidad médica según la literatura internacional. Entre los resultados se observa que los alumnos provenientes de zonas rurales o ciudades pequeñas tenderían a elegir Medicina Familiar. Los hombres tendrían mayor predilección por especialidades como Cirugía. También influiría ser hijos de médicos especialistas en un área específica. En cuanto a los intereses y preferencias, éstas se conjugan con rasgos de personalidad y algunas variables biológicas, que podrían predecir la elección de especialidades hacia el tipo clínico o quirúrgico. En lo académico, el curriculum, una buena experiencia y modelos docentes favorecen la elección, mientras el maltrato sufrido podría contribuir al descarte. Además, tanto el prestigio como el potencial económico de ciertas especialidades ha sido un factor influyente en la elección: se prefieren especialidades como Cirugía. La elección de especialidad debería encontrar un equilibrio entre los intereses del estudiante y las necesidades de salud pública. Identificar todos estos factores, permitiría una mejor comprensión de la actual escasez o sobrecarga en determinados campos, y contribuiría en la construcción de políticas y toma de decisiones, con el fin de mejorar el reclutamiento de estudiantes.

14-3-3ß isoform is specifically acetylated at Lys51 during differentiation to the osteogenic lineage.

The 14-3-3 protein family binds and regulates hundreds of serine/threonine phosphorylated proteins as an essential component of many signaling networks. Specific biological functions are currently been discovered for each of its seven isoforms in mammals. These proteins have been traditionally considered unregulated; however, its acetylation in an essential lysine residue, causing its inactivation, was recently published. Here, we studied the acetylation state of this lysine 49/51 during the osteogenic differentiation of human adipose-derived stem cells. We found that during this process, the levels of 14-3-3ß (but not its isoform 14-3-3γ) acK49/51 increase, representing the first report linking this PTM to a specific isoform and a cellular process. Our results suggested that this posttranslational modification could be catalyzed by the HBO1 acetyltransferase, as overexpression of HBO1 increased specifically 14-3-3 acK49/51 acetylation. Acetylated 14-3-3 proteins are located primarily in the nucleus, where their active state has been described to bind H3 histones and many transcription factors. The inhibition of the expression of different isoforms showed that the specific silencing of the 14-3-3ß gene, but not γ, increased significantly the osteogenic potential of the cells. This result correlated to the increase in acetylation of 14-3- 3ß Lys 49/51 during osteogenesis. The possible role of this PTM in osteogenesis is discussed.

Analysis of SARS-CoV-2 nucleocapsid phosphoprotein N variations in the binding site to human 14-3-3 proteins.

The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of ∼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.

When it is time to decide: factors associated to the choice of a medical specialty.

After finishing their initial education, physicians can enter post-graduate training programs. In Chile, there are multiple medical specialties options. This review synthesizes the factors associated with the choice of specialty, according to international publications. Students coming from rural areas or small towns would tend to choose Family Medicine. Men prefer specialties such as Surgery. Having relatives in the medical profession, specializing in a specific field, also influence the choice. Concerning interests and preferences, these are linked to personal traits and some biological variables. The inclination towards clinical or surgical fields could be predicted. Academically, the curriculum, good learning experiences and teacher models favor choice, while the harassment suffered could contribute to avoidance. Besides, both prestige and economic prospects are influential factors in the choice and specialties such as Surgery are preferred. The choice of specialty must reach a balance between the students' interests and public health needs. Identifying these factors would allow a better understanding of the present shortfall (or surplus) in certain fields and contribute to the definition of policies and decision-making.

Combined methods of optical spectroscopy and artificial intelligence in the assessment of experimentally induced non-alcoholic fatty liver.

BACKGROUND AND OBJECTIVE: Due to the existing prevalence of nonalcoholic fatty liver disease (NAFLD) and its relation to the epidemic of obesity in the general population, it is imperative to develop detection and evaluation methods of the early stages of the disease with improved efficacy over the current diagnostic approaches. We aimed to obtain an improved diagnosis, combining methods of optical spectroscopy -diffuse reflectance and fluorescence- with statistical data analysis applied to detect early stages of NAFLD. METHODS: Statistical analysis scheme based on quadratic discriminant analysis followed by canonical discriminant analysis were applied to the diffuse reflectance data combined with endogenous fluorescence spectral data excited at one of these wavelengths: 330, 365, 385, 405 or 415 nm. The statistical scheme was also applied to the combinations of fluorescence spectrum (405 nm) with each one of the other fluorescence spectra. Details of the developed software, including the application of machine learning algorithms to the combination of spectral data followed by classification statistical schemes, are discussed. RESULTS: Steatosis progression was differentiated with little classification error (≤1.3%) by using diffuse reflectance and endogenous fluorescence at different wavelengths. Similar results were obtained using fluorescence at 405 nm and one of the other fluorescence spectra (classification error ≤1.0%). Adding the corresponding areas under the curves to the above combinations of spectra diminished errors to 0.6% and 0.3% or less, respectively. The best results for the compounded reflectance-plus-fluorescence spectra were obtained with fluorescence spectra excited at 415 nm with a total classification error of 0.2%; for the combination of the 405nm-excited fluorescence spectrum with another fluorescence spectrum, the best results were achieved for 385 nm, for which total relative classification error amounted 0.4%. The consideration of the area under the spectral curves further improved both classifiers, reducing the error to 0.0% in both cases. CONCLUSION: Spectrometric techniques combined with statistical processing are a promising tool to improve steatosis classification through a label free approach. However, statistical schemes here applied, might result complex for the everyday medical practice, the designed software including machine learning algorithms is able to render automatic classification of samples according to their steatosis grade with low error.

14-3-3ε protein-loaded 3D hydrogels favor osteogenesis.

3D printing has emerged as vanguard technique of biofabrication to assemble cells, biomaterials and biomolecules in a spatially controlled manner to reproduce native tissues. In this work, gelatin methacrylate (GelMA)/alginate hydrogel scaffolds were obtained by 3D printing and 14-3-3ε protein was encapsulated in the hydrogel to induce osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASC). GelMA/alginate-based grid-like structures were printed and remained stable upon photo-crosslinking. The viscosity of alginate allowed to control the pore size and strand width. A higher viscosity of hydrogel ink enhanced the printing accuracy. Protein-loaded GelMA/alginate-based hydrogel showed a clear induction of the osteogenic differentiation of hASC cells. The results are relevant for future developments of GelMA/alginate for bone tissue engineering given the positive effect of 14-3-3ε protein on both cell adhesion and proliferation.

Engineering Saccharomyces cerevisiae for the Overproduction of ß-Ionone and Its Precursor ß-Carotene.

ß-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment ß-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current ß-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of ß-carotene and its subsequent conversion to ß-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on ß-carotene accumulation, and later, on ß-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For ß-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest ß-carotene-producing strains, reaching up to 33 mg/L of ß-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing ß-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Fluorescence spectroscopy on paraffin-preserved human liver samples to classify several grades of fibrosis.

Nowadays, it is well established that biopsy is the gold standard for medical diagnosis of liver disease; however, recent studies have shown numerous discrepancies in biopsy assessment, even when it is evaluated by senior pathologists. Fluorescence spectroscopy is a tool that has been of utility in the diagnosis of different diseases based on biopsy analysis. Thus, fluorescence study of liver samples with five different degrees of fibrosis is presented. Paraffin-preserved human liver tissue was provided on white plastic cassettes by the Hospital General de Mexico "Dr. Eduardo Liceaga". Specimens were diagnosed by two independent-senior pathologists in a double-blind test and classified into five different groups: F0, F1, F2, F3, and F4, according to the METAVIR scale for liver fibrosis. Fluorescence spectroscopy measurements were performed using three different excitation wavelengths: 385, 405, and 450 nm. Besides, diffuse reflectance spectroscopy (DRS) measurements were taken with white light to determine morphological changes in the tissue and to compare the results with medical diagnosis. The spectral analysis at excitation wavelengths of 385 nm and 405 nm showed poor correlation with medical diagnosis. Likewise, in order to discard all possible error-sources involved in the measurements, an exhaustive study was carried out; it included the determination of the fluorescence noise produced by paraffin, cassette, and the tissue itself. At 450 nm excitation wavelength, no fluorescence by the cassette was detected and noise-subtraction methods were not required, this allows a high correlation of hepatic fibrosis stages between pathological diagnosis and spectroscopic analysis. For this excitation wavelength, 89.87% correlation with DRS measurements and 82.00% with medical diagnosis were obtained. This work demonstrates that fluorescence spectroscopy using 450 nm excitation wavelength might work as a complementary tool to grade hepatic fibrosis in human liver specimens.

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.

Order-disorder skewness in alpha-synuclein: a key mechanism to recognize membrane curvature.

Currently, membrane curvature is understood as an active mechanism to control cells spatial organization and activity. Protein processes involved in sensing and generating curvature are therefore of major interest. In this work, we have studied α-synuclein interactions with a model lipid bilayer, inducing curvature in a controlled manner and describing protein responses at molecular level. We show that the intrinsically disordered region of α-synuclein binds to the bilayer as an acknowledgment to the induced curvature, a mechanism used by the interacting protein-membrane assembly to relieve free energy. We have calculated free energies for bending the bilayer with α-synuclein adsorbed on the surface and we have established the crucial role of the intrinsically disordered region, suggesting that a dynamic order/disorder interplay takes place as the bilayer reorganizes to bend.

Fiber-optic pulseoximeter for local oxygen saturation determination using a Monte Carlo multi-layer model for calibration.

BACKGROUND AND OBJECTIVE: Local tissue oxygenation determines the relationship between the supply and the demand for oxygen by the tissue and it is an important indicator of the physiological or pathological condition of the tissue. Moreover, some therapeutic methods strongly depend on the oxygen content of the tissue. In photodynamic therapy, when molecular oxygen is present, the irradiation of the photosensitizer with light triggers the generation of reactive oxygen species that kill the target diseased cells within the treated tissue. To ensure the best possible therapy response, the tissue must be well oxygenated; hence, oxygen concentration measurement becomes a decisive factor. In this work, the design, construction and calibration of a module to locally measure the blood oxygen saturation in tissue is presented. METHODS: The system is built using a red (660-nm) and an infrared (940-nm) light emitting diodes as light sources, a photodiode as a detector, and a homemade handheld fiber optic-based reflectance pulse oximetry sensor. In addition, the developed sensor was modeled by means of multilayered Monte Carlo simulations, to study its behavior when used in different thickness and melanin content skin. RESULTS: From the simulation reflectance values, the oxygen saturation calibration curves considering different melanin concentrations and skin thicknesses were obtained for two different skin models, one comprising three skin layers and the second, assuming seven different layers for the skin. A comparison of the performances of the developed pulse oximeter sensor with a commercial one is also presented. CONCLUSIONS: A new pulseoximeter for the measurement of local oxygenation in tissue was developed. Its calibration strongly depends on the site of measurement due to the influence of tissue thickness, vascularization, and melanin content. A three-layer skin model is proved to be suitable for the calibration of the pulseoximeter in thin and medium thickness skin.

Intrinsic disorder associated with 14-3-3 proteins and their partners.

Protein-protein interactions (PPIs) mediate a variety of cellular processes and form complex networks, where connectivity is achieved owing to the "hub" proteins whose interaction with multiple protein partners is facilitated by the intrinsically disordered protein regions (IDPRs) and posttranslational modifications (PTMs). Universal regulatory proteins of the eukaryotic 14-3-3 family nicely exemplify these concepts and are the focus of this chapter. The extremely wide interactome of 14-3-3 proteins is characterized by high levels of intrinsic disorder (ID) enabling protein phosphorylation and consequent specific binding to the well-structured 14-3-3 dimers, one of the first phosphoserine/phosphothreonine binding modules discovered. However, high ID enrichment also challenges structural studies, thereby limiting the progress in the development of small molecule modulators of the key 14-3-3 PPIs of increased medical importance. Besides the well-known structural flexibility of their variable C-terminal tails, recent studies revealed the strong and conserved ID propensity hidden in the N-terminal segment of 14-3-3 proteins (~40 residues), normally forming the α-helical dimerization region, that may have a potential role for the dimer/monomer dynamics and recently reported moonlighting chaperone-like activity of these proteins. We review the role of ID in the 14-3-3 structure, their interactome, and also in selected 14-3-3 complexes. In addition, we discuss approaches that, in the future, may help minimize the disproportion between the large amount of known 14-3-3 partners and the small number of 14-3-3 complexes characterized with atomic precision, to unleash the whole potential of 14-3-3 PPIs as drug targets.

Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier's performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively.

A connection between reversible tyrosine phosphorylation and SNARE complex disassembly activity of N-ethylmaleimide-sensitive factor unveiled by the phosphomimetic mutant N-ethylmaleimide-sensitive factor-Y83E.

N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor (SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF is constitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF-Y83E inhibits secretion in the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our model is human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosine phosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitation increased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomal domain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF-Y83E bound PTP1B and thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complex disassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF-Y83E prevents endogenous PTP1B from dephosphorylating sperm NSF, thus maintaining NSF's activity in a repressed mode and the SNARE complex unable to dissociate. The contribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosine phosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generated direct evidence that explains the dominant-negative role of the phosphomimetic mutant NSF-Y83E.