In vivo CAR T-cell generation in non-human primates using lentiviral vectors displaying a multi-domain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high cost and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVecTM platform, a lentiviral vector capable of generating CAR T-cells in vivo. Here we describe the incorporation of T cell activation and costimulatory signals onto the surface of VivoVecTM particles (VVPs) in the form of a multi-domain fusion protein and show enhanced in vivo transduction and improved CAR-T cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into non-human primates resulted in the robust generation of anti-CD20 CAR T-cells and the complete depletion of B cells for more than 10 weeks. These data validate the VivoVecTM platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Quantification of Vitamin A in Edible Oils: Comparison of Portable Device iCheck Chroma3 to High-Performance Liquid Chromatography.

Fortification of edible oil with vitamin A is a widely adopted intervention to minimize the effects of vitamin A deficiency in vulnerable groups and mitigate some of its deleterious consequences. Regulatory monitoring is an important prerequisite to ensure that the fortification program is implemented effectively. Standard laboratory analysis methods for vitamin A in oils to assess adequate addition levels remain expensive and time-consuming. Portable testing devices are relatively less expensive in terms of capital investment and cost per test. However, the reliability of results needs to be assured to ensure acceptability and confidence. This study compared a portable device to high-performance liquid chromatography (HPLC) in terms of quantification of vitamin A in both spiked and commercially fortified oils. Nine oils (soybean, palm, cottonseed, rapeseed, corn, peanut, coconut, sunflower, and rice bran oils) were selected and spiked with retinyl palmitate at six different concentrations, and 112 commercially fortified oils were quantified for their vitamin A content using both methods. A good indicator of intra-day and inter-day repeatability (< 10% CV) was obtained for the measurement of vitamin A in the spiked oils for both methods, which denotes a high agreement between them. Vitamin A recoveries were 97-132% for HPLC and 74-127% for the portable device. A strong positive correlation, r = 0.88, is observed between the two methods for the quantification of vitamin A in the commercially fortified oils. The portable device provides a relatively low-cost, quick, and user-friendly alternative to HPLC.

Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease.

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Comparison between the KARVI scale and the Child Development Evaluation test (EDI) as a screening tool for suspected neurodevelopmental delay.

BACKGROUND: Early detection of suspected neurodevelopmental delay allows for timely diagnosis and appropriate intervention, for which numerous screening tests have been developed. However, most are complex and impractical for health-care workers at the community level. This study aimed to validate the KARVI scale in the neurodevelopment assessment of children under 1 year of age. METHODS: We conducted an observational, longitudinal, comparative, inferential, and prospective study. Healthy children without risk factors for developing neurodevelopmental delay from 0 to 12 months of age were evaluated remotely using the Zoom® application. The Child Development Evaluation Test and the KARVI scale were applied once a month for four consecutive months. RESULTS: Fifty individuals were analyzed, with a predominance of males in 52%. Adequate percentages for a screening test were obtained in the first evaluation with a sensitivity of 70% (confidence interval [CI] 95% 34.75-93.33) and a specificity of 75% (CI 95% 58.8-87.31), and in the fourth evaluation with a sensitivity of 100% (CI 95% 29.4-100) and a specificity of 78.72% (CI 95% 64.34-89.3), being significant in both evaluations (p = 0.007 and p = 0.001, respectively). CONCLUSIONS: The KARVI scale has the elements to be an effective screening test for suspected neurodevelopmental delay, but more extensive studies are needed to obtain more reliable results.

INTRODUCCIÓN: La identificación temprana de retraso en el neurodesarrollo permite un diagnóstico oportuno y una intervención apropiada. Para ello, se han creado diversas pruebas de tamizaje; sin embargo, la mayoría son complejas y poco prácticas para el personal de la salud a nivel comunitario. El objetivo del estudio fue realizar la validación de la escala KARVI en la valoración del neurodesarrollo en niños menores de un año. MÉTODOS: Se realizó un estudio observacional, longitudinal, comparativo inferencial y prospectivo, en el cual se evaluaron, vía remota mediante la aplicación Zoom®, niños sanos de 0 a 12 meses de edad sin factores de riesgo para desarrollar retraso en el neurodesarrollo. Se aplicaron la prueba EDI (Evaluación del Desarrollo Infantil) y la escala KARVI una vez al mes por cuatro meses consecutivos. RESULTADOS: Se analizaron 50 individuos, con predominio del sexo masculino en el 52%. Se obtuvieron porcentajes adecuados para una prueba de tamizaje tanto en la primera evaluación, con sensibilidad de 70% (IC 95% 34.75-93.33) y especificidad de 75% (IC 95% 58.8-87.31), como en la cuarta, con sensibilidad de 100% (IC 95% 29.4-100) y especificidad de 78.72% (IC 95% 64.34-89.3), con significación estadística en ambas evaluaciones (p = 0.007 y p = 0.001, respectivamente). CONCLUSIONES: Se considera que la escala KARVI cuenta con los elementos para considerarla como una prueba de tamizaje efectiva para detectar retraso del neurodesarrollo, sin embargo. Sin requieren estudios más extensos para obtener resultados más confiables.

Comparison between the KARVI scale and the Child Development Evaluation test (EDI) as a screening tool for suspected neurodevelopmental delay / Comparación entre la escala KARVI y la prueba de Evaluación del Desarrollo Infantil (EDI) como tamizaje para la sospecha de retraso en el neurodesarrollo

Abstract Background: Early detection of suspected neurodevelopmental delay allows for timely diagnosis and appropriate intervention, for which numerous screening tests have been developed. However, most are complex and impractical for health-care workers at the community level. This study aimed to validate the KARVI scale in the neurodevelopment assessment of children under 1 year of age. Methods: We conducted an observational, longitudinal, comparative, inferential, and prospective study. Healthy children without risk factors for developing neurodevelopmental delay from 0 to 12 months of age were evaluated remotely using the Zoom® application. The Child Development Evaluation Test and the KARVI scale were applied once a month for four consecutive months. Results: Fifty individuals were analyzed, with a predominance of males in 52%. Adequate percentages for a screening test were obtained in the first evaluation with a sensitivity of 70% (confidence interval [CI] 95% 34.75-93.33) and a specificity of 75% (CI 95% 58.8-87.31), and in the fourth evaluation with a sensitivity of 100% (CI 95% 29.4-100) and a specificity of 78.72% (CI 95% 64.34-89.3), being significant in both evaluations (p = 0.007 and p = 0.001, respectively). Conclusions: The KARVI scale has the elements to be an effective screening test for suspected neurodevelopmental delay, but more extensive studies are needed to obtain more reliable results.

Resumen Introducción: La identificación temprana de retraso en el neurodesarrollo permite un diagnóstico oportuno y una intervención apropiada. Para ello, se han creado diversas pruebas de tamizaje; sin embargo, la mayoría son complejas y poco prácticas para el personal de la salud a nivel comunitario. El objetivo del estudio fue realizar la validación de la escala KARVI en la valoración del neurodesarrollo en niños menores de un año. Métodos: Se realizó un estudio observacional, longitudinal, comparativo inferencial y prospectivo, en el cual se evaluaron, vía remota mediante la aplicación Zoom®, niños sanos de 0 a 12 meses de edad sin factores de riesgo para desarrollar retraso en el neurodesarrollo. Se aplicaron la prueba EDI (Evaluación del Desarrollo Infantil) y la escala KARVI una vez al mes por cuatro meses consecutivos. Resultados: Se analizaron 50 individuos, con predominio del sexo masculino en el 52%. Se obtuvieron porcentajes adecuados para una prueba de tamizaje tanto en la primera evaluación, con sensibilidad de 70% (IC 95% 34.75-93.33) y especificidad de 75% (IC 95% 58.8-87.31), como en la cuarta, con sensibilidad de 100% (IC 95% 29.4-100) y especificidad de 78.72% (IC 95% 64.34-89.3), con significación estadística en ambas evaluaciones (p = 0.007 y p = 0.001, respectivamente). Conclusiones: Se considera que la escala KARVI cuenta con los elementos para considerarla como una prueba de tamizaje efectiva para detectar retraso del neurodesarrollo, sin embargo. Sin requieren estudios más extensos para obtener resultados más confiables.

Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Identification and distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in Astragalus garbancillo tissues.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

Interactions of variants of human apolipoprotein A-I with biopolymeric model matrices. Effect of collagen and heparin.

BACKGROUND: The extracellular matrix (ECM) is a complex tridimensional scaffold that actively participates in physiological and pathological events. The objective of this study was to test whether structural proteins of the ECM and glycosaminoglycans (GAGs) may favor the retention of human apolipoprotein A-I (apoA-I) variants associated with amyloidosis and atherosclerosis. METHODS: Biopolymeric matrices containing collagen type I (Col, a main macromolecular component of the ECM) with or without heparin (Hep, a model of GAGs) were constructed and characterized, and used to compare the binding of apoA-I having the native sequence (Wt) or Arg173Pro, a natural variant inducing cardiac amyloidosis. Protein binding was observed by fluorescence microscopy and unbound proteins quantified by a colorimetric assay. RESULTS: Both, Wt and Arg173Pro bound to the scaffolds containing Col, but the presence of Hep diminished the binding efficiency. Col-Hep matrices retained Arg173Pro more than the Wt. The retained protein was only partially removed from the matrices with saline solutions, indicating that electrostatic interactions may occur but are not the main driving force. Using in addition thermodynamic molecular simulations and size exclusion chromatography approaches, we suggest that the binding of apoA-I variants to the biopolymeric matrices is driven by many low affinity interactions. CONCLUSIONS: Under this scenario Col-Hep scaffolds contribute to the binding of Arg173Pro, as a cooperative platform which could modify the native protein conformation affecting protein folding. GENERAL SIGNIFICANCE: We show that the composition of the ECM is key to the protein retention, and well characterized biosynthetic matrices offer an invaluable in vitro model to mimic the hallmark of pathologies with interstitial infiltration such as cardiac amyloidosis.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Interaction time with conspecifics induces food preference or aversion in the wild Algerian mouse.

The social transmission of a novel food preference can avoid unnecessary costs arising from tasting nonedible foods. This type of social learning has been demonstrated in laboratory rats and mice. However, among wild animals, there may be several constraints that make it less effective. Using wild Algerian mice (Mus spretus) tested in the laboratory, we demonstrate that a preference for a novel food can be transmitted between Observer and Demonstrator individuals and that it is maintained for at least 30 days. However, only half of the Observers acquired a preference for the same food as the Demonstrators, and only when the duration of oronasal investigation was above a certain threshold (≥122 s); below this threshold (<122 s), Observers acquired a preference for the alternative food offered, which was maintained for a shorter time. Sex, size, and identity of individuals did not influence the transmission of social information. The results show that different interaction times will result in animals copying or avoiding the food choices of others. This suggests that the transmission of social information among wild animals is complex and probably influenced by many factors (e.g., dominance, familiarity, and health condition), ultimately conditioning the type of interaction between individuals and its outcome. Testing wild animals and the ecological and social constraints they face is, therefore, an important step in our understanding of how effectively social information is transmitted in nature.

Trypanosoma cruzi Secreted Cyclophilin TcCyP19 as an Early Marker for Trypanocidal Treatment Efficiency.

Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30-38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency.

Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.

Morphometrical Identification and Phylogenetic Analysis of Rhinonyssidae (Acari: Mesostigmata) Parasitizing Avian Hosts: New Molecular Data.

Members of the family Rhinonyssidae are tiny hematophagous endoparasitic mites that inhabit the nasal cavities of birds and can cause trauma to their hosts. Traditionally, identifying species in this group has relied on observing their morphometrical characteristics. Nevertheless, determining species within this particular group has become more challenging due to the rising number of newly discovered species. Moreover, the morphometrical traits vary depending on the specific genus or group of species being studied. In this study, the complete internal transcribed spacer ITS1, 5.8S rDNA, and ITS2 regions of the ribosomal DNA from eighteen species of rhinonyssid mites belonging to four genera were sequenced to assess the utility of this genomic region in resolving taxonomic questions in this group and to estimate the phylogenetic relationships among the species. Mites were collected by dissecting the nasal cavities of birds under a stereomicroscope. Specimens used for morphometrical analyses were cleared in 85% lactic acid for 1-48 h and mounted in Hoyer's medium. Other specimens were preserved at -20 °C for molecular studies. From the data obtained in this study, it can be concluded that a thorough review and an accurate morphometrical identification and determination of the discriminatory traits are needed in this group of mites. Moreover, although the ITS1-5.8S-ITS2 fragment solves different taxonomic and phylogenetic problems at the species level, it would be necessary to test new molecular markers, or even a combination of nuclear and mitochondrial markers or different domains of the nuclear 28S rDNA, to discover a reliable taxonomic situation for rhinonyssids.

Quantitative PCR-Based Diagnosis and Follow-up of Chagas Disease Primary Infection After Solid Organ Transplant: A Multicentre Study.

Chagas disease in solid organ transplant recipients may present as a primary infection (PI). Early detection is crucial for timely treatment. This is the largest observational multicentre study evaluating qPCR for early diagnosis and treatment monitoring of PI in seronegative recipients of organs from seropositive donors. Of 34 patients admitted at 5 health centers, PI was detected by qPCR in 8 (23.5%) within a posttransplant period of 40 days (interquartile range [IQR], 31-50 days). No PI was detected by the Strout test or clinical symptoms/signs. All patients had favorable treatment outcome with negative qPCR 31 days (IQR, 18-35 days) after treatment, with no posttreatment relapse episodes.

Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization.

Intracellular organelle organization is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule post-translational modifications (PTMs) can contribute to microtubule diversity and differentially regulate motor-mediated transport. Here, we show that centrosome amplification, commonly observed in cancer and shown to promote aneuploidy and invasion, induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganization requires kinesin-1 and is analogous to the loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM that could enhance kinesin-1-mediated transport. Depletion of α-tubulin acetyltransferase 1 (αTAT1) to block tubulin acetylation rescues the displacement of centrosomes, mitochondria, and vimentin but not Golgi or endosomes. Analyses of the distribution of total and acetylated microtubules indicate that the polarized distribution of modified microtubules, rather than levels alone, plays an important role in the positioning of specific organelles, such as the centrosome. We propose that increased tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement to regulate intracellular organization.

Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases.

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Prevention of Neurological Sequelae in Preterm Infants.

BACKGROUND: Preterm birth is one of the world's critical health problems, with an incidence of 5% to 18% of living newborns according to various countries. White matter injuries due to preoligodendrocytes deficits cause hypomyelination in children born preterm. Preterm infants also have multiple neurodevelopmental sequelae due to prenatal and perinatal risk factors for brain damage. The purpose of this work was to explore the effects of the brain risk factors and MRI volumes and abnormalities on the posterior motor and cognitive development at 3 years of age. METHODS: A total of 166 preterm infants were examined before 4 months and clinical and MRI evaluations were performed. MRI showed abnormal findings in 89% of the infants. Parents of all infants were invited to receive the Katona neurohabilitation treatment. The parents of 128 infants accepted and received Katona's neurohabilitation treatment. The remaining 38 infants did not receive treatment for a variety of reasons. At the three-year follow-up, Bayley's II Mental Developmental Index (MDI) and the Psychomotor Developmental Index (PDI) were compared between treated and untreated subjects. RESULTS: The treated children had higher values of both indices than the untreated. Linear regression showed that the antecedents of placenta disorders and sepsis as well as volumes of the corpus callosum and of the left lateral ventricle significantly predicted both MDI and PDI, while Apgar < 7 and volume of the right lateral ventricle predicted the PDI. CONCLUSIONS: (1) The results indicate that preterm infants who received Katona's neurohabilitation procedure exhibited significantly better outcomes at 3 years of age compared to those who did not receive the treatment. (2) The presence of sepsis and the volumes of the corpus callosum and lateral ventricles at 3-4 months were significant predictors of the outcome at 3 years of age.