Amplification of Protein Expression by Self-Amplifying mRNA Delivered in Lipid Nanoparticles Containing a ß-Aminoester Ionizable Lipid Correlates with Reduced Innate Immune Activation.

Self-amplifying mRNA (saRNA) is witnessing increased interest as a platform technology for protein replacement therapy, gene editing, immunotherapy, and vaccination. saRNA can replicate itself inside cells, leading to a higher and more sustained production of the desired protein at a lower dose. Controlling innate immune activation, however, is crucial to suppress unwanted inflammation upon delivery and self-replication of RNA in vivo. In this study, we report on a class of ß-aminoester lipids (ßAELs) synthesized through the Michael addition of an acrylate to diethanolamine, followed by esterification with fatty acids. These lipids possessed one or two ionizable amines, depending on the use of nonionic or amine-containing acrylates. We utilized ßAELs for encapsulating saRNA in lipid nanoparticles (LNPs) and evaluated their transfection efficiency in vitro and in vivo in mice, while comparing them to LNPs containing ALC-0315 as an ionizable lipid reference. Among the tested lipids, OC7, which comprises two unsaturated oleoyl alkyl chains and an ionizable azepanyl motif, emerged as a ßAEL with low cytotoxicity and immunogenicity relative to ALC-0315. Interestingly, saRNA delivered via the OC7 LNP exhibited a distinct in vivo transfection profile. Initially, intramuscular injection of OC7 LNP resulted in low protein expression shortly after administration, followed by a gradual increase over a period of up to 7 days. This pattern is indicative of successful self-amplification of saRNA. In contrast, saRNA delivered via ALC-0315 LNP demonstrated high protein translation initially, which gradually declined over time and lacked the amplification seen with OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA ALC-0315 LNP induced potent innate immune activation by triggering cytoplasmic RIG-I-like receptors (RLRs), likely due to the highly efficient endosomal membrane rupturing properties of ALC-0315 LNP. Consequently, the massive production of type I interferons quickly hindered the amplification of the saRNA. Our findings highlight the critical role of the choice of ionizable lipid for saRNA formulation in LNPs, particularly in shaping the qualitative profile of protein expression. For applications where minimizing inflammation is desired, the use of ionizable lipids, such as the ßAEL reported in this study, that elicit a low type I interferon response in saRNA LNP is crucial.

Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

Dual-ligand PROTACS mediate superior target protein degradation in vitro and therapeutic efficacy in vivo.

Proteolysis targeting chimeras (PROTACs) are revolutionizing the drug development landscape due to their unique ability to selectively degrade disease-associated proteins. Conventional PROTACs are bivalent entities that induce ubiquitination and subsequent proteolysis of a chosen protein of interest (POI) by forming a ternary complex with an E3 ligase. We hypothesized that dual-ligand PROTACs, featuring two copies each of a POI ligand and an E3 ligase ligand, would facilitate the formation of high-avidity, long-lived ternary complexes inside cells, thereby increasing POI degradation potency. To this end, we developed a convergent synthesis route, using l-aspartic acid as a building block for homodimer synthesis, followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to conjugate both dimers through a flexible linker. Dual-ligand PROTACs achieved up to a tenfold increase in degradation efficiency and a hundredfold increase in cytotoxicity in vitro across various cancer cell lines compared to their single-ligand counterparts. Furthermore, dual-ligand PROTACs sustain prolonged protein degradation, up to 60 hours after pulsing and washout. In vivo, in a mouse tumor model, the superior therapeutic activity of dual ligand PROTACs was observed.

Effect of single parenteral administration of marbofloxacin on bacterial load and selection of resistant Enterobacteriaceae in the fecal microbiota of healthy pigs.

BACKGROUND: Antimicrobial resistance (AMR) is a global concern impacting both humans, animals and their environment. The use of oral antimicrobials in livestock, particularly in pigs, has been identified as a driver in the selection of AMR bacteria. The aim of the present study was to evaluate the effects of a single intramuscular (IM) dose of marbofloxacin (8 mg/kg) on Enterobacteriaceae and E. coli populations, as well as on fluoroquinolone resistance within the fecal microbiota of pigs. Twenty healthy pigs, 60-days old, were divided into two groups: a treated group (n = 13) and a control group (n = 7) and were monitored over a 28-day experimental period. Fecal samples were collected from all animals for the isolation of E. coli and Salmonella strains. The minimum inhibitory concentration (MIC) of marbofloxacin for the isolates recovered on MacConkey agar supplemented with 1 or 4 µg/mL of marbofloxacin and for some generic E. coli isolates (recovered from MacConkey agar not supplemented with marbofloxacin) was determined using the broth microdilution method. Genomic DNA was extracted from the confirmed bacterial strains and sequenced using the Sanger method to identify mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes. RESULTS: The single IM administration of marbofloxacin resulted in a significant decrease in Enterobacteriaceae and E. coli fecal populations from days 1 to 3 post- treatment. No Salmonella isolates were detected in either group, and no marbofloxacin-resistant E. coli isolates were identified. The MIC of the selected generic E. coli strains (n = 100) showed an increase to up to 0.5 µg/mL between days 1 and 3 post-treatment but remained below the clinical breakpoint of marbofloxacin resistance (4 µg/mL). Sequencing of these isolates revealed no mutations in gyrA and parC genes. CONCLUSIONS: The present study showed that this dosing regimen of marbofloxacin significantly decreases the fecal shedding of Enterobacteriaceae and E. coli populations in pigs, while limiting the selection of marbofloxacin-resistant E. coli isolates. These findings warrant validation in sick pigs to support the selective use of this antibiotic solely in cases of clinical disease, thereby minimizing the reliance on conventional (metaphylactic) group treatments in pigs.

Loperamide induced acute pancreatitis in pediatrics.

Presentation: A 13-year-old boy was admitted to the hospital following an accidental ingestion of 12 mg of loperamide hydrochloride. His main complaint was abdominal pain, especially in his right hypochondrium and epigastrium. Diagnosis: He was diagnosed with acute pancreatitis secondary to the use of loperamide. His lipase and amylase were indeed raised showing an acute and reversible trend. Infections and cholelithiasis were ruled out by blood tests and US abdomen. Treatment: The patient was treated with a single dose of activated charcoal and improved with no further treatment. Discussion: Loperamide is an opioid-receptor agonist and acts on the µ-opioid receptor. This causes spasms and dysfunction of the sphincter of Oddi resulting in obstruction of the pancreatic drainage and leading to acute pancreatitis. This case report shows the first case of loperamide induced acute pancreatitis in pediatrics.

Enhancement of early proximal caries annotations in radiographs: introducing the Diagnostic Insights for Radiographic Early-caries with micro-CT (ACTA-DIRECT) dataset.

BACKGROUND: Proximal caries datasets for training artificial intelligence (AI) algorithms commonly include clinician-annotated radiographs. These conventional annotations are susceptible to observer variability, and early caries may be missed. Micro-computed tomography (CT), while not feasible in clinical applications, offers a more accurate imaging modality to support the creation of a reference-standard dataset for caries annotations. Herein, we present the Academic Center for Dentistry Amsterdam-Diagnostic Insights for Radiographic Early-caries with micro-CT (ACTA-DIRECT) dataset, which is the first dataset pairing dental radiographs and micro-CT scans to enable higher-quality annotations. METHODS: The ACTA-DIRECT dataset encompasses 179 paired micro-CT scans and radiographs of early proximal carious teeth, along with three types of annotations: conventional annotations on radiographs, micro-CT-assisted annotations on radiographs, and micro-CT annotations (reference standard). Three dentists independently annotated proximal caries on radiographs, both with and without micro-CT assistance, enabling determinations of interobserver agreement and diagnostic accuracy. To establish a reference standard, one dental radiologist annotated all caries on the related micro-CT scans. RESULTS: Micro-CT support improved interobserver agreement (Cohen's Kappa), averaging 0.64 (95% confidence interval [CI]: 0.59-0.68) versus 0.46 (95% CI: 0.44-0.48) in its absence. Likewise, average sensitivity and specificity increased from 42% (95% CI: 34-51%) to 63% (95% CI: 54-71%) and from 92% (95% CI: 88-95%) to 95% (95% CI: 92-97%), respectively. CONCLUSION: The ACTA-DIRECT dataset offers high-quality images and annotations to support AI-based early caries diagnostics for training and validation. This study underscores the benefits of incorporating micro-CT scans in lesion assessments, providing enhanced precision and reliability.

Defective regulation of the eIF2-eIF2B translational axis underlies depressive-like behavior in mice and correlates with major depressive disorder in humans.

Major depressive disorder (MDD) is a significant cause of disability in adults worldwide. However, the underlying causes and mechanisms of MDD are not fully understood, and many patients are refractory to available therapeutic options. Impaired control of brain mRNA translation underlies several neurodevelopmental and neurodegenerative conditions, including autism spectrum disorders and Alzheimer's disease (AD). Nonetheless, a potential role for mechanisms associated with impaired translational control in depressive-like behavior remains elusive. A key pathway controlling translation initiation relies on the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P) which, in turn, blocks the guanine exchange factor activity of eIF2B, thereby reducing global translation rates. Here we report that the expression of EIF2B5 (which codes for eIF2Bε, the catalytic subunit of eIF2B) is reduced in postmortem MDD prefrontal cortex from two distinct human cohorts and in the frontal cortex of social isolation-induced depressive-like behavior model mice. Further, pharmacological treatment with anisomycin or with salubrinal, an inhibitor of the eIF2α phosphatase GADD34, induces depressive-like behavior in adult C57BL/6J mice. Salubrinal-induced depressive-like behavior is blocked by ISRIB, a compound that directly activates eIF2B regardless of the phosphorylation status of eIF2α, suggesting that increased eIF2α-P promotes depressive-like states. Taken together, our results suggest that impaired eIF2-associated translational control may participate in the pathophysiology of MDD, and underscore eIF2-eIF2B translational axis as a potential target for the development of novel approaches for MDD and related mood disorders.

Exploration of Solid Phase Peptoid Synthesis for the Design of Trifunctional Hapten-Lipid-TLR7/8 Agonist Antibody-Recruiting Oligomers That Combine Innate Effector with Innate Activation Function.

The strategic engagement of innate immunity is a promising avenue for cancer treatment. Antibody-recruiting molecules (ARMs) direct endogenous antibodies to target tumor sites, eliciting innate immune effector killing responses. In this study, we report the synthesis of ARMs by employing solid-phase peptoid synthesis to construct three libraries of antibody-recruiting oligomers. Using dinitrophenyl (DNP) as a model hapten and alkyl lipid chains for cell surface anchoring, we tailored oligomers with variations in valency and spatial configuration. Among these, an oligomer design featuring DNP connected to the oligomer backbone through an extended PEG linker and flanked by two lipid motifs emerged as the most effective in antibody recruitment in vitro. This oligomer was further functionalized to include an imidazoquinoline, creating a trifunctional hapten-lipid-TLR7/8 agonist oligomer, and a parallel variant was conjugated with rhodamine, resulting in a trifunctional hapten-lipid-dye oligomer. Upon intratumorally administration in a murine model, these oligomers induced localized immune activation within tumors. Subsequent ex vivo analysis of single-cell suspensions from excised tumors confirmed the enhanced binding of anti-DNP antibodies. These findings underscore the potential of custom-designed ARMs in orchestrating precise immune-mediated tumor targeting and highlight the adaptability of solid-phase synthesis in oligomer design for the design of multifunctional antibody recruiting molecules.

PH-Triggered, Lymph Node Focused Immunodrug Release by Polymeric 2-Propionic-3-Methyl-maleic Anhydrides with Cholesteryl End Groups.

Gaining spatial control over innate immune activation is of great relevance during vaccine delivery and anticancer therapy, where one aims at activating immune cells at draining lymphoid tissue while avoiding systemic off-target innate immune activation. Lipid-polymer amphiphiles show high tendency to drain to lymphoid tissue upon local administration. Here, pH-sensitive, cholesteryl end group functionalized polymers as stimuli-responsive carriers are introduced for controlled immunoactivation of draining lymph nodes. Methacrylamide-based monomers bearing pendant 2-propionic-3-methylmaleic anhydride groups are polymerized by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization using a cholesterol chain-transfer agent (chol-CTA). The amine-reactive anhydrides are conjugated with various amines, however, while primary amines afforded irreversible imides, secondary amines provided pH-responsive conjugates that are released upon acidification. This can be applied to fluorescent dyes for irreversibly carrier labeling or immunostimulatory Toll-like receptor (TLR) 7/8 agonists as cargos for pH-responsive delivery. Hydrophilization of remaining anhydride repeating units with short PEG-chains yielded cholesteryl-polymer amphiphiles that showed efficient cellular uptake and increased drug release at endosomal pH. Moreover, reversibly conjugated TLR 7/8 agonist amphiphiles efficiently drained to lymph nodes and increased the number of effectively maturated antigen-presenting cells after subcutaneous injection in vivo. Consequently, cholesteryl-linked methacrylamide-based polymers with pH-sensitive 2-propionic-3-methylmaleic anhydride side groups provide ideal features for immunodrug delivery.

Comparison of objectively measured and estimated cardiorespiratory fitness to predict all-cause and cardiovascular disease mortality in adults: A systematic review and meta-analysis of 42 studies representing 35 cohorts and 3.8 million observations.

BACKGROUND: Cardiorespiratory fitness (CRF) is a powerful health marker recommended by the American Heart Association as a clinical vital sign. Comparing the predictive validity of objectively measured CRF (the "gold standard") and estimated CRF is clinically relevant because estimated CRF is more feasible. Our objective was to meta-analyze cohort studies to compare the associations of objectively measured, exercise-estimated, and non-exercise-estimated CRF with all-cause and cardiovascular disease (CVD) mortality in adults. METHODS: Systematic searches were conducted in 9 databases (MEDLINE, SPORTDiscus, Embase, Scopus, PsycINFO, Web of Science, PubMed, CINAHL, and the Cochrane Library) up to April 11, 2024. We included full-text refereed cohort studies published in English that quantified the association (using risk estimates with 95% confidence intervals (95%CIs)) of objectively measured, exercise-estimated, and non-exercise-estimated CRF with all-cause and CVD mortality in adults. CRF was expressed as metabolic equivalents (METs) of task. Pooled relative risks (RR) for all-cause and CVD mortality per 1-MET (3.5 mL/kg/min) higher level of CRF were quantified using random-effects models. RESULTS: Forty-two studies representing 35 cohorts and 3,813,484 observations (81% male) (362,771 all-cause and 56,471 CVD deaths) were included. The pooled RRs for all-cause and CVD mortality per higher MET were 0.86 (95%CI: 0.83-0.88) and 0.84 (95%CI: 0.80-0.87), respectively. For both all-cause and CVD mortality, there were no statistically significant differences in RR per higher MET between objectively measured (RR range: 0.86-0.90) and maximal exercise-estimated (RR range: 0.85-0.86), submaximal exercise-estimated (RR range: 0.91-0.94), and non-exercise-estimated CRF (RR range: 0.81-0.85). CONCLUSION: Objectively measured and estimated CRF showed similar dose-response associations for all-cause and CVD mortality in adults. Estimated CRF could provide a practical and robust alternative to objectively measured CRF for assessing mortality risk across diverse populations. Our findings underscore the health-related benefits of higher CRF and advocate for its integration into clinical practice to enhance risk stratification.

SGLT2 inhibitors and kidney outcomes across the spectrum of kidney disease: a systematic review and meta-analysis.

BACKGROUND: SGLT2 inhibitors have shown to reduce clinically meaningful kidney outcomes in individuals with chronic kidney disease at high risk for adverse outcomes. The effect of these agents in preventing clinically meaningful kidney outcomes in populations at lower risk remain uncertain. We aim to evaluate the effect of SGLT2 inhibitors on kidney outcomes across the Kidney Disease: Improving Global Outcomes (KDIGO) classification and urinary albumin-to-creatinine ratio (UACR) levels. METHODS: We have searched MEDLINE (PubMed), EMBASE and the Cochrane Central Register of Controlled Trials from inception up to August 8th, 2023. In pairs, researchers selected large (≥500 subjects per arm) randomized placebo-controlled trials of SGLT2 inhibitors, with a minimum duration of one year. Researchers independently extracted study-level data and assessed within-study risk of bias with the RoB 2.0 tool and quality of evidence with GRADE. RESULTS: We included 10 trials, encompassing 78,184 participants and a median follow-up of 2.7 years. Risk of bias was overall low. We performed meta-analyses summarizing individual study hazard ratios (HR) and 95% confidence intervals (CI) using a random-effects model. SGLT2 inhibitors reduced the composite kidney outcome across all KDIGO (HR [95% CI]: low 0.48 [0.32-0.71], moderate 0.60 [0.39-0.93], high 0.59 [0.47-0.74], very high 0.59 [0.49-0.72]) and UACR (HR [95% CI]: <30 mg/g 0.62 [0.50-0.78], ≥30 ≤300 mg/g 0.80 [0.67-0.96], >300 mg/g 0.61 [0.52-0.73]) groups, without evidence of heterogeneity between groups. LIMITATIONS: Small proportion of subjects without diabetes in low-risk groups and lack of standardization of composite outcomes. CONCLUSIONS: SGLT2 inhibitors consistently reduce kidney outcomes across the spectrum of KDIGO classes and UACR levels.

MSK1 is required for the experience- and ampakine-dependent enhancement of spatial reference memory and reversal learning and for the induction of Arc and BDNF.

There is considerable interest in the development of nootropics, pharmacological agents that can improve cognition across a range of both cognitive modalities and cognitive disabilities. One class of cognitive enhancers, the ampakines, has attracted particular attention by virtue of improving cognition associated with animal models of neurodevelopmental, neurodegenerative, and psychiatric conditions, as well as in age-related cognitive impairment. Ampakines elevate CNS levels of BDNF, and it is through this elevation that their beneficial actions are believed to occur. However, what transduces the elevation of BDNF into long-lasting cognitive enhancement is not known. We have previously shown that MSK1, by virtue of its ability to regulate gene transcription, converts the elevation of BDNF associated with environmental enrichment into molecular, synaptic, cognitive and genomic adaptations that underlie enrichment-induced enhanced synaptic plasticity and learning and memory, a property that MSK1 retains across the lifespan. To establish whether MSK1 similarly converts ampakine-induced elevations of BDNF into cognitive enhancement we tested an ampakine (CX929) in male WT mice and in male mice in which the kinase activity of MSK1 was inactivated. We found that MSK1 is required for the ampakine-dependent improvement in spatial reference memory and cognitive flexibility, and for the elevations of BDNF and the plasticity-related protein Arc associated with ampakines and experience. These observations implicate MSK1 as a key enabler of the beneficial effects of ampakines on cognitive function, and furthermore identify MSK1 as a hub for BDNF-elevating nootropic strategies.

The XENONnT dark matter experiment.

The multi-staged XENON program at INFN Laboratori Nazionali del Gran Sasso aims to detect dark matter with two-phase liquid xenon time projection chambers of increasing size and sensitivity. The XENONnT experiment is the latest detector in the program, planned to be an upgrade of its predecessor XENON1T. It features an active target of 5.9 tonnes of cryogenic liquid xenon (8.5 tonnes total mass in cryostat). The experiment is expected to extend the sensitivity to WIMP dark matter by more than an order of magnitude compared to XENON1T, thanks to the larger active mass and the significantly reduced background, improved by novel systems such as a radon removal plant and a neutron veto. This article describes the XENONnT experiment and its sub-systems in detail and reports on the detector performance during the first science run.

CO-DELIVERY of glutamic acid-extended peptide antigen and imidazoquinoline TLR7/8 agonist via ionizable lipid nanoparticles induces protective anti-tumor immunity.

Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.

Perinatal protein malnutrition alters maternal behavior and leads to maladaptive stress response, neurodevelopmental delay and disruption on DNA methylation machinery in female mice offspring.

Deficiencies in maternal nutrition have long-term consequences affecting brain development of the progeny and its behavior. In the present work, female mice were exposed to a normal-protein or a low-protein diet during gestation and lactation. We analyzed behavioral and molecular consequences of malnutrition in dams and how it affects female offspring at weaning. We have observed that a low-protein diet during pregnancy and lactation leads to anxiety-like behavior and anhedonia in dams. Protein malnutrition during the perinatal period delays physical and neurological development of female pups. Glucocorticoid levels increased in the plasma of malnourished female offspring but not in dams when compared to the control group. Interestingly, the expression of glucocorticoid receptor (GR) was reduced in hippocampus and amygdala on both malnourished dams and female pups. In addition, malnourished pups exhibited a significant increase in the expression of Dnmt3b, Gadd45b, and Fkbp5 and a reduction in Bdnf VI variant mRNA in hippocampus. In contrast, a reduction on Dnmt3b has been observed on the amygdala of weaned mice. No changes have been observed on global methylation levels (5-methylcytosine) in hippocampal genomic DNA neither in dams nor female offspring. In conclusion, deregulated behaviors observed in malnourished dams might be mediated by a low expression of GR in brain regions associated with emotive behaviors. Additionally, low-protein diet differentially deregulates the expression of genes involved in DNA methylation/demethylation machinery in female offspring but not in dams, providing an insight into regional- and age-specific mechanisms due to protein malnutrition.

Chitosan-Tricarbocyanine-Based Nanogels Were Able to Cross the Blood-Brain Barrier Showing Its Potential as a Targeted Site Delivery Agent.

Targeting drugs to the central nervous system (CNS) is challenging due to the presence of the blood-brain barrier (BBB). The cutting edge in nanotechnology generates optimism to overcome the growing challenges in biomedical sciences through the effective engineering of nanogels. The primary objective of the present report was to develop and characterize a biocompatible natural chitosan (CS)-based NG that can be tracked thanks to the tricarbocyanine (CNN) fluorescent probe addition on the biopolymer backbone. FTIR shed light on the chemical groups involved in the CS and CNN interactions and between CNN-CS and tripolyphosphate, the cross-linking agent. Both in vitro and in vivo experiments were carried out to determine if CS-NGs can be utilized as therapeutic delivery vehicles directed towards the brain. An ionic gelation method was chosen to generate cationic CNN-CS-NG. DLS and TEM confirmed that these entities' sizes fell into the nanoscale. CNN-CS-NG was found to be non-cytotoxic, as determined in the SH-SY5Y neuroblastoma cell line through biocompatibility assays. After cellular internalization, the occurrence of an endo-lysosomal escape (a crucial event for an efficient drug delivery) of CNN-CS-NG was detected. Furthermore, CNN-CS-NG administered intraperitoneally to female CF-1 mice were detected in different brain regions after 2 h of administration, using fluorescence microscopy. To conclude, the obtained findings in the present report can be useful in the field of neuro-nanomedicine when designing drug vehicles with the purpose of delivering drugs to the CNS.

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance.

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Effects of Delaying the Storage of 'Hass' Avocados under a Controlled Atmosphere on Skin Color, Bioactive Compounds and Antioxidant Capacity.

During ripening, 'Hass' avocado skin changes from green to purple/black. Low-temperature storage with a controlled atmosphere (CA) is the most widely used method for avocado storage; however, few studies have simulated this technology and considered the days of regular air (RA) storage prior to CA storage. Herein, the effect of delaying the storage of 'Hass' avocado (>30% dry matter) in a CA was examined. Long-term storage conditions (5 °C for 50 days) corresponded to (i) regular air storage (RA), (ii) CA (4 kPa O2 and 6 kPa CO2) and (iii) 10 days in RA + 40 days in a CA and (iv) 20 days in RA + 30 days in a CA. Evaluations were performed during storage and at the ready-to-eat (RTE) stage. Skin color remained unchanged during storage, but at the RTE stage, more color development was observed for fruits stored under CA conditions, as these fruits were purple/black (>50%). At the RTE stage, the anthocyanin content increased, and compared to fruit under RA, fruit under a CA contained a five-fold greater content. A 20-day delay between harvest and CA storage increased the fruit softening rate and skin color development after cold storage, reducing the effectiveness of CA as a postharvest technology for extending storage life.

Comparison of the Accuracy and Efficiency of Two Dynamic Navigation System Workflow for Fiber-post Removal: Small versus Large Field-of-view Registration Workflows.

INTRODUCTION: This study investigates the feasibility of a dynamic navigation system (DNS) small field of view workflow (DNS-SFOVw) for fiber-post removal and compares its accuracy and efficiency to the conventional large field of view workflow (DNS-LFOVw). METHODS: Fifty-six extracted human maxillary molars were divided into DNS-SFOVw (n = 28) and DNS-LFOVw (n = 28). The palatal canal was restored with an intraradicular RelyX fiber post and luted with RelyX Unicem; a core buildup was used. Teeth were mounted in a 3D-printed surgical jaw. A preoperative cone-beam computed tomography (CBCT) scan was taken with a 40 × 40 mm FOV for the DNS-SFOVw and a single arch CBCT scan for the DNS-LFOVw. The drilling entry point, trajectory, angle, and depth were planned in the X-guide software. The DNS registration method for the DNS-SFOVw was virtual-based registration on teeth, and the marker point-based method was used for the DNS-LFOVw. The fiber posts were drilled out under DNS guidance. A postoperative CBCT scan was taken. Three-dimensional deviations, angular deflection, number of mishaps, registration, and total operation time were calculated. RESULTS: The DNS-SFOVw was as accurate as DNS-LFOVw (P > .05). The DNS-LFOVw registration time was less than DNS-SFOVw (P < .05). There was no difference in the number of mishaps (P > .05). Both DNS-SFOVw and DNS-LFOVw were time-efficient, with DNS-LFOVw taking less total operational time (P < .05). CONCLUSION: Within the limitations of this in-vitro study, the DNS-SFOVw was as accurate as the DNS-LFOVw for fiberpost removal. Both DNS-LFOVw and DNS-SFOVw were time-efficient in removing fiber-posts.

Epigenetic mechanisms linking early-life adversities and mental health.

Early-life adversities, whether prenatal or postnatal exposure, have been linked to adverse mental health outcomes later in life increasing the risk of several psychiatric disorders. Research on its neurobiological consequences demonstrated an association between exposure to adversities and persistent alterations in the structure, function, and connectivity of the brain. Consistent evidence supports the idea that regulation of gene expression through epigenetic mechanisms are involved in embedding the impact of early-life experiences in the genome and mediate between social environments and later behavioral phenotypes. In addition, studies from rodent models and humans suggest that these experiences and the acquired risk factors can be transmitted through epigenetic mechanisms to offspring and the following generations potentially contributing to a cycle of disease or disease risk. However, one of the important aspects of epigenetic mechanisms, unlike genetic sequences that are fixed and unchangeable, is that although the epigenetic markings are long-lasting, they are nevertheless potentially reversible. In this review, we summarize our current understanding of the epigenetic mechanisms involved in the mental health consequences derived from early-life exposure to malnutrition, maltreatment and poverty, adversities with huge and pervasive impact on mental health. We also discuss the evidence about transgenerational epigenetic inheritance in mammals and experimental data suggesting that suitable social and pharmacological interventions could reverse adverse epigenetic modifications induced by early-life negative social experiences. In this regard, these studies must be accompanied by efforts to determine the causes that promote these adversities and that result in health inequity in the population.