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The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

Alzheimer's Disease (AD) is physiopathologically marked by an accumulation of beta-amyloid peptide (Aβ), hyperphosphorylation of tau protein, inflammation, and oxidative stress in the brain tissue. While new drugs for AD have been approved, novel treatments are still needed. Eplingiella fruticosa (EF) has demonstrated anti-inflammatory and antioxidant properties, which may be beneficial against AD. This study aimed to evaluate the effects of EF leaf essential oil complexed with β-cyclodextrin in a sporadic AD model induced by streptozotocin (STZ). Male Wistar rats (5-6 months old) received an intracerebroventricular STZ injection (3 mg/kg) or vehicle, and were orally treated with vehicle, EF (5 mg/kg), or donepezil (5 mg/kg) for 14 days. Behavioral tests included olfactory discrimination, open field, novel object recognition, sucrose preference, and spontaneous alternation. Upon completion, rats were euthanatized, and their brains were analyzed for Aβ, tau, and IL-1β via immunohistochemistry, and for oxidative stress markers. STZ-treated rats showed memory deficits and anhedonia, accompanied by increased Aβ, tau, and IL-1β immunoreactivity in the olfactory bulb, cortex, hippocampus, and increased TBARS levels in the hippocampus. On the other hand, EF treatment improved short-term and working memory (p < 0.001), and reduced depressive-like behavior (p = 0.02). Additionally, EF treatment decreased Aβ, tau, and IL-1β immunoreactivity in the olfactory bulb, hippocampus and cortex (p < 0.05), and reduced TBARS levels (p = 0.04) and total oxidant status in the hippocampus (p = 0.03), and increased total antioxidant status in the cortex (p = 0.04). These findings suggest EF has neuroprotective effects against STZ-induced damage, indicating its potential as a novel compound for AD treatment., Competing Interests: Declarations. Ethical approval: All procedures involving animals followed the Brazilian law for the use of animals in scientific research (Law number 11.794) of October 8, 2008, Decree 6899 of July 15, 2009, as well as with the rules issued by the National Council for Control of Animal Experimentation (CONCEA), and was approved by the Ethic Committee on Animal Use of the Federal University of Sao Paulo (CEUA/UNIFESP Protocol No. 5135090620) in the meeting of 08/03/2020. Consent for publication: Not applicable. Financial interests: The authors declare they have no financial interests. Consent to participate: Not applicable. Conflict of interest: The authors declare that they have no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency., Methods: We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation., Results: Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect., Conclusions: Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background/Objectives: Mental disorders pose a significant public health challenge, affecting millions worldwide. Given the limitations of current therapies, many patients experience inadequate responses and adverse effects. Intermittent hypoxia (IH) has demonstrated anxiolytic, antidepressant, and neuroprotective properties in various protocols. This study investigated the effects of acute IH (13% O 2 , 1 h), fluoxetine (FLX) and their combination on depression-like behavior, serum corticosterone, and inflammatory cytokine levels induced by acute restraint stress in C57BL/6 female mice. Methods: Behavioral assessments included the tail suspension test, forced swim test, and open field test. Results: The combined IH + FLX treatment exhibited a synergistic effect, reducing immobility time and increasing latency time, respectively, in the tail suspension test (46%, p = 0.0014; 73%, p = 0.0033) and forced swim test (56%, p = 0.0082; 48%, p = 0.0322) compared to the ARS group. Biochemical analysis revealed that individual and combined treatments significantly reduced most inflammatory interleukins by up to 96%. Corticosterone levels were reduced by 30% only in the IH group. Conclusions: These findings highlight the potential of a one-hour IH session, particularly when combined with fluoxetine, to alleviate depressive-like behaviors and exert anti-inflammatory effects, suggesting a promising therapeutic approach for depression.

Background: Spondyloarthritis (SpA) encompasses a spectrum of immune-mediated inflammatory conditions primarily affecting the axial skeleton, including sacroiliitis and spondylitis, each with distinct features. This study aimed to investigate imaging disparities, focusing on sacroiliac magnetic resonance and spine radiography, across phenotypes and between males and females in axial SpA., Method: A cross-sectional study was conducted to assess clinical data, laboratory findings, magnetic resonance imaging (MRI) scores of sacroiliac joints using the Spondyloarthritis Research Consortium of Canada (SPARCC) and Sacroiliac Joint Structural Score (SSS), and cervical and lumbar spine radiographs utilizing the Modified Stoke Ankylosing Spondylitis Spine Score (mSASSS). The study aimed to compare these parameters between two groups: axial spondyloarthritis (axSpA, radiographic and non-radiographic) and axial psoriatic arthritis (axPsA), as well as between males and females., Results: Ninety-four patients were included, with 62 patients in the axSpA group and 32 patients in the axPsA group. There were no differences in disease activity, mobility, radiographic damage in the spine (Modified Stoke Ankylosing Spondylitis Spine Score- mSASSS), or sacroiliac magnetic resonance imaging (MRI) scores (Spondyloarthritis Research Consortium of Canada Magnetic Resonance Imaging Index - SPARCC and Sacroiliac Joint Structural Score - SSS) between the two phenotypes. Regarding sex, in imaging exams, men had higher mSASSS (p = 0.008), SSS (p = 0.001), and fat metaplasia (MG) score based on SSS (p = 0.001), while women had significantly higher SPARCC scores (p = 0.039). In the male group, the presence of HLA-B27 allele had an impact on more structural lesions on MRI (SSS), p = 0.013., Conclusion: In this study, imaging of sacroiliac joints and spine in patients with axial SpA did not show differences in phenotypes but did reveal differences based on sex, which may have an impact on future diagnostic recommendations. Further studies are needed to confirm these findings., (© 2024. The Author(s).)

Despite efforts to elucidate the cellular adaptations induced by obesity, cellular bioenergetics is currently considered a crucial target. New strategies to delay the onset of the hazardous adaptations induced by obesity are needed. Therefore, we evaluated the effects of 4 weeks of melatonin treatment on mitochondrial function and lipid metabolism in the livers of leptin-deficient mice. Our results revealed that the absence of leptin increased lipid storage in the liver and induced significant mitochondrial alterations, which were ultimately responsible for defective ATP production and reactive oxygen species overproduction. Moreover, leptin deficiency promoted mitochondrial biogenesis, fusion, and outer membrane permeabilization. Melatonin treatment reduced the bioenergetic deficit found in ob/ob mice, alleviating some mitochondrial alterations in the electron transport chain machinery, biogenesis, dynamics, respiration, ATP production, and mitochondrial outer membrane permeabilization. Given the role of melatonin in maintaining mitochondrial homeostasis, it could be used as a therapeutic agent against adipogenic steatosis.

This study aimed to compare the effects of High-Intensity Interval Training (HIIT) performed in a single session(1xHIIT) versus three daily sessions (3xHIIT) on fitness level and behavior of aged rats. Eighteen-month-old Wistar rats were assigned to Untrained (UN), 1xHIIT, or 3xHIIT (n = 12/group). Both groups, 1xHIIT and 3xHIIT, performed 15 min of a treadmill running HIIT protocol during 8 weeks. 1xHIIT protocol consisted of a single daily session of 15 min, while the 3xHIIT performed three daily sessions of 5 min with a 4 h interval between the sessions. Morris Water Maze (MWM) task was used to evaluate spatial learning and memory. Splash test, Forced Swim test, and Elevated Plus Maze task (EPM) were used to evaluate anhedonic, depressive-like, and anxious behaviors, respectively. Rats were euthanized, and the hippocampus was harvested for western blot analyses (CaMKII and BDNF). Both HIIT protocols improved VO 2 max and spatial memory. Notably, only the 3xHIIT protocol attenuated anxious and depressive-like behaviors. Western blot analyses of the hippocampus revealed that both HIIT protocols increased BDNF levels. BDNF levels were higher in the 3xHIIT when compared with 1xHIIT group, and we observed increasement of the CamKII levels just in the 3x HIIT group. Therefore, this study provides evidence indicating that accumulated HIIT sessions is more effective than traditional daily HIIT sessions in improving fitness level, cognitive function, memory, inhibiting the development of mood disorders, and enhancing BDNF and CaMKII levels in the hippocampus of aged rats., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cardiovascular diseases account for a significant portion of the worldwide mortality rate. This aroused interest among the specialised scientific community, seeking for solutions based on non-clinical and clinical investigations, to shed light onto the physio-pathology of cardiovascular impairment. It is proven challenging managing chronic cardiovascular illnesses like atherosclerosis, arrhythmias, and diverse cardiomyopathies. In certain cases, there is no approved treatment. In other cases, the need for combining therapeutic components, when dealing with co-morbidities, may increase the risk of toxicity-driven cardiovascular impairment. In this case, because the risk of cardiac events correlates with the QT prolongation rates, the QT or QTc interval prolongation has become an important biomarker to access drug-related cardio-toxicity. Several approaches have been found in the current literature, aiming at improving physiological acceptance, i.e., to reduce toxicity. Nanotechnology has increasingly appeared as a promising ally to modulate active substances, preserving cardiovascular function and optimising drug effectiveness, i.e., acting as a cardio-protective mechanism, leveraging the effects of drug-driven cardio-toxicity. In this manuscript, the author combines plant active compounds and nanotechnological strategies, e.g., nano-encapsulation, nano-enzymes, magnetically driven nano-delivery systems, applied in regenerative medicine, and assesses their effects on the cardiovascular system, e.g., as cardio-protective factors, reducing cardio-toxicity. The aim is to propose a new strategy to tackle atherosclerosis initiation and progression, in a drug design that targets ROS-removal and reduces inflammation, using auto-immunity biomarkers to select key atheroma-related signalling cascades. To analyse physiological phenomena related to atherosclerosis initiation and progression, the author proposes both experimental observations and a new haemorheological computational model of arterial constriction. The results of such analysis are used as motivators in the design of the here presented strategy to tackle atheroma. This novel design is based on degradable polyethylene glycol (PEG) superparamagnetic iron oxide capsule coupled with a polyphenolic nano-enzymatic conjugate (PSPM-NE)., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Pereira.)

COVID-19, caused by SARS-CoV-2, affects neuronal cells, causing several symptoms such as memory loss, anosmia and brain inflammation. Curcuminoids (Me08 e Me23) and curcumin (CUR) are derived from Curcuma Longa extract (EXT). Many therapeutic actions have been linked to these compounds, including antiviral action. Given the severe implications of COVID-19, especially within the central nervous system, our study aims to shed light on the therapeutic potential of curcuminoids against SARS-CoV-2 infection, particularly in neuronal cells. Here, we investigated the effects of CUR, EXT, Me08 and Me23 in human neuroblastoma SH-SY5Y. We observed that Me23 significantly decreased the expression of plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and TMPRSS11D, consequently mitigating the elevated ROS levels induced by SARS-CoV-2. Furthermore, Me23 exhibited antioxidative properties by increasing NRF2 gene expression and restoring NQO1 activity following SARS-CoV-2 infection. Both Me08 and Me23 effectively reduced SARS-CoV-2 replication in SH-SY5Y cells overexpressing ACE2 (SH-ACE2). Additionally, all of these compounds demonstrated the ability to decrease proinflammatory cytokines such as IL-6, TNF-α, and IL-17, while Me08 specifically reduced INF-γ levels. Our findings suggest that curcuminoid Me23 could serve as a potential agent for mitigating the impact of COVID-19, particularly within the context of central nervous system involvement., (© 2024. The Author(s).)

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cell's energy supplies - particularly troublesome for energy-demanding cells of nervous tissue and muscle. Unsurprisingly then, import breakdown is implicated in disease. Here, we explore the consequences of import failure in mammalian cells; wherein, blocking the import machinery impacts mitochondrial ultra-structure and dynamics, but, surprisingly, does not affect import. Our data are consistent with a response involving intercellular mitochondrial transport via tunnelling nanotubes to import healthy mitochondria and jettison those with blocked import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial dysfunction., (© 2024. The Author(s).)

The emergence and continued geographic expansion of arboviruses and the growing number of infected people have highlighted the need to develop and improve multiplex methods for rapid and specific detection of pathogens. Sequencing technologies are promising tools that can help in the laboratory diagnosis of conditions that share common symptoms, such as pathologies caused by emerging arboviruses. In this study, we integrated nanopore sequencing and the advantages of reverse transcription polymerase chain reaction (RT-PCR) to develop a multiplex RT-PCR protocol for the detection of Chikungunya virus (CHIKV) and several orthoflaviviruses (such as dengue ( Orthoflavivirus dengue ), Zika ( Orthoflavivirus zikaense ), yellow fever ( Orthoflavivirus flavi ), and West Nile ( Orthoflavivirus nilense ) viruses) in a single reaction, which provides data for sequence-based differentiation of arbovirus lineages.

The spread of Chikungunya virus is a major public health concern in the Americas. There were >120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiologic techniques, we characterized the ongoing large chikungunya epidemic in Paraguay.

The emergence and reemergence of mosquito-borne diseases in Brazil such as yellow fever, zika, chikungunya, and dengue have had serious impacts on public health. Concerns have been raised due to the rapid dissemination of the chikungunya virus across the country since its first detection in 2014 in Northeast Brazil. In this work, we carried out on-site training activities in genomic surveillance in partnership with the National Network of Public Health Laboratories that have led to the generation of 422 chikungunya virus genomes from 12 Brazilian states over the past two years (2021-2022), a period that has seen more than 312 thousand chikungunya fever cases reported in the country. These genomes increased the amount of available data and allowed a more comprehensive characterization of the dispersal dynamics of the chikungunya virus East-Central-South-African lineage in Brazil. Tree branching patterns revealed the emergence and expansion of two distinct subclades. Phylogeographic analysis indicated that the northeast region has been the leading hub of virus spread towards other regions. Increased frequency of C > T transitions among the new genomes suggested that host restriction factors from the immune system such as ADAR and AID/APOBEC deaminases might be driving the genetic diversity of the chikungunya virus in Brazil., (© 2023. The Author(s).)

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Goodier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase-providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Obesity is one of the modifiable risk factors for dementia. Insulin resistance, the abundance of advanced glycated end-products, and inflammation are some of the mechanisms associated with the lower cognitive performance observed in obesity. This study aims to evaluate the cognitive function of subjects with distinct degrees of obesity, comparing class I and II obesity (OBI/II) to class III obesity (OBIII), and to investigate metabolic markers that can distinguish OBIII from OBI/II. Study Design. This is a cross-sectional study, in which 45 females with BMI varying from 32.8 to 51.9 kg/m 2 completed a set of 4 cognitive tests (verbal paired-associate test, stroop color, digit span, and Toulouse-Pieron cancellation test) and their plasma metabolites, enzymes, and hormones related to glycemia, dyslipidemia, and liver function, as well as the biomarkers of iron status, were concomitantly analyzed., Results: OBIII showed lower scores in the verbal paired-associate test compared to OBI/II. In other cognitive tests, both groups showed similar performance. OBIII presented a lower iron status compared to OBI/II based on total iron binding capacity, degree of transferrin saturation, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. The levels of indicators for glycemia, liver function, and lipid metabolism were similar in both groups. Analysis of plasma metabolites showed that OBIII had lower levels of pyroglutamic acid, myoinositol, and aspartic acid and higher levels of D-ribose than OBI/II., Conclusion: Iron is an essential micronutrient for several metabolic pathways. Thus, iron dyshomeostasis observed in severe obesity may aggravate the cognitive impairment by altering metabolic homeostasis and enhancing oxidative stress. These findings can contribute to searching for biomarkers that indicate cognitive performance in the population with obesity., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Jessica M. V. Pino et al.)

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma 1 . Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate 2 . However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response., (© 2023. The Author(s).)

Different plant products and co-products have been studied as wall materials for the microencapsulation of probiotics due to the need for new lost-cost, abundant, and natural materials. In this study, microparticles were developed by spray drying using different combinations of conventional materials such as maltodextrin, pectin, gelatin, and agar-agar with unconventional materials such as sweet potato flour to microencapsulate Lactiplantibacillus plantarum. The microparticles obtained were evaluated for encapsulation efficiency, thermal resistance, and rupture test. The most resistant microparticles were characterized and evaluated for probiotic viability during storage and survival to in vitro gastrointestinal conditions. Microparticles A (10 % maltodextrin, 5 % sweet potato flour, and 1 % pectin) and B (10 % maltodextrin, 4 % sweet potato flour, and 2 % gelatin) showed high thermal resistance (>59 %) and survival in acidic conditions (>80 %). L. plantarum in microparticles A and B remained viable with counts > 6 log CFU.g -1 for 45 days at 8 °C and -18 °C and resisted in vitro gastrointestinal conditions after processing with counts of 8.38 and 9.10 log CFU.g -1 , respectively. Therefore, the selected microparticles have great potential for application in different products in the food industry, as they promote the protection and distribution of probiotic microorganisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nucleic acids are paving the way for advanced therapeutics. Unveiling the genome enabled a better understanding of unique genotype-phenotype profiling. Methods for engineering and analysis of nucleic acids, from polymerase chain reaction to Cre-Lox recombination, are contributing greatly to biomarkers' discovery, mapping of cellular signaling cascades, and smart design of therapeutics in precision medicine. Investigating the different subtypes of DNA and RNA via sequencing and profiling is empowering the scientific community with valuable information, to be used in advanced therapeutics, tracking epigenetics linked to disease. Recent results from the application of nucleic acids in novel therapeutics and precision medicine are very encouraging, demonstrating great potential to treat cancer, viral infections via inoculation (e.g., SAR-COV-2 mRNA vaccines), along with metabolic and genetic disorders. Limitations posed by challenges in delivery mode are being addressed to enable efficient guided-gene-programmed precision therapies. With the focus on genetic engineering and novel therapeutics, more precisely, in precision medicine, this chapter discusses the advance enabled by knowledge derived from these innovative branches of biotechnology., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanotechnology and genomics are the newest allies of inoculation design. In recent years, nucleic acids have been targeted as sources of therapeutics to stimulate immune responses, to both fight disease and create memory to trigger further responses to threat. A myriad of promising findings in cancer research and virology has been reported in the current literature. Nanosystems are demonstrating their capabilities as efficient carriers, improving the efficacy of drug delivery, including nucleic acids as therapeutics, at focal sites, in living systems. This chapter approaches major elements involved in the successful use of nanotechnology as delivery platforms to optimise the efficacy of nucleic acids-driven therapeutics, particularly mRNA vectors as coding engines for targeted viral proteins. Latest findings in nanotechnological design are highlighted, key discoveries associated with the success of nanodelivery platforms are presented, and key characteristics of nanodelivery systems in nucleic acids-based vaccine technology are discussed, to illustrate their distinct advantages and disadvantages., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A fully automated strategy to handle antigenic variability in immunisation protocols is here presented. The method comprises of (1) nanopore sequencing of infectious agent variants, with focus on the SARS-CoV-2 and its variants, followed by (2) in-vitro transcribed mRNA vector design for immunotherapy. This chapter introduces the mRNA vector design protocol and Chapter 16 presents the nano-pore sequencing step., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rationale: Major depressive disorder (MDD) is one of the most diagnosed mental disorders. Despite this, its pathophysiology remains poorly understood. In this context, basic research aims to unravel the pathophysiological mechanisms of MDD as well as investigate new targets and substances with therapeutic potential. Transient receptor potential ankyrin 1 (TRPA1) is a transmembrane channel considered a sensor for inflammation and oxidative stress. Importantly, both inflammation and oxidative stress have been suggested as participants in the pathophysiology of MDD. However, the potential participation of TRPA1 in depressive disorder remains poorly investigated., Objective: To investigate the involvement of the TRPA1 channel in the behavioral changes induced by chronic corticosterone administration (CCA) in male mice., Methods: Swiss male mice were exposed to 21 days of CCA protocol and then treated with HC-030031 or A-967079, TRPA1 antagonists. Behavioral tests, analyzes of oxidative parameters and TRPA1 immunocontent were performed in the prefrontal cortex (PFC) and hippocampus (HIP)., Results: CCA induced despair-like behavior in mice accompanied by an increase in the levels of hydrogen peroxide (H 2 O 2 ), a TRPA1 agonist, which was reversed by TRPA1 antagonists and ketamine (positive control). In addition, CCA protocol reduced the immunocontent of this channel in the HIP and showed a tendency to increase the TRPA1 protein expression in the PFC., Conclusion: Our work suggests that TRPA1 channel appears crucial to mediate the behavioral impairment induced by CCA in male Swiss mice., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Meeting medical challenges posed by global burdens is proven to be of primary interest. One example is the COVID-19 epidemic that humankind is currently experiencing, since around December 2019. Innovation is key to respond rapidly and effectively to sanitary and health emergencies, when human lives are severely threatened. In this scenery, medical devices that can be rapidly launched in the market and manufactured at scale are crucial for saving lives. One example is a lifesaving respiratory device launched in about 10 days (Mercedes F1 team's new device based on continuous positive airway pressure devices) and rapidly approved by international agencies responsible for assuring drug and medical devices safety, in response to the COVID-19 burden. Remarkably, it is the first time in history that mankind observes disease spread reaching such high proportions, globally, in such short time scale. However, while this epidemic had, in March 2020, reached the critical figures of about 38,000 deaths and c. 738,000 infected, organ donation and transplantation patients are suffering for years, accounting for an increasing number of affected, annually. These patients are invisible for the general public. Therefore, this chapter approaches the organ donation and transplantation burden, proposing effective solutions to leverage the suffering, improving life quality of patients enduring several underlying issues, from hemodialysis complications and critical organ failure to lacking compatible donors. This, on the basis of technology repurposing, to speed up approval processes followed by international agencies responsible for assuring drug and medical devices safety, while adding innovative methods to existing technology and reducing invasiveness., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Infectious agents often challenge therapeutics, from antibiotics resistance to antigenic variability affecting inoculation measures. Over the last decades, genome sequencing arose as an important ally to address such challenges. In bacterial infection, whole-genome-sequencing (WGS) supports tracking pathogenic alterations affecting the human microbiome. In viral infection, the analysis of the relevant sequence of nucleotides helps with determining historical variants of a virus and elucidates details about infection clusters and their distribution. Additionally, genome sequencing is now an important step in inoculation protocols, isolating target genes to design more robust immunisation assays. Ultimately, genetic engineering has empowered repurposing at scale, allowing long-lasting repeating clinical trials to be automated within a much shorter time-frame, by adjusting existing protocols. This is particularly important during sanitary emergencies as the ones caused by the 2014 West African Ebola outbreak, the Zika virus rapid spread in both South and North America in 2015, followed by Asia in 2016, and the pandemic caused by the SARS-CoV-2, which has infected more than 187 million people and caused more than 4 million deaths, worldwide, as per July 2021 statistics. In this scenery, this chapter presents a novel fully automated strategy to handle antigenic variability in immunisation protocols. The methodology comprises of two major steps (1) nanopore sequencing of infectious agent variants - the focus is on the SARS-CoV-2 and its variants; followed by (2) mRNA vector design for immunotherapy. This chapter presents the nanopore sequencing step and Chapter 17 introduces a protocol for mRNA vector design., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gender-bias in COVID-19 severity has been suggested by clinical data. Experimental data in cell and animal models have demonstrated the role of sex hormones, particularly estrogens, in viral infections such as in COVID-19. SARS-CoV-2 uses ACE2 as a receptor to recognize host cells, and the protease TMPRSS2 for priming the Spike protein, facilitating virus entry into cells. However, the involvement of estrogenic receptors in SARS-CoV-2 infection are still being explored. Thus, in order to investigate the role of estrogen and its receptors in COVID-19, the estrogen receptors ERα, ERβ and GPER1 were overexpressed in bronchial BEAS-2B cell, and then infected with SARS-CoV-2. Interestingly, the levels of ACE2 and TMPRSS2 mRNA were higher in SARS-CoV-2-infected cells, but no difference was observed in cells with estrogen receptors overexpression. GPER1 can be involved in virus infection or replication, since its higher levels reduces SARS-CoV-2 load. On the other hand, pharmacological antagonism of GPER1 enhanced viral load. Those data suggest that GPER1 has an important role in SARS-CoV-2 infection., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest in this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Migraine represents one of the major causes of disability worldwide and is more prevalent in women; it is also related to anxiety symptoms. Stress, such as sound stress, is a frequently reported trigger in migraine patients, but the underlying mechanisms are not fully understood. However, it is known that patients with migraine have higher levels of plasma inflammatory cytokines and calcitonin gene-related peptide (CGRP). Stress mediated by unpredictable sound is already used as a model of painful sensitization, but migraine-like behaviors and sexual dimorphism have not yet been evaluated. This study characterized nociception and anxiety-related symptoms after the induction of sound stress in mice. C57BL/6 mice (20-30 g) were exposed to unpredictable sound stress for 3 days, nonconsecutive days. We observed enhanced plasma corticosterone levels on day 1 after stress induction. First, 7 days after the last stress session, mice developed hind paw and periorbital mechanical allodynia, grimacing pain behavior, anxiety-like symptoms, and reduced exploratory behavior. The nociceptive and behavioral alterations detected in this model were mostly shown in female stressed mice at day 7 post-stress. In addition, on day 7 post-stress nociception, these behaviors were consistently abolished by the CGRP receptor antagonist olcegepant (BIBN4096BS, 100 mg/kg by intraperitoneal route) in female and male stressed mice. We also demonstrated an increase in interleukine-6 (IL-6), tumor necrosis factor (TNF-α), and CGRP levels in stressed mice plasma, with female mice showing higher levels compared to male mice. This stress paradigm allows further preclinical investigation of mechanisms contributing to migraine-inducing pain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Viero, Rodrigues, Frare, Da Silva, Ferreira, Da Silva, Pereira, Ferreira, Pillat, Bocchi, Nassini, Geppetti and Trevisan.)

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness., Competing Interests: HF, WA, GP, XL, MD, IC No competing interests declared, (© 2022, Ford et al.)

Major depression is a leading contributor to the global burden of disease. This is mainly related to the disorder chronic and recurrent nature, and to high rates of refractoriness to treatment. Limited efficacy with currently available antidepressants highlights the need for more effective options for treating drug-resistant patients and emphasizes the importance of developing specific preclinical models for treatment-resistant populations. Treatment-resistant depression (TRD) is commonly defined as failure to respond to two or more trials of antidepressants. In this study, we investigated the effect of fluoxetine treatment for fourteen days on the depressive-like behavior and the oxidative and inflammatory parameters of mice submitted to chronic corticosterone administration. After 21 days of subcutaneous corticosterone administration (20 mg/kg/day) and 14 days of oral fluoxetine treatment (10 mg/kg/day, started on day 7 of induction protocol), we separated animals into two groups according to the tail suspension test (TST) results: antidepressant responders (good response to antidepressant, GRA) and non-responders (resistance to antidepressant, AR). Forced swimming test (FST), elevated plus maze test (EPMT), and open field test (OFT) were performed. We found that animals classified as AR (i.e., those with higher immobility values in the TST) demonstrated anxiety-like behavior in the EPMT, increased H 2 O 2 levels, and decreased catalase activity in the hippocampus, as well as increased serum levels of IL-17 and IFN-γ. Our findings suggest that a redox imbalance in the hippocampus, combined with increased levels of peripheral IL-17 and INF-γ, may be involved with an impaired response to fluoxetine., (Copyright © 2022 Elsevier B.V. All rights reserved.)