A Review on the Current Knowledge on ZIKV Infection and the Interest of Organoids and Nanotechnology on Development of Effective Therapies against Zika Infection.

Zika virus (ZIKV) acquired a special relevance due to the pandemic that occurred in the Americas in 2015, when an important number of fetal microcephaly cases occurred. Since then, numerous studies have tried to elucidate the pathogenic mechanisms and the potential therapeutic approaches to combat the virus. Cellular and animal models have proved to be a basic resource for this research, with the more recent addition of organoids as a more realistic and physiological 3D culture for the study of ZIKV. Nanotechnology can also offer a promising therapeutic tool, as the nanoparticles developed by this field can penetrate cells and deliver a wide array of drugs in a very specific and controlled way inside the cells. These two state-of-the-art scientific tools clearly provide a very relevant resource for the study of ZIKV, and will help researchers find an effective treatment or vaccine against the virus.

High Preventive Effect of G2-S16 Anionic Carbosilane Dendrimer against Sexually Transmitted HSV-2 Infection.

Anionic carbosilane dendrimers such as G2-S16 are very effective in preventing HSV-2 infection both in vitro and in vivo. We present the main achievements obtained for the G2-S16 dendrimer in vivo, especially related to its efficacy against HSV-2 infection. Moreover, we discuss the mechanisms by which the G2-S16 dendrimer applied vaginally as a topical microbicide has been demonstrated to be safe and harmless for the vaginal microbiome balance, as both conditions present an essential step that has to be overcome during microbicide development. This review points to the marked protective effect of the G2-S16 dendrimer against sexually transmitted HSV-2 infection, supporting its role as a possible microbicide against HSV-2 infection.

Uses for humanised mouse models in precision medicine for neurodegenerative disease.

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer's Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Comparative study of hematopoietic stem and progenitor cells between sexes in mice under physiological conditions along time.

Hematopoietic stem and progenitor cells (HSPCs) are attractive targets in regenerative medicine, although the differences in their homeostatic maintenance between sexes along time are still under debate. We accurately monitored hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs) frequencies by flow cytometry, by performing serial peripheral blood extractions from male and female B6SJL wild-type mice and found no significant differences. Only modest differences were found in the gene expression profile of Slamf1 and Gata2. Our findings suggest that both sexes could be used indistinctly to perform descriptive studies in the murine hematopoietic system, especially for flow cytometry studies in peripheral blood. This would allow diminishing the number of animals needed for the experimental procedures. In addition, the use of serial extractions in the same animals drastically decreases the number of animals needed.

Granulocyte Colony-Stimulating Factor Ameliorates Skeletal Muscle Dysfunction in Amyotrophic Lateral Sclerosis Mice and Improves Proliferation of SOD1-G93A Myoblasts in vitro.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) causes loss of upper and lower motor neurons as well as skeletal muscle (SKM) dysfunction and atrophy. SKM is one of the tissues involved in the development of ALS pathology, and studies in a SOD1-G93A mouse model of ALS have demonstrated alterations in SKM degeneration/regeneration marker expression in vivo and defective mutant myoblast proliferation in vitro. Granulocyte colony-stimulating factor (G-CSF) has been shown to alleviate SOD1-G93A pathology. However, it is unknown whether G-CSF may have a direct effect on SKM or derived myoblasts. OBJECTIVE: To investigate effects of G-CSF and its analog pegfilgrastim (PEGF) on SOD1-G93A- associated SKM markers in vivo and those of G-CSF on myoblast proliferation in vitro. METHODS: The effect of PEGF treatment on hematopoietic stem cell mobilization, survival, and motor function was determined. RNA expression of SKM markers associated with mutant SOD1 expression was quantified in response to PEGF treatment in vivo, and the effect of G-CSF on the proliferation of myoblasts derived from mutant and control muscles was determined in vitro. RESULTS: Positive effects of PEGF on hematopoietic stem cell mobilization, survival, and functional assays in SOD1-G93A animals were confirmed. In vivo PEGF treatment augmented the expression of its receptor Csf3r and alleviated typical markers for mutant SOD1 muscle. Additionally, G-CSF was found to directly increase the proliferation of SOD1-G93A, but not wild-type primary myoblasts in vitro. CONCLUSION: Our results support the beneficial role of the G-CSF analog PEGF in a SOD1-G93A model of ALS. Thus, G-CSF and its analogs may be directly beneficial in diseases where the SKM function is compromised.

Hematopoietic stem and progenitor cells as novel prognostic biomarkers of longevity in a murine model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a difficult diagnosis and prognosis. In this regard, new and more reliable biomarkers for the disease are needed. We propose peripheral blood, and, more specifically, the hematopoietic stem and progenitor cells (HSPCs) as potential prognostic biomarkers in the SOD1G93A murine model of ALS. We accurately and serially studied three HSPCs-hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and common myeloid progenitors (CMPs)-in both control and SOD1G93A mice along the disease's progression by RT-PCR and flow cytometry analysis. We found interesting differences for every HSPC type in the transgenic mice compared with the control mice at every time point selected, as well as differences along the disease course. The results showed a maintained compensatory increase of HSCs along disease progression. However, the downregulated levels of CLPs and CMPs suggested an exit of these cell populations to the peripheral tissues, probably due to their supporting role to the damaged tissues. In addition, a positive correlation of the percentage of CLPs and CMPs with the longevity was found, as well as a positive correlation of HSCs and CMPs with motor function and weight, thus reinforcing the idea that HSPCs play a relevant role in the longevity of the SOD1G93A mice. On the basis of these results, both CLPs and CMPs could be considered prognostic biomarkers of longevity in this animal model, opening the door to future studies in human patients for their potential clinical use.

Erratum: Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

[This corrects the article DOI: 10.1016/j.isci.2021.103463.].

Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy.

miRNAs have been extensively studied in pathological conditions, including viral infections, such as those provoked by HIV-1. Several cellular and circulating miRNAs are altered during HIV-1 infection, with either beneficial effects on host defenses or enhanced virus infectivity. Blood samples were collected in sterile EDTA tubes and plasma was separated and stored, as were PBMCs. RNA was isolated and reverse-transcribed. Finally, the miRNA gene expression profile was assessed using TaqMan Array Human microRNA Card A v2.0. A comprehensive statistical analysis was performed on the results obtained. This is the first study on miRNAs in HIV-1 paediatric patients, and a miRNA profile differentiating patients starting combination antiretroviral therapy (cART) at different times after HIV-1 diagnosis was established. Thirty-four miRNAs were observed to have different expression levels between the control group and the cART group. The data indicates the need to start cART as soon as possible after the establishment of HIV-1 infection to assure the best outcome possible. Finally, the selected 34 miRNAs may be used as biomarkers for prognosis and assessing therapy effectiveness. However, more research must be conducted to establish adequate quantitative correlations.

Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

Age-Associated Differences in Hematopoietic Stem and Progenitor Cells of Mice.

Establishing the appropriate yet minimal number of control mice for experiments is a critical step in experimental design. Thisdecision is particularly important regarding the study of the hematopoietic system over time, given various age-associated changes in murine hematopoietic cell populations. Here we used flow cytometry to serially monitor the frequencies of hematopoietic stem cells, common lymphoid progenitor cells, and common myeloid progenitor cells and RT-PCR assays to study the levels of Ly6a(Sca1), Slamf1, Ikzf1, and Cebpa-4 genes that control the hematopoietic process-in wildtype male and female mice with a B6SJL genetic background. These analyses revealed many differences, both at the cellular and mRNA levels, between immature and mature mice at various developmental stages. In conclusion, although it is necessary to minimize the number of mice possible insofar as possible to reduce animal use and meet animal welfare requirements, the numerous differences shown by our findings highlight the need to establish controls for every time point selected for the study of the hematopoietic system cells. This need is especially crucial when comparing immature and mature stages of mouse development.

Inflammatory and non-inflammatory monocytes as novel prognostic biomarkers of survival in SOD1G93A mouse model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) has lately become a suitable scenario to study the interplay between the hematopoietic system and disease progression. Recent studies in C9orf72 null mice have demonstrated that C9orf72 is necessary for the normal function of myeloid cells. In this study, we aimed to analyze in depth the connection between the hematopoietic system and secondary lymphoid (spleen) and non-lymphoid (liver and skeletal muscle) organs and tissues along the disease progression in the transgenic SOD1G93A mice. Our findings suggested that the inflammatory response due to the neurodegeneration in this animal model affected all three organs and tissues, especially the liver and the skeletal muscle. However, the liver was able to compensate this inflammatory response by means of the action of non-inflammatory monocytes, while in the skeletal muscle inflammatory monocytes prompted a further inflammation process until the terminal state of the animals. Interestingly, in blood, a positive correlation was found between non-inflammatory monocytes and survival of the transgenic SOD1G93A mice, while the contrary (a negative correlation) was found in the case of inflammatory monocytes, supporting their potential role as biomarkers of disease progression and survival in this animal model. These findings could prompt future translational studies in ALS patients, promoting the identification of new reliable biomarkers of disease progression.

Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.