The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

The autophagy protein RUBCNL/PACER represses RIPK1 kinase-dependent apoptosis and necroptosis.

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.

Porphyromonas gingivalis, a bridge between oral health and immune evasion in gastric cancer.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative oral pathogen associated with chronic periodontitis. Previous studies have linked poor oral health and periodontitis with oral cancer. Severe cases of periodontal disease can result in advanced periodontitis, leading to tissue degradation, tooth loss, and may also correlate with higher gastric cancer (GC) risk. In fact, tooth loss is associated with an elevated risk of cancer. However, the clinical evidence for this association remains inconclusive. Periodontitis is also characterized by chronic inflammation and upregulation of members of the Programmed Death 1/PD1 Ligand 1 (PD1/PDL1) axis that leads to an immunosuppressive state. Given that chronic inflammation and immunosuppression are conditions that facilitate cancer progression and carcinogenesis, we hypothesize that oral P. gingivalis and/or its virulence factors serve as a mechanistic link between oral health and gastric carcinogenesis/GC progression. We also discuss the potential impact of P. gingivalis' virulence factors (gingipains, lipopolysaccharide (LPS), and fimbriae) on inflammation and the response to immune checkpoint inhibitors in GC which are part of the current standard of care for advanced stage patients.

The Autophagy Protein Pacer Positively Regulates the Therapeutic Potential of Mesenchymal Stem Cells in a Mouse Model of DSS-Induced Colitis.

Mesenchymal stem cells (MSC) have emerged as a promising tool to treat inflammatory diseases, such as inflammatory bowel disease (IBD), due to their immunoregulatory properties. Frequently, IBD is modeled in mice by using dextran sulfate sodium (DSS)-induced colitis. Recently, the modulation of autophagy in MSC has been suggested as a novel strategy to improve MSC-based immunotherapy. Hence, we investigated a possible role of Pacer, a novel autophagy enhancer, in regulating the immunosuppressive function of MSC in the context of DSS-induced colitis. We found that Pacer is upregulated upon stimulation with the pro-inflammatory cytokine TNFα, the main cytokine released in the inflammatory environment of IBD. By modulating Pacer expression in MSC, we found that Pacer plays an important role in regulating the autophagy pathway in this cell type in response to TNFα stimulation, as well as in regulating the immunosuppressive ability of MSC toward T-cell proliferation. Furthermore, increased expression of Pacer in MSC enhanced their ability to ameliorate the symptoms of DSS-induced colitis in mice. Our results support previous findings that autophagy regulates the therapeutic potential of MSC and suggest that the augmentation of autophagic capacity in MSC by increasing Pacer levels may have therapeutic implications for IBD.

Proteogenomic analysis in an early onset diffuse gastric cancer patient revealed alterations in PIK3R1, TP53, SMAD4 and a potential role of the PI3K-AKT and EGFR pathways: a case report.

Background: Early-onset gastric cancers (EOGC) are poor prognosis hard-to treat malignancies that affect young individuals (<45 years old). Case Description: Herein we describe the case of a 26-year-old female EOGC patient that initially displayed stable disease after first-line CAPOX plus immunotherapy. However, patient eventually developed progressive disease and was consecutively switched to paclitaxel plus ramucirumab, and palliative irinotecan. In search for therapeutic alternatives a proteo-genomic analysis was performed in a tissue biopsy taken after the first progression. Our analyses found a total of 18 somatic mutations, including TP53 and PIK3R1, and a previously unreported germline alteration in the tumor suppressor SMAD4. Also, our proteomic analysis found 62 proteins previously documented as "enriched in stomach cancer" and AKT/mTOR and EGFR as pathways with therapeutic potential. Unfortunately, the clinical utility of AKT/mTOR inhibitors or EGFR targeted therapies could not be assessed. Conclusions: As explained above EOGC is a growing health concern that affects young individuals. Furthermore, the reported case displayed a poor response to standard therapy including checkpoint inhibitors and chemotherapy despite the presence of biomarkers that predict a favorable outcome. Future studies should adopt alternative approaches to find novel, more effective therapies.

Mutational Landscape and Actionable Target Rates on Advanced Stage Refractory Cancer Patients: A Multicenter Chilean Experience.

Major advances in sequencing technologies and targeted therapies have accelerated the incorporation of oncology into the era of precision medicine and "biomarker-driven" treatments. However, the impact of this approach on the everyday clinic has yet to be determined. Most precision oncology reports are based on developed countries and usually involve metastatic, hard-to-treat or incurable cancer patients. Moreover, in many cases race and ethnicity in these studies is commonly unreported and real-world evidence in this topic is scarce. Herein, we report data from a total of 202 Chilean advanced stage refractory cancer patients. Retrospectively, we collected patient data from NGS tests and IHC in order to determine the proportion of patients that would benefit from targeted treatments. Overall >20 tumor types were included in our cohort and 37% of patients (n = 74) displayed potentially actionable alterations, including on-label, off-label and immune checkpoint inhibitor recommendations. Our findings were in-line with previous reports such as the cancer genome atlas (TCGA). To our knowledge, this is the first report of its kind in Latin America delivering real-world evidence to estimate the percentage of refractory tumor patients that might benefit from precision oncology. Although this approach is still in its infancy in Chile, we strongly encourage the implementation of mutational tumor boards in our country in order to provide more therapeutic options for advanced stage refractory patients.

Neuronal Rubicon Represses Extracellular APP/Amyloid ß Deposition in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.

Trypanosoma cruzi infection induces a global host cell response in cardiomyocytes.

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.

DEF8 and Autophagy-Associated Genes Are Altered in Mild Cognitive Impairment, Probable Alzheimer's Disease Patients, and a Transgenic Model of the Disease.

BACKGROUND: Disturbances in the autophagy/endolysosomal systems are proposed as early signatures of Alzheimer's disease (AD). However, few studies are available concerning autophagy gene expression in AD patients. OBJECTIVE: To explore the differential expression of classical genes involved in the autophagy pathway, among them a less characterized one, DEF8 (Differentially expressed in FDCP 8), initially considered a Rubicon family member, in peripheralblood mononuclear cells (PBMCs) from individuals with mild cognitive impairment (MCI) and probable AD (pAD) and correlate the results with the expression of DEF8 in the brain of 5xFAD mice. METHOD: By real-time PCR and flow cytometry, we evaluated autophagy genes levels in PBMCs from MCI and pAD patients. We evaluated DEF8 levels and its localization in brain samples of the 5xFAD mice by real-time PCR, western blot, and immunofluorescence. RESULTS: Transcriptional levels of DEF8 were significantly reduced in PBMCs of MCI and pAD patients compared with healthy donors, correlating with the MoCA and MoCA-MIS cognitive tests scores. DEF8 protein levels were increased in lymphocytes from MCI but not pAD, compared to controls. In the case of brain samples from 5xFAD mice, we observed a reduced mRNA expression and augmented protein levels in 5xFAD compared to age-matched wild-type mice. DEF8 presented a neuronal localization. CONCLUSION: DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially expressed in PBMCs of MCI and pAD and neurons of 5xFAD mice. These results suggest a potential role for DEF8 in the pathophysiology of AD.

The genome of Cryptosporidium hominis.

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens.

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

A genome-scale metabolic model of Cryptosporidium hominis.

The apicomplexan Cryptosporidium is a protozoan parasite of humans and other mammals. Cryptosporidium species cause acute gastroenteritis and diarrheal disease in healthy humans and animals, and cause life-threatening infection in immunocompromised individuals such as people with AIDS. The parasite has a one-host life cycle and commonly invades intestinal epithelial cells. The current genome annotation of C. hominis, the most serious human pathogen, predicts 3884 genes of which ca. 1581 have predicted functional annotations. Using a combination of bioinformatics analysis, biochemical evidence, and high-throughput data, we have constructed a genome-scale metabolic model of C. hominis. The model is comprised of 213 gene-associated enzymes involved in 540 reactions among the major metabolic pathways and provides a link between the genotype and the phenotype of the organism, making it possible to study and predict behavior based upon genome content. This model was also used to analyze the two life stages of the parasite by integrating the stage-specific proteomic data for oocyst and sporozoite stages. Overall, this model provides a computational framework to systematically study and analyze various functional behaviors of C. hominis with respect to its life cycle and pathogenicity.

Implications of Selective Autophagy Dysfunction for ALS Pathology.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder that progressively affects motor neurons in the brain and spinal cord. Due to the biological complexity of the disease, its etiology remains unknown. Several cellular mechanisms involved in the neurodegenerative process in ALS have been found, including the loss of RNA and protein homeostasis, as well as mitochondrial dysfunction. Insoluble protein aggregates, damaged mitochondria, and stress granules, which contain RNA and protein components, are recognized and degraded by the autophagy machinery in a process known as selective autophagy. Autophagy is a highly dynamic process whose dysregulation has now been associated with neurodegenerative diseases, including ALS, by numerous studies. In ALS, the autophagy process has been found deregulated in both familial and sporadic cases of the disease. Likewise, mutations in genes coding for proteins involved in the autophagy machinery have been reported in ALS patients, including selective autophagy receptors. In this review, we focus on the role of selective autophagy in ALS pathology.

Decoding Gene Networks Modules That Explain the Recovery of Hymenoglossum cruentum Cav. After Extreme Desiccation.

Hymenoglossum cruentum (Hymenophyllaceae) is a poikilohydric, homoiochlorophyllous desiccation-tolerant (DT) epiphyte fern. It can undergo fast and frequent dehydration-rehydration cycles. This fern is highly abundant at high-humidity/low-light microenvironments within the canopy, although rapid changes in humidity and light intensity are frequent. The objective of this research is to identify genes associated to desiccation-rehydration cycle in the transcriptome of H. cruentum to better understand the genetic dynamics behind its desiccation tolerance mechanism. H. cruentum plants were subjected to a 7 days long desiccation-rehydration process and then used to identify key expressed genes associated to its capacity to dehydrate and rehydrate. The relative water content (RWC) and maximum quantum efficiency (F v/F m) of H. cruentum fronds decayed to 6% and 0.04, respectively, at the end of the desiccation stage. After re-watering, the fern showed a rapid recovery of RWC and F v/F m (ca. 73% and 0.8, respectively). Based on clustering and network analysis, our results reveal key genes, such as UBA/TS-N, DYNLL, and LHC, orchestrating intracellular motility and photosynthetic metabolism; strong balance between avoiding cell death and defense (CAT3, AP2/ERF) when dehydrated, and detoxifying pathways and stabilization of photosystems (GST, CAB2, and ELIP9) during rehydration. Here we provide novel insights into the genetic dynamics behind the desiccation tolerance mechanism of H. cruentum.

T-bet-dependent regulation of CD8+ T-cell expansion during experimental Trypanosoma cruzi infection.

The transcription factor T-bet (T-box, expressed in T cells), promotes type I immunity to pathogens through effects involving T cells and dendritic cells. In CD8(+) T cells, many of the functions of T-bet are redundant with those of eomesodermin (Eomes), a paralogue of T-bet. We therefore investigated the role of T-bet in immunity to Trypanosoma cruzi, an intracellular pathogen that causes Chagas disease, and which requires CD8(+) T cells for resistance. T-bet-deficient mice (tbx21(-/-)) were highly susceptible to T. cruzi infection, marked by severe liver pathology. CD8(+) T cells from infected tbx21(-/-) mice expressed typical markers of activation, including CD44 and CD25. In striking contrast, there was a 10-fold reduction in the number of antigen-specific CD8(+) T cells in tbx21(-/-) mice. This reduction was not a consequence of increased apoptosis or altered tissue-specific migration. Further, antigen-presenting cell (APC) functions in tbx21(-/-) mice were normal as we observed comparable levels of B7-1, B7-2 and CD40 expression as well as normal antigen-driven proliferation of wild-type CD8(+) T cells in infected tbx21(-/-) mice. However, adoptive transfer of naïve T cells from tbx21(-/-) donors into infected Rag-2-deficient mice (tbx21(+/+)) demonstrated a similar quantitative defect in CD8(+) T-cell expansion. These data demonstrate that T-bet facilitates immunity to T. cruzi by promoting the expansion of T. cruzi-specific CD8(+) T cells in a T cell-intrinsic manner. They also serve to further illustrate the multifaceted functions of T-box proteins in regulating quantitative aspects of T-cell immunity, in addition to qualitative components such as cytokine production.

Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs.

Exosomes are small (30-150 nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.

Genome-wide circulating microRNA expression profiling reveals potential biomarkers for amyotrophic lateral sclerosis.

The occurrence of mutations of TDP-43, FUS, and C9ORF72 in amyotrophic lateral sclerosis (ALS) suggests pathogenic alterations to RNA metabolism and specifically to microRNA (miRNA) biology. Moreover, several ALS-related proteins impact stress granule dynamics affecting miRNA biogenesis and cellular miRNA levels. miRNAs are present in different biological fluids and have been proposed as potential biomarkers. Here we used next-generation sequencing to perform a comparative analysis of the expression profile of circulating miRNAs in the serum of 2 mutant superoxide dismutase 1 transgenic mice. Top hit candidates were then validated using quantitative real-time polymerase chain reaction, confirming significant changes for 6 miRNAs. In addition, one of these miRNAs was also altered in mutant TDP-43 mice. Then, we tested this set of miRNAs in the serum from sporadic ALS patients, observing a significant deregulation of hsa-miR-142-3p and hsa-miR-1249-3p. A negative correlation between the revised ALS functional rating scale and hsa-miR-142-3p levels was found. Bioinformatics analysis of the regulatory network governed by hsa-miR-142-3p identified TDP-43 and C9orf72 as possible targets, suggesting a connection with ALS pathogenesis. This study identifies miRNAs that are altered in ALS that may serve as potentials biomarkers.

The Enigmatic Role of C9ORF72 in Autophagy.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the loss of motor neurons resulting in a progressive and irreversible muscular paralysis. Advances in large-scale genetics and genomics have revealed intronic hexanucleotide repeat expansions in the gene encoding C9ORF72 as a main genetic cause of ALS and frontotemporal dementia (FTD), the second most common cause of early-onset dementia after Alzheimer's disease. Novel insights regarding the underlying pathogenic mechanisms of C9ORF72 seem to suggest a synergy of loss and gain of toxic function during disease. C9ORF72, thus far, has been found to be involved in homeostatic cellular pathways, such as actin dynamics, regulation of membrane trafficking, and macroautophagy. All these pathways have been found compromised in the pathogenesis of ALS. In this review, we aim to summarize recent findings on the function of C9ORF72, particularly in the macroautophagy pathway, hinting at a requirement to maintain the fine balance of macroautophagy to prevent neurodegeneration.

Axonal Degeneration during Aging and Its Functional Role in Neurodegenerative Disorders.

Aging constitutes the main risk factor for the development of neurodegenerative diseases. This represents a major health issue worldwide that is only expected to escalate due to the ever-increasing life expectancy of the population. Interestingly, axonal degeneration, which occurs at early stages of neurodegenerative disorders (ND) such as Alzheimer's disease, Amyotrophic lateral sclerosis, and Parkinson's disease, also takes place as a consequence of normal aging. Moreover, the alteration of several cellular processes such as proteostasis, response to cellular stress and mitochondrial homeostasis, which have been described to occur in the aging brain, can also contribute to axonal pathology. Compelling evidence indicate that the degeneration of axons precedes clinical symptoms in NDs and occurs before cell body loss, constituting an early event in the pathological process and providing a potential therapeutic target to treat neurodegeneration before neuronal cell death. Although, normal aging and the development of neurodegeneration are two processes that are closely linked, the molecular basis of the switch that triggers the transition from healthy aging to neurodegeneration remains unrevealed. In this review we discuss the potential role of axonal degeneration in this transition and provide a detailed overview of the literature and current advances in the molecular understanding of the cellular changes that occur during aging that promote axonal degeneration and then discuss this in the context of ND.

High quality RNA extraction from Maqui berry for its application in next-generation sequencing.

Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and ß-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.