Sustained Nrf2 Overexpression-Induced Metabolic Deregulation Can Be Attenuated by Modulating Insulin/Insulin-like Growth Factor Signaling.

The modulation of insulin/insulin-like growth factor signaling (IIS) is associated with altered nutritional and metabolic states. The Drosophila genome encodes eight insulin-like peptides, whose activity is regulated by a group of secreted factors, including Ecdysone-inducible gene L2 (ImpL2), which acts as a potent IIS inhibitor. We recently reported that cncC (cncC/Nrf2), the fly ortholog of Nrf2, is a positive transcriptional regulator of ImpL2, as part of a negative feedback loop aiming to suppress cncC/Nrf2 activity. This finding correlated with our observation that sustained cncC/Nrf2 overexpression/activation (cncCOE; a condition that signals organismal stress) deregulates IIS, causing hyperglycemia, the exhaustion of energy stores in flies' tissues, and accelerated aging. Here, we extend these studies in Drosophila by assaying the functional implication of ImpL2 in cncCOE-mediated metabolic deregulation. We found that ImpL2 knockdown (KD) in cncCOE flies partially reactivated IIS, attenuated hyperglycemia and restored tissue energetics. Moreover, ImpL2 KD largely suppressed cncCOE-mediated premature aging. In support, pharmacological treatment of cncCOE flies with Metformin, a first-line medication for type 2 diabetes, restored (dose-dependently) IIS functionality and extended cncCOE flies' longevity. These findings exemplify the effect of chronic stress in predisposition to diabetic phenotypes, indicating the potential prophylactic role of maintaining normal IIS functionality.

Predictive Factors for Neutralizing Antibody Levels Nine Months after Full Vaccination with BNT162b2: Results of a Machine Learning Analysis.

Vaccination against SARS-CoV-2 with BNT162b2 mRNA vaccine plays a critical role in COVID-19 prevention. Although BNT162b2 is highly effective against COVID-19, a time-dependent decrease in neutralizing antibodies (NAbs) is observed. The aim of this study was to identify the individual features that may predict NAbs levels after vaccination. Machine learning techniques were applied to data from 302 subjects. Principal component analysis (PCA), factor analysis of mixed data (FAMD), k-means clustering, and random forest were used. PCA and FAMD showed that younger subjects had higher levels of neutralizing antibodies than older subjects. The effect of age is strongest near the vaccination date and appears to decrease with time. Obesity was associated with lower antibody response. Gender had no effect on NAbs at nine months, but there was a modest association at earlier time points. Participants with autoimmune disease had lower inhibitory levels than participants without autoimmune disease. K-Means clustering showed the natural grouping of subjects into five categories in which the characteristics of some individuals predominated. Random forest allowed the characteristics to be ordered by importance. Older age, higher body mass index, and the presence of autoimmune diseases had negative effects on the development of NAbs against SARS-CoV-2, nine months after full vaccination.

Comparison of Neutralizing Antibody Responses at 6 Months Post Vaccination with BNT162b2 and AZD1222.

Along with their level of protection against COVID-19, SARS-CoV-2-specific antibodies decline over time following vaccination with BNT162b2. However, relevant data on AZD1222 are scarce. In this context, the aim of this study was to compare SARS-CoV-2 neutralizing antibody (NAb) levels at one, three and six months after second vaccination with the BNT162b2 mRNA vaccine and the ChAdOx1 (AZD1222) viral vector vaccine (NCT04743388). The measurements were performed with the GenScript's cPassTM SARS-CoV-2 NAbs Detection Kit (GenScript, Inc.; Piscataway, NJ, USA). Overall, data from 282 individuals were included (BNT162b2 n = 83, AZD1222 n = 199). Both vaccines induced strong NAbs responses at 1 month following vaccination. Interestingly, NAb activity seemed superior with BNT162b2 compared with AZD1222. A gradual decline in NAbs titers was evident at 3 and 6 months post vaccination with both vaccines. However, the superiority of NAb response with BNT162b2 over AZD1222 remained consistent at all time points examined. Furthermore, the elimination rate of the NAb titer was higher throughout during the study period for those vaccinated with AZD1222 compared with BNT162b2. Age, gender, body mass index or comorbidities did not have a significant impact on NAbs levels over time. Our results may inform public health policies regarding the timing of booster COVID-19 vaccine shots.

Differential Dose- and Tissue-Dependent Effects of foxo on Aging, Metabolic and Proteostatic Pathways.

Aging is the gradual deterioration of physiological functions that culminates in death. Several studies across a wide range of model organisms have revealed the involvement of FOXO (forkhead box, class O) transcription factors in orchestrating metabolic homeostasis, as well as in regulating longevity. To study possible dose- or tissue-dependent effects of sustained foxo overexpression, we utilized two different Drosophila transgenic lines expressing high and relatively low foxo levels and overexpressed foxo, either ubiquitously or in a tissue-specific manner. We found that ubiquitous foxo overexpression (OE) accelerated aging, induced the early onset of age-related phenotypes, increased sensitivity to thermal stress, and deregulated metabolic and proteostatic pathways; these phenotypes were more intense in transgenic flies expressing high levels of foxo. Interestingly, there is a defined dosage of foxo OE in muscles and cardiomyocytes that shifts energy resources into longevity pathways and thus ameliorates not only tissue but also organismal age-related defects. Further, we found that foxo OE stimulates in an Nrf2/cncC dependent-manner, counteracting proteostatic pathways, e.g., the ubiquitin-proteasome pathway, which is central in ameliorating the aberrant foxo OE-mediated toxicity. These findings highlight the differential dose- and tissue-dependent effects of foxo on aging, metabolic and proteostatic pathways, along with the foxo-Nrf2/cncC functional crosstalk.

An enriched polyphenolic extract obtained from the by-product of Rosa damascena hydrodistillation activates antioxidant and proteostatic modules.

BACKGROUND: Prolonged maintenance of proteome stability and functionality (proteostasis) is of emerging significance in aging retardation and healthspan. PURPOSE: An enriched polyphenolic extract obtained from the hydrodistillation of rose petals was tested for its capacity to activate the proteostasis network modules, and thus modulate health- and/or lifespan at the cellular and whole organism level. METHODS: The aqueous extract that remained after the hydrodistillation of Rosa damascena petals, was processed with a polystyrene-FPX66 adsorption resin and sequentially fractionated by FCPC. NMR and UHPLC-HRMS analyses revealed the presence of 28 metabolites, mainly glycosides of kaempferol and quercetin. RESULTS: The extract showed high in vitro antioxidant activity and was not toxic in normal human skin fibroblasts, while it promoted the upregulation of NRF2-induced antioxidant genes and main proteostatic modules. Consistently, supplementation of this extract in Drosophila flies' culture medium induced a cncC/NRF2-mediated upregulation of antioxidant and proteostatic modules. Prolonged administration of the extract in flies' culture medium was not toxic and did not affect food intake rate or fecundity; also, it delayed the age-related decline of stress tolerance and locomotion performance (neuromuscular functionality) and dose-dependently extended flies' lifespan. CONCLUSION: Our findings indicate that the enriched polyphenolic extract obtained from the residue of R. damascena hydrodistillation activates cytoprotective cellular modules that, likely, contribute to its potential anti-aging properties.

Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes.

The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies' tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.

Amyloid toxicity in a Drosophila Alzheimer's model is ameliorated by autophagy activation.

Alzheimer's disease (AD) is the prevailing form of dementia. Protein degradation and antioxidant pathways have a critical role in preventing the accumulation of protein aggregation; thus, failure of proteostasis in neurons along with redox imbalance mark AD. Herein, we exploited an AD Drosophila model expressing human amyloid precursor (hAPP) and beta-secretase 1 (hBACE1) proteins, to better understand the role of proteostatic or antioxidant pathways in AD. Ubiquitous expression of hAPP, hBACE1 in flies caused more severe degenerative phenotypes versus neuronal targeted expression; it also, suppressed proteasome activity, increased oxidative stress and significantly enhanced stress-sensitivity. Overexpression of Prosß5 proteasomal subunit or Nrf2 transcription factor in AD Drosophila flies partially restored proteasomal activity but did not rescue hAPP, hBACE1 induced neurodegeneration. On the other hand, expression of autophagy-related Atg8a in AD flies decelerated neurodegeneration, increased stress-resistance, and improved flies' health-/lifespan. Overall, our data suggest that the noxious effects of amyloid-beta aggregates can be alleviated by enhanced autophagy, thus dietary or pharmacological interventions that target autophagy should be considered in AD therapeutic approaches.

SARS-CoV-2 Infection Is Asymptomatic in Nearly Half of Adults with Robust Anti-Spike Protein Receptor-Binding Domain Antibody Response.

Between June and November 2020, we assessed plasma antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein in 4996 participants (aged 18-82 years, 34.5% men) from the National and Kapodistrian University of Athens. The weighted overall prevalence was 1.6% and monthly prevalence correlated with viral RNA-confirmed SARS-CoV-2 infections in Greece, in the same period. Notably, 49% of seropositive cases reported no history of SARS-CoV-2 infection-related clinical symptoms and 33% were unsuspected of their previous infection. Additionally, levels of anti-SARS-CoV-2 antibodies against the spike-protein receptor-binding domain were similar between symptomatic and asymptomatic individuals, irrespective of age and gender. Using Food and Drug Administration Emergency Use Authorization-approved assays, these results support the need for such studies on pandemic evaluation and highlight the development of robust humoral immune responses even among asymptomatic individuals. The high percentage of unsuspected/asymptomatic active cases, which may contribute to community transmission for more days than that of cases who are aware and self-isolate, underscores the necessity of measures across the population for the efficient control of the pandemic.

Isolation of an Extract from the Soft Coral Symbiotic Microorganism Salinispora arenicola Exerting Cytoprotective and Anti-Aging Effects.

Cells have developed a highly integrated system responsible for proteome stability, namely the proteostasis network (PN). As loss of proteostasis is a hallmark of aging and age-related diseases, the activation of PN modules can likely extend healthspan. Here, we present data on the bioactivity of an extract (SA223-S2BM) purified from the strain Salinispora arenicola TM223-S2 that was isolated from the soft coral Scleronephthya lewinsohni; this coral was collected at a depth of 65 m from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel). Treatment of human cells with SA223-S2BM activated proteostatic modules, decreased oxidative load, and conferred protection against oxidative and genotoxic stress. Furthermore, SA223-S2BM enhanced proteasome and lysosomal-cathepsins activities in Drosophila flies and exhibited skin protective effects as evidenced by effective inhibition of the skin aging-related enzymes, elastase and tyrosinase. We suggest that the SA223-S2BM extract constitutes a likely promising source for prioritizing molecules with anti-aging properties.

Seroprevalence of Antibodies against SARS-CoV-2 among the Personnel and Students of the National and Kapodistrian University of Athens, Greece: A Preliminary Report.

Due to early implementation of public health measures, Greece had low number of SARS-CoV-2 infections and COVID-19 severe incidents in hospitalized patients. The National and Kapodistrian University of Athens (ΝΚUA), especially its health-care/medical personnel, has been actively involved in the first line of state responses to COVID-19. To estimate the prevalence of antibodies (Igs) against SARS-CoV-2 among NKUA members, we designed a five consecutive monthly serosurvey among randomly selected NKUA consenting volunteers. Here, we present the results from the first 2500 plasma samples collected during June-July 2020. Twenty-five donors were tested positive for anti-SARS-CoV-2 Igs; thus, the overall seroprevalence was 1.00%. The weighted overall seroprevalence was 0.93% (95% CI: 0.27, 2.09) and varied between males [1.05% (95% CI: 0.18, 2.92)] and females [0.84% (95% CI: 0.13, 2.49)], age-groups and different categories (higher in participants from the School of Health Sciences and in scientific affiliates/faculty members/laboratory assistants), but no statistical differences were detected. Although focused on the specific population of NKUA members, our study shows that the prevalence of anti-SARS-CoV-2 Igs for the period June-July 2020 remained low and provides knowledge of public health importance for the NKUA members. Given that approximately one in three infections was asymptomatic, continuous monitoring of the progression of the pandemic by assessing Ig seroprevalence is needed.

Alterations in Organismal Physiology, Impaired Stress Resistance, and Accelerated Aging in Drosophila Flies Adapted to Multigenerational Proteome Instability.

Being an assembly of highly sophisticated protein machines, cells depend heavily on proteostatic modules functionality and on adequate supply of energetic molecules for maintaining proteome stability. Yet, our understanding of the adaptations induced by multigenerational proteotoxic stress is limited. We report here that multigenerational (>80 generations) proteotoxic stress in OregonR flies induced by constant exposure to developmentally nonlethal doses of the proteasome inhibitor bortezomib (BTZ) (G80-BTZ flies) increased proteome instability and redox imbalance, reduced fecundity and body size, and caused neuromuscular defects; it also accelerated aging. G80-BTZ flies were mildly resistant to increased doses of BTZ and showed no age-related loss of proteasome activity; these adaptations correlated with sustained upregulation of proteostatic modules, which however occurred at the cost of minimal responses to increased BTZ doses and increased susceptibility to various types of additional proteotoxic stress, namely, autophagy inhibition or thermal stress. Multigenerational proteome instability and redox imbalance also caused metabolic reprogramming being evidenced by altered mitochondrial biogenesis and suppressed insulin/IGF-like signaling (IIS) in G80-BTZ flies. The toxic effects of multigenerational proteome instability could be partially mitigated by a low-protein diet that extended G80-BTZ flies' longevity. Overall, persistent proteotoxic stress triggers a highly conserved adaptive metabolic response mediated by the IIS pathway, which reallocates resources from growth and longevity to somatic preservation and stress tolerance. Yet, these trade-off adaptations occur at the cost of accelerated aging and/or reduced tolerance to additional stress, illustrating the limited buffering capacity of survival pathways.

Nutrigenomics as a tool to study the impact of diet on aging and age-related diseases: the Drosophila approach.

Aging is a complex phenomenon caused by the time-dependent loss of cellular homeodynamics and consequently of physiological organismal functions. This process is affected by both genetic and environmental (e.g., diet) factors, as well as by their constant interaction. Consistently, deregulation of nutrient sensing and signaling pathways is considered a hallmark of aging. Nutrigenomics is an emerging scientific discipline that studies changes induced by diet on the genome and thus it considers the intersection of three topics, namely health, diet, and genomics. Model organisms, such as the fruit fly Drosophila melanogaster, have been successfully used for in vivo modeling of higher metazoans aging and for nutrigenomic studies. Drosophila is a well-studied organism with sophisticated genetics and a fully annotated sequenced genome, in which ~ 75% of human disease-related genes have functional orthologs. Also, flies have organs/tissues that perform the equivalent functions of most mammalian organs, while discrete clusters of cells maintain insect carbohydrate homeostasis in a way similar to pancreatic cells. Herein, we discuss the mechanistic connections between nutrition and aging in Drosophila, and how this model organism can be used to study the effect of different diets (including natural products and/or their derivatives) on higher metazoans longevity.

Correction: Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

[This corrects the article DOI: 10.1371/journal.pone.0201651.].

Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

Pore forming proteins such as those belonging to the membrane attack/perforin (MACPF) family have important functions in many organisms. Of the five MACPF proteins found in Plasmodium parasites, three have functions in cell passage and one in host cell egress. Here we report an analysis of the perforin-like protein 4, PPLP4, in the rodent parasite Plasmodium berghei. We found that the protein is expressed only in the ookinete, the invasive stage of the parasite formed in the mosquito midgut. Transcriptional analysis revealed that expression of the pplp4 gene commences during ookinete development. The protein was detected in retorts and mature ookinetes. Using two antibodies, the protein was found localized in a dotted pattern, and 3-D SIM super-resolution microcopy revealed the protein in the periphery of the cell. Analysis of a C-terminal mCherry fusion of the protein however showed mainly cytoplasmic label. A pplp4 null mutant formed motile ookinetes, but these were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice. However, when in vitro cultured ookinetes were injected into the thorax of the mosquito, thus by-passing midgut passage, sporozoites were formed and the mutant parasites were able to infect naïve mice. Taken together, our data show that PPLP4 is required only for ookinete invasion of the mosquito midgut. Thus PPLP4 has a similar role to the previously studied PPLP3 and PPLP5, raising the question why three proteins with MACPF domains are needed for invasion by the ookinete of the mosquito midgut epithelium.

Targeting Protein Quality Control Mechanisms by Natural Products to Promote Healthy Ageing.

Organismal ageing is associated with increased chance of morbidity or mortality and it is driven by diverse molecular pathways that are affected by both environmental and genetic factors. The progression of ageing correlates with the gradual accumulation of stressors and damaged biomolecules due to the time-dependent decline of stress resistance and functional capacity, which eventually compromise cellular homeodynamics. As protein machines carry out the majority of cellular functions, proteome quality control is critical for cellular functionality and is carried out through the curating activity of the proteostasis network (PN). Key components of the PN are the two main degradation machineries, namely the ubiquitin-proteasome and autophagy-lysosome pathways along with several stress-responsive pathways, such as that of nuclear factor erythroid 2-related factor 2 (Nrf2), which mobilises cytoprotective genomic responses against oxidative and/or xenobiotic damage. Reportedly, genetic or dietary interventions that activate components of the PN delay ageing in evolutionarily diverse organisms. Natural products (extracts or pure compounds) represent an extraordinary inventory of highly diverse structural scaffolds that offer promising activities towards meeting the challenge of increasing healthspan and/or delaying ageing (e.g., spermidine, quercetin or sulforaphane). Herein, we review those natural compounds that have been found to activate proteostatic and/or anti-stress cellular responses and hence have the potential to delay cellular senescence and/or in vivo ageing.