Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia.

SPG15 is a hereditary spastic paraplegia subtype caused by mutations in Spastizin, a protein encoded by the ZFYVE26 gene. Spastizin is involved in autophagosome maturation and autophagic lysosome reformation and SPG15-related mutations lead to autophagic lysosome reformation defects with lysosome enlargement, free lysosome depletion and autophagosome accumulation. Symptomatic and rehabilitative treatments are the only therapy currently available for patients. Here, we targeted autophagy and lysosomes in SPG15 patient-derived cells by using a library of autophagy-modulating compounds. We identified a rose of compounds affecting intracellular calcium levels, the calcium-calpain pathway or lysosomal functions, which reduced autophagosome accumulation. The six most effective compounds were tested in vivo in a new SPG15 loss of function Drosophila model that mimicked the reported SPG15 phenotype, with autophagosome accumulation, enlarged lysosomes, reduced free lysosomes, autophagic lysosome reformation defects and locomotor deficit. These compounds, namely verapamil, Bay K8644, 2',5'-dideoxyadenosine, trehalose, Small-Molecule Enhancer of Rapamycin 28 and trifluoperazine, improved lysosome biogenesis and function in vivo, demonstrating that lysosomes are a key pharmacological target to rescue SPG15 phenotype. Among the others, the Small-Molecule Enhancer of Rapamycin 28 was the most effective, rescuing both autophagic lysosome reformation defects and locomotor deficit, and could be considered as a potential therapeutic compound for this hereditary spastic paraplegia subtype.

Immune response and adverse events after vaccination against SARS-CoV-2 in adult patients with transfusion-dependent thalassaemia.

Patients with transfusion-dependent thalassaemia (TDT) are considered an at increased-risk population for severe and/or morbid coronavirus disease 2019 (COVID-19) infection. Timely vaccination is the main preventive method for severe COVID-19. Different adverse events and reactions after vaccination have been reported, with severe ones being extremely rare. Patients with TDT may have altered immunity due to chronic transfusions, iron overload and chelation therapy, and splenic dysfunction. Here, we show that adult patients with TDT following vaccination with the novel messenger RNA vaccines have mild adverse events and can produce protective antibodies comparable to the healthy population.

Myeloma patients with COVID-19 have superior antibody responses compared to patients fully vaccinated with the BNT162b2 vaccine.

Patients with multiple myeloma (MM) have a suboptimal antibody response following vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and lower seroconversion rates following coronavirus disease 2019 (COVID-19) compared with healthy individuals. In this context, we evaluated the development of neutralising antibodies (NAbs) against SARS-CoV-2 in non-vaccinated patients with MM and COVID-19 compared with patients after vaccination with two doses of the BNT162b2 vaccine. Serum was collected either four weeks post confirmed diagnosis or four weeks post a second dose of BNT162b2. NAbs were measured with a Food and Drug Administration-approved enzyme-linked immunosorbent assay methodology. Thirty-five patients with COVID-19 and MM along with 35 matched patients were included. The two groups did not differ in age, sex, body mass index, prior lines of therapy, disease status, lymphocyte count, immunoglobulin levels and comorbidities. Patients with MM and COVID-19 showed a superior humoral response compared with vaccinated patients with MM. The median (interquartile range) NAb titre was 87·6% (71·6-94%) and 58·7% (21·4-91·8%) for COVID-19-positive and vaccinated patients, respectively (P = 0·01).Importantly, there was no difference in NAb production between COVID-19-positive and vaccinated patients who did not receive any treatment (median NAb 85·1% vs 91·7%, P = 0·14). In conclusion, our data indicate that vaccinated patients with MM on treatment without prior COVID-19 should be considered for booster vaccine doses.

Comparative kinetics of SARS-CoV-2 anti-spike protein RBD IgGs and neutralizing antibodies in convalescent and naïve recipients of the BNT162b2 mRNA vaccine versus COVID-19 patients.

BACKGROUND: Coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused a still evolving global pandemic. Given the worldwide vaccination campaign, the understanding of the vaccine-induced versus COVID-19-induced immunity will contribute to adjusting vaccine dosing strategies and speeding-up vaccination efforts. METHODS: Anti-spike-RBD IgGs and neutralizing antibodies (NAbs) titers were measured in BNT162b2 mRNA vaccinated participants (n = 250); we also investigated humoral and cellular immune responses in vaccinated individuals (n = 21) of this cohort 5 months post-vaccination and assayed NAbs levels in COVID-19 hospitalized patients (n = 60) with moderate or severe disease, as well as in COVID-19 recovered patients (n = 34). RESULTS: We found that one (boosting) dose of the BNT162b2 vaccine triggers robust immune (i.e., anti-spike-RBD IgGs and NAbs) responses in COVID-19 convalescent healthy recipients, while naïve recipients require both priming and boosting shots to acquire high antibody titers. Severe COVID-19 triggers an earlier and more intense (versus moderate disease) immune response in hospitalized patients; in all cases, however, antibody titers remain at high levels in COVID-19 recovered patients. Although virus infection promotes an earlier and more intense, versus priming vaccination, immune response, boosting vaccination induces antibody titers significantly higher and likely more durable versus COVID-19. In support, high anti-spike-RBD IgGs/NAbs titers along with spike (vaccine encoded antigen) specific T cell clones were found in the serum and peripheral blood mononuclear cells, respectively, of vaccinated individuals 5 months post-vaccination. CONCLUSIONS: These findings support vaccination efficacy, also suggesting that vaccination likely offers more protection than natural infection.

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor.

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC50 value of 63 µg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2-7), four diketopiperazines (8-11), two butyrolactones (12-13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 µM and six times stronger anti-tyrosinase activity (IC50 2.18 µΜ) than kojic acid (IC50 14.07 µΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC50 19.18 µΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 µg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC50 >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.

Biological Evaluation and In Silico Study of Benzoic Acid Derivatives from Bjerkandera adusta Targeting Proteostasis Network Modules.

Abstract: A main cellular functional module that becomes dysfunctional during aging is the proteostasis network. In the present study, we show that benzoic acid derivatives isolated from Bjerkandera adusta promote the activity of the two main protein degradation systems, namely the ubiquitin-proteasome (UPP) and especially the autophagy-lysosome pathway (ALP) in human foreskin fibroblasts. Our findings were further supported by in silico studies, where all compounds were found to be putative binders of both cathepsins B and L. Among them, compound 3 (3-chloro-4-methoxybenzoic acid) showed the most potent interaction with both enzymes, which justifies the strong activation of cathepsins B and L (467.3 ± 3.9%) on cell-based assays. Considering that the activity of both the UPP and ALP pathways decreases with aging, our results suggest that the hydroxybenzoic acid scaffold could be considered as a promising candidate for the development of novel modulators of the proteostasis network, and likely of anti-aging agents.

Osmanicin, a Polyketide Alkaloid Isolated from Streptomyces osmaniensis CA-244599 Inhibits Elastase in Human Fibroblasts.

The strain Streptomyces osmaniensis CA-244599 isolated from the Comoros islands was submitted to liquid-state fermentation coupled to in situ solid-phase extraction with amberlite XAD-16 resin. Elution of the trapped compounds on the resin beads by ethyl acetate afforded seven metabolites, osmanicin (1), streptazolin (2), streptazone C (3), streptazone B1 (4), streptenol C (5), nocardamine (6) and desmethylenylnocardamine (7). Osmanicin (1) is a newly reported unusual scaffold combining streptazolin (2) and streptazone C (3) through a Diels-Alder type reaction. Experimental evidence excluded the spontaneous formation of 1 from 2 and 3. The isolated compounds were evaluated for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 1 exhibited the most potent activity with an IC50 of 3.7 µM.

A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence.

BACKGROUND: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. METHODS: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. RESULTS: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.

Spastin binds to lipid droplets and affects lipid metabolism.

Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP.

Proteome Stability as a Key Factor of Genome Integrity.

DNA damage is constantly produced by both endogenous and exogenous factors; DNA lesions then trigger the so-called DNA damaged response (DDR). This is a highly synchronized pathway that involves recognition, signaling and repair of the damage. Failure to eliminate DNA lesions is associated with genome instability, a driving force in tumorigenesis. Proteins carry out the vast majority of cellular functions and thus proteome quality control (PQC) is critical for the maintenance of cellular functionality. PQC is assured by the proteostasis network (PN), which under conditions of proteome instability address the triage decision of protein fold, hold, or degrade. Key components of the PN are the protein synthesis modules, the molecular chaperones and the two main degradation machineries, namely the autophagy-lysosome and the ubiquitin-proteasome pathways; also, part of the PN are a number of stress-responsive cellular sensors including (among others) heat shock factor 1 (Hsf1) and the nuclear factor erythroid 2-related factor 2 (Nrf2). Nevertheless, the lifestyle- and/or ageing-associated gradual accumulation of stressors results in increasingly damaged and unstable proteome due to accumulation of misfolded proteins and/or protein aggregates. This outcome may then increase genomic instability due to reduced fidelity in processes like DNA replication or repair leading to various age-related diseases including cancer. Herein, we review the role of proteostatic machineries in nuclear genome integrity and stability, as well as on DDR responses.

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.

Longitudinal Analysis of Antibody Response Following SARS-CoV-2 Infection Depending on Disease Severity: A Prospective Cohort Study.

OBJECTIVE: Severe coronavirus disease 19 (COVID-19) is characterized by a dysregulated inflammatory response, with humoral immunity playing a central role in the disease course. The objective of this study was to assess the immune response and the effects of vaccination in recovered individuals with variable disease severity up to one year following natural infection. METHODS: A prospective cohort study was conducted including patients with laboratory-confirmed COVID-19. Disease severity was classified as mild, moderate, and severe based on clinical presentation and outcomes. Anti-RBD (receptor binding domain) and neutralizing antibodies were evaluated at multiple timepoints during the first year after COVID-19 diagnosis. RESULTS: A total of 106 patients were included; of them, 28 were diagnosed with mild, 38 with moderate, and 40 with severe disease. At least one vaccine dose was administered in 58 individuals during the follow-up. Participants with mild disease presented significantly lower anti-RBD and neutralizing antibodies compared to those with moderate and severe disease up to the 3rd and 6th months after the infection, respectively. After adjusting for covariates, in the third month, severe COVID-19 was associated with significantly higher anti-RBD (ß: 563.09; 95% confidence intervals (CI): 257.02 to 869.17) and neutralizing (ß: 21.47; 95% CI: 12.04 to 30.90) antibodies. Among vaccinated individuals, at the 12th month, a history of moderate disease was associated with significantly higher anti-RBD levels (ß: 5615.19; 95% CI: 657.92 to 10,572.46). CONCLUSIONS: Severe COVID-19 is associated with higher anti-RBD and neutralizing antibodies up to 6 months after the infection. Vaccination of recovered patients is associated with a remarkable augmentation of antibody titers up to one year after COVID-19 diagnosis, regardless of disease severity.

Patients With Autoimmune Thyroiditis Present Similar Immunological Response to COVID-19 BNT162b2 mRNA Vaccine With Healthy Subjects, While Vaccination May Affect Thyroid Function: A Clinical Study.

Background: This is the first study, that aimed: a) to compare immune response, namely the kinetics of neutralizing antibodies (Nabs), after vaccination with BNT162b2 mRNA vaccine (Comirnaty, Pfizer/BioNTech) between patients with autoimmune thyroiditis and controls, and b) to investigate changes in thyroid function in healthy subjects with no history of thyroid dysfunction before and after vaccination with BNT162b2 mRNA vaccine (Comirnaty, Pfizer/BioNTech). Methods: The entire study consisted of two sub-studies. In the first sub-study, NAbs levels after BNT162b2 mRNA vaccination were compared between 56 patients with autoimmune thyroiditis and 56 age and gender-matched healthy controls from the day of the first dose until a period of up to three months after the second dose. In the second sub-study, thyroid hormones (T3, T4, TSH) and thyroid auto-antibodies levels (anti-TG, anti-TPO) of 72 healthy subjects with no history of thyroid disease were examined before (D1) and one month after completion of the second dose (D50). Results: Among patients with autoimmune thyroiditis, the median neutralizing inhibition on D22, immediately before second dose, was 62.5%. One month later (D50), values increased to 96.7%, while three months after the second dose NAbs titers remained almost the same (94.5%). In the healthy group, median NAbs levels at D22 were 53.6%. On D50 the median inhibition values increased to 95.1%, while after three months they were 89.2%. The statistical analysis did not show significant differences between two groups (p-values 0.164, 0.390, 0.105 for D22, D50 and three months). Regarding changes in thyroid function, the mean value for T4 before vaccination was 89.797 nmol/L and one month after the second dose was 89.11 nmol/L (p-value=0.649). On D1 the mean T3 value was 1.464 nmol/L, which dropped to 1.389 nmol/L on D50 (p-value = 0.004). For TSH, mean levels were 2.064 mIU/ml on D1 and fell to 1.840 mIU/ml one month after the second dose (p-value=0.037). Despite decrease, all thyroid hormone levels remained within the normal range. No changes were found for anti-TPO or anti-TG. Conclusions: This study provided evidence that patients with autoimmune thyroiditis present similar immunological response to COVID-19 BNT162b2 mRNA vaccine (Comirnaty, Pfizer/BioNTech) with healthy subjects, while vaccination may affect thyroid function.

Comoclathrin, a novel potent skin-whitening agent produced by endophytic Comoclathris strains associated with Andalusia desert plants.

As part of our screening program for the discovery of molecules of microbial origin with skin-whitening activity, 142 diverse fungal endophytes from a wide variety of Andalusia arid plants were screened, applying the OSMAC approach. The fungal strains CF-090361 and CF-090766, isolated from xerophytic plants, were selected as the most promising, while phylogenetic analysis revealed that both strains could represent a new species within the genus Comoclathris. The effect of different fermentation conditions on the production of tyrosinase inhibitory activity was examined, in order to identify the optimum cultivation conditions. LCMS based metabolomics was applied to determine significant differences between the strains and fermentation conditions, and to identify potential bioactive secondary metabolites. Bioassay-guided purification of the main active components led to the isolation of three new compounds (1-3), along with the known compounds graphostrin B (4) and brevianamide M (5). Compound 1 (Comoclathrin) demonstrated the strongest anti-tyrosinase activity (IC50 0.16 µΜ), which was 90-times higher than kojic acid (IC50 14.07 µΜ) used as positive control. Additionally, comoclathrin showed no significant cytotoxicity against a panel of cancer cell lines (HepG2, A2058, A549, MCF-7 and MIA PaCa-2) and normal BJ fibroblasts. These properties render comoclathrin an excellent development candidate as whitening agent.

Treatment with abiraterone or enzalutamide does not impair immunological response to COVID-19 vaccination in prostate cancer patients.

Data regarding the safety and efficacy of COVID-10 vaccines among cancer patients are lacking. Factors such as age, underlying disease and antineoplastic treatment confer negatively to the immune response due to vaccination. The degree of immunosuppression though may be lessen by targeted treatments like the androgen receptor-targeted agents (ARTA) that are commonly used in patients with metastatic prostate cancer. Herein, we report our data on 25 patients with prostate cancer under treatment with ARTA who were vaccinated for COVID-19. Our data suggest that these patients develop neutralizing antibodies against SARS-CoV-2 similarly to healthy volunteers. No safety issues were noted.

Elastase inhibitory activity of secondary metabolites from the fungus Virgaria nigra CF-231658.

Three known compounds were isolated from Virgaria nigra CF-231658; 2,7-dihydroxy naphthalene (1), virgaricin B (2) and virgaricin (3). The isolated compounds was obtained from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. Their structures were elucidated on the basis of 1D and 2D NMR as well as HRMS spectroscopic analyses. The isolated compounds were examined for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 2 displayed the most potent activity with 76.7 ± 2.12% inhibition of the enzyme activity at 5 µM concentration.