Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events.

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

High concentrations of phthalates affect the early development of the sea urchin Paracentrotus lividus.

The toxicity of three phthalates (PAEs) - butylbenzyl phthalate (BBP), diethyl phthalate (DEP), and di-(2-ethylhexyl) phthalate (DEHP) - was tested on the Mediterranean sea urchin Paracentrotus lividus. Fertilized eggs were exposed to environmental and high PAE concentrations for 72 h. The potential toxic effects on larval development and any morphological anomalies were then assessed to estimate PAEs impact. Environmental concentrations never affected development, while high concentrations induced toxic effects in larvae exposed to BBP (EC50: 2.9 ×103 µg/L) and DEHP (EC50: 3.72 ×103 µg/L). High concentrations caused skeletal anomalies, with a slight to moderate impact for DEP/DEHP and BBP, respectively. PAE toxicity was: BBP>DEHP>DEP. In conclusion, the three PAEs at environmental concentrations do not pose a risk to sea urchins. However, PAE concentrations should be further monitored in order not to constitute a concern to marine species, especially at their early developmental stages.

Transvaginal ultrasound in evaluation and follow-up of ovarian granulosa cell tumors.

OBJECTIVE: Ultrasound features of granulosa cell tumors of the ovary are still poorly defined. The aim of this study is to widen current knowledge on the role of sonographic gray scale and pattern recognition in the characterization of these tumors and to compare the ultrasound characteristics of primary diagnosis and recurrences. METHODS: Transvaginal ultrasound images of primary diagnosis or recurrences of histologically-confirmed granulosa cell tumors of the ovary were retrospectively retrieved from a dedicated database designed for the collection of clinical and ultrasound data from January 2001 to January 2019. All patients included were treated at San Raffaele and Santa Chiara Hospitals. Women with a concomitant diagnosis of another malignancy other than endometrial carcinoma were excluded from the study. All ultrasound images were described according to International Ovarian Tumor Analysis terminology and examined by experienced ultrasound examiners. RESULTS: A total of 27 patients were included: 24 with adult and 3 with juvenile ovarian granulosa cell tumors. At primary diagnosis, mean ovarian mass size was 103.8 mm (range 30-200). On ultrasound evaluation at primary diagnosis, 12 patients presented with a multilocular solid lesion (48%), 9 with a solid lesion (36%), and 4 with a multilocular lesion(16%). The echogenicity of the cyst was low level or anechoic, mixed, or hemorrhagic in 56.3%, 31.2%, and 12.5% of cases, respectively. Most tumors (45.1%), including first diagnosis and relapses, had a moderate to high color score on doppler evaluation. CONCLUSIONS: Our study showed that sonographic features and pattern recognition of relapses were comparable to those of tumors at primary diagnosis. In order to highlight the importance of transvaginal ultrasound evaluation during follow-up, further studies based on a standardized ultrasound characterization of ovarian masses are recommended.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a "partial threading" model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.

Reduced Trichomonas vaginalis viability in mice pretreated with parasite DNA.

Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract leading to trichomoniasis, the most common sexually-transmitted non-viral disease worldwide. The immune response plays a critical role in the host defense against this parasite. Trichomonas' DNA contains unmethylated CpG motifs (CpGDNA) that in other microorganisms act as modulators of the immune response. However, the molecular mechanisms responsible for CpGDNA immune modulation are still unclear. As macrophages participate in the first line of defense against infection, we investigated the type of immune response of murine macrophages to T. vaginalis DNA (TvDNA). We observed high expression of the proinflammatory cytokines IL-6 and IL-12p40 in macrophages stimulated with TvDNA. In contrast, the anti-inflammatory response, assessed by IL-10 and IL-13 mRNA expression was delayed. This suggests that the immune response induced by TvDNA is modulated through cytokine production, mediated partly by NADPH-oxidase activity, as TvDNA induced reactive species of oxygen production and a rounded morphology in macrophages indicative of an M1 phenotype. Furthermore, infected mice pretreated with TvDNA displayed persistent vulvar inflammation and decreased parasite viability consistent with higher proinflammatory cytokine levels during infection compared to untreated mice. Overall, our findings suggest that TvDNA pretreatment modulates the immune response favouring parasite elimination.

Trichoderma and Mycosynthesis of Metal Nanoparticles: Role of Their Secondary Metabolites.

Nanocompounds are widely used in many fields such as environmental, medicine, or agriculture. Nowadays, these nanocompounds are mainly synthesized by chemical methods, causing environmental pollution and potential health problems. Thus, microorganisms have been investigated as potential nanoparticle green biosynthesizers. The main research is focused on the synthesis of nanoparticles (NPs) using algae, yeast, bacteria, and fungi. Among them, fungi have been the most used, due to their simple and effective mycosynthesis. Fungi as well as other organisms involved in green synthesis of NPs use their secondary metabolites (SMs) to mediate and catalyze the reactions to produce metal nanoparticles (MNPs) as well as being able to act as capping agents producing different physicochemical characteristics and biological activities in the MNPs. Among the various fungi used for mycosynthesis are Trichoderma species, which mediate the production of Ag, Cu, CuO, Zn, ZnO, and other MNPs. Here, we review the main SMs from Trichoderma that have been reported or suggested to contribute to synthesize or act as capping agents and their applications, as well as present the main challenges faced by this type of synthesis.

Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individuals m- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.

Blunt-End Driven Re-entrant Ordering in Quasi Two-Dimensional Dispersions of Spherical DNA Brushes.

We investigate the effects of crowding on the conformations and assembly of confined, highly charged, and thick polyelectrolyte brushes in the osmotic regime. Particle tracking experiments on increasingly dense suspensions of colloids coated with ultralong double-stranded DNA (dsDNA) fragments reveal nonmonotonic particle shrinking, aggregation, and re-entrant ordering. Theory and simulations show that aggregation and re-entrant ordering arise from the combined effect of shrinking, which is induced by the osmotic pressure exerted by the counterions absorbed in neighbor brushes and of a short-range attractive interaction competing with electrostatic repulsion. An unconventional mechanism gives origin to the short-range attraction: blunt-end interactions between stretched dsDNA fragments of neighboring brushes, which become sufficiently intense for dense and packed brushes. The attraction can be tuned by inducing free-end backfolding through the addition of monovalent salt. Our results show that base stacking is a mode parallel to hybridization to steer colloidal assembly in which attractions can be fine-tuned through salinity and, potentially, grafting density and temperature.

Gel dressing based on type I collagen modified with oligourethane and silica for skin wound healing.

Cutaneous wound healing is a complex process that leads the skin reparation with the formation of scar tissue that typically lacks skin appendages. This fact drives us to find new strategies to improve regenerative healing of the skin. This study outlines, the contribution of colloidal silica particles and oligourethane crosslinking on the collagen material properties and the effect on skin wound healing in rats. We characterized the gel properties that are key forin-situgelation, which is accomplished by the latent reactivity of oligourethane bearing blocked isocyanate groups to crosslink collagen while entrapping silica particles. The swelling/degradation behavior and the elastic modulus of the composite gel were consistent with the modification of collagen type I with oligourethane and silica. On the other hand, these gels were characterized as scaffold for murine macrophages and human stem cells. The application of a composite gel dressing on cutaneous wounds showed a histological appearance of the recovered skin as intact skin; featured by the epidermis, hair follicles, sebaceous glands, subcutaneous adipose layer, and dermis. The results suggest that the collagen-based composite dressings are promising modulators in skin wound healing to achieve a regenerative skin closure with satisfactory functional and aesthetic scars.

Concordance and interaction of guanine nucleotide dissociation inhibitor (RhoGDI) with RhoA in oogenesis and early development of the sea urchin.

Rho GTPases are Ras-related GTPases that regulate a variety of cellular processes. In the sea urchin Strongylocentrotus purpuratus, RhoA in the oocyte associates with the membrane of the cortical granules and directs their movement from the cytoplasm to the cell cortex during maturation to an egg. RhoA also plays an important role regulating the Na(+) -H(+) exchanger activity, which determines the internal pH of the cell during the first minutes of embryogenesis. We investigated how this activity may be regulated by a guanine-nucleotide dissociation inhibitor (RhoGDI). The sequence of this RhoA regulatory protein was identified in the genome on the basis of its similarity to other RhoGDI species, especially for key segments in the formation of the isoprenyl-binding pocket and in interactions with the Rho GTPase. We examined the expression and the subcellular localization of RhoGDI during oogenesis and in different developmental stages. We found that RhoGDI mRNA levels were high in eggs and during cleavage divisions until blastula, when it disappeared, only to reappear in gastrula stage. RhoGDI localization overlaps the presence of RhoA during oogenesis and in embryonic development, reinforcing the regulatory premise of the interaction. By use of recombinant protein interactions in vitro, we also find that these two proteins selectively interact. These results support the hypothesis of a functional relationship in vivo and now enable mechanistic insight for the cellular and organelle rearrangements that occur during oogenesis and embryonic development.

Parallel anxiolytic-like effects and upregulation of neuronal nicotinic acetylcholine receptors following chronic nicotine and varenicline.

INTRODUCTION: Clinical and preclinical studies suggest that regulation of nicotinic acetylcholine receptors (nAChR) maybe involved in the etiology of withdrawal symptoms. METHODS: We evaluated heteromeric nAChR regulation via [³H]epibatidine binding following cessation of chronic nicotine or varenicline treatment. Animals were concurrently tested in the marble-burying test to evaluate treatment-related effects. RESULTS: We found that both nicotine (18 mg/kg/day, free base) and varenicline (1.8 mg/kg/day) chronically administered for 14 days upregulated nAChRs significantly in the cortex, hippocampus, striatum, and thalamus. The duration of upregulation (up to 72 hr) was both drug and region specific. In addition to nAChR upregulation, chronic administration of both nicotine and varenicline had anxiolytic-like effects in the marble-burying test. This effect was maintained for 48 hr following cessation of varenicline but was absent 24 hr following cessation from nicotine. Additionally, marble-burying behavior positively correlated to the regulation of cortical nAChRs following cessation of either treatment. CONCLUSIONS: Varenicline has been shown to be an efficacious smoking cessation aid, with a proposed mechanism of action that includes modulation of dopamine release in reward areas of the brain. Our studies show that varenicline elicits both anxiolytic effects in the marble-burying test as well as region- and time-specific receptor upregulation. These findings suggest receptor upregulation as a mechanism for its efficacy as a smoking cessation therapy.

Molecular characterization of five novel Wx-A1 alleles in common wheat including one silent allele by transposon insertion.

Wheat starch is composed of two glucose polymers, amylose and amylopectin. Although several starch synthases are responsible for its synthesis, only the waxy protein is associated with the amylose synthesis. The waxy protein composition of 45 Spanish common wheat landraces from Andalusia (southern Spain) was evaluated. Within these materials, five novel alleles for the Wx-A1 gene were detected. Four of them showed functional proteins (Wx-A1p, Wx-A1q, Wx-A1r and Wx-A1s), although some amino acid changes were found in the mature protein sequence. However, one of them (Wx-A1t) exhibited loss of the Wx-A1 protein, and its base sequence contained one large insert (1,073 bp) in the tenth exon, that interrupted the ORF of the Wx-A1 gene. This insert exhibited the characteristics of a Class II transposon of the Mutator superfamily, which had not been described previously, and has been named Baetica. The conservation of such inserts could be related to their low effect on vital properties of the plants, as occurs with most of the genes associated with technological quality. In conclusion, the evaluation of old wheat landraces showed that, in addition to their use as alternative crops, these materials could be a useful source of interesting genes in wheat quality improvement.

Antibiotic resistance in diabetic foot infection: how it changed with COVID-19 pandemic in a tertiary care center.

AIM: To investigate the rate of antibiotic resistance and its main risk factors in a population of patients with diabetic foot infection (DFI) during the COVID-19 pandemic, in comparison with the population of 2019. METHODS: Two hundred and twenty-five patients with DFI were admitted in a tertiary care center from January 2019 to December 2020. Antibiotic resistance was evaluated by microbiological examination of soft tissues' or bone's biopsy. RESULTS: Compared with 2019 group (n = 105), 2020 group (n = 120) had a significantly higher prevalence of antibiotic resistance [2019 vs 2020, 36% vs 63%, P <0.001] and more often was admitted with recent or current antibiotic therapy (18% vs 52%, P <0.001), which was frequently self-administered (5% vs 30%, P = 0.032). The risk of antibiotic resistance was also higher in 2020 group [OR 95% CI, 2.90 (1.68 to 4.99)]. Prior hospitalization, antibiotic self-administration and antibiotic prescription by general practitioners resulted as independent predictors of antibiotic resistance. CONCLUSIONS: In a population of people with DFI admitted in a tertiary care center during the COVID-19 pandemic the prevalence of antibiotic resistance was higher than 2019. Previous hospitalization, antibiotic self-administration /prescription by general practitioners were related to higher risk of antibiotic resistant infections.

Nicotinic partial agonists varenicline and sazetidine-A have differential effects on affective behavior.

Clinical and preclinical studies suggest that nicotinic acetylcholine receptors are involved in affective disorders; therefore, the potential therapeutic value of nicotinic partial agonists as treatments of these disorders is of growing interest. This study evaluated the effects of acute and chronic administration of nicotine and the alpha4beta2 nicotinic partial agonists varenicline and sazetidine-A in mouse models of anxiety and depression. Acutely, only nicotine and varenicline had anxiolytic effects in the marble-burying test and in the novelty-induced hypophagia (NIH) test. In contrast, in animal models of antidepressant efficacy, such as the forced swim and the tail suspension test, only acute sazetidine-A had significant antidepressant-like effects. The NIH test provides an anxiety-related measure that is sensitive to the effects of chronic but not acute antidepressant treatment. Chronic nicotine and chronic sazetidine-A treatment were effective in this paradigm, but varenicline was ineffective. These results suggest that the partial agonists varenicline and sazetidine-A may have diverse therapeutic benefits in affective disorders.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.

Role of the novel protein TmcR in regulating the expression of the cel9-cel48 operon from Myxobacter sp. AL-1.

Northern-blot analysis revealed that cel9 and cel48, which encode family 9 and 48 glycosyl hydrolases, respectively, were expressed as a bicistronic mRNA in the soil bacterium Myxobacter sp. AL-1. The two cistrons of the cel9-cel48 mRNA as well as their encoded products were detected in stationary phase cultures of Myxobacter sp. AL-1, suggesting that a mechanism delayed the transcription of cel9-cel48 until this growth phase. Interestingly, in the same strand and orientation as cel48 a different reading frame was found fully embedded within another ORF encoding a novel DNA-binding protein termed TmcR (Temporal cellulase regulator). Results of Western-blot analysis revealed that although TmcR occurred in growing cells, its concentration decreased during the late stationary growth phase. A possible regulatory role of TmcR during cel9-cel48 expression was studied in E. coli. Results showed that in comparison with E. coli cells expressing cel9-cel48 cloned in pBR322, deletion of tmcR from this plasmid increased not only the cellulase activity but also the amount of Cel9 secreted to the culture medium. Moreover, both, the cellulase activity and Cel9 production decreased in E. coli cells when tmcR was cloned back in the plasmid lacking tmcR. These results suggest that TmcR has the properties required to repress the expression of the cel9-cel48 cluster from Myxobacter sp. AL-1 and suggest the existence of a mechanism involved in regulating the expression of cellulase genes in soil bacteria.

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.

Development and characterization of an immunomodulatory and injectable system composed of collagen modified with trifunctional oligourethanes and silica.

Immunomodulatory biomaterials have emerged as a promising approach to engineer wound healing. To achieve this task, the bioactivity of the biomaterials and an easy application are two key desirable characteristics. This work reports an injectable gel system containing immune cells primed for wound healing. By combining the self-assembly of type I collagen, cross-linked with trifunctional oligourethanes, and silica particle entrapment, the structured collagen network acts as a delivery vehicle for macrophages. This structured collagen network primes the macrophages for an anti-inflammatory response. Rheological measurements suggest that the mixture of liquid precursors can be safely stored at low temperatures and low pH (4 °C, pH 3) for at least one month. After pH neutralization and injection, gels with a storage modulus of 50-80 Pa are obtained in five minutes. Several immunocytochemistry and ELISA tests strongly suggest that mouse and human macrophages are stimulated by the material to up-regulate the production of anti-inflammatory cytokines, while down-regulating the production of pro-inflammatory cytokines. The injection of gel in an ex vivo inflammation model of intervertebral discs demonstrated that it is possible to transit from a pro-inflammatory to an anti-inflammatory microenvironment. Altogether, the results suggest that this gel can polarize the macrophage response and promote a surrounding anti-inflammatory microenvironment ready for injection for wound healing applications.