Inhibitors of Transthyretin Amyloidosis: How to Rank Drug Candidates Using X-ray Crystallography Data.

Amyloidosis is a group of protein misfolding diseases, which include spongiform encephalopathies, Alzheimer's disease and transthyretin (TTR) amyloidosis; all of them are characterized by extracellular deposits of an insoluble fibrillar protein. TTR amyloidosis is a highly debilitating and life-threatening disease. Patients carry less stable TTR homotetramers that are prone to dissociation into non-native monomers, which in turn rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Liver transplantation to induce the production of wild-type TTR was the only therapeutic strategy until recently. A promising approach to ameliorate transthyretin (TTR) amyloidosis is based on the so-called TTR kinetic stabilizers. More than 1000 TTR stabilizers have already been tested by many research groups, but the diversity of experimental techniques and conditions used hampers an objective prioritization of the compounds. One of the most reliable and unambiguous techniques applied to determine the structures of the TTR/drug complexes is X-ray diffraction. Most of the potential inhibitors bind in the TTR channel and the crystal structures reveal the atomic details of the interaction between the protein and the compound. Here we suggest that the stabilization effect is associated with a compaction of the quaternary structure of the protein and propose a scoring function to rank drugs based on X-ray crystallography data.

Crystal structures of Streptomyces tsukubaensis sigma factor SigG1 and anti-sigma RsfG.

Transcription of specific genes in bacteria under environmental stress is frequently initiated by extracytoplasmic function (ECF) σ factors. ECFs σ factors harbour two conserved domains, σ2 and σ4, for transcription initiation by recognition of the promoter region and recruitment of RNA polymerase (RNAP). The crystal structure of Streptomyces tsukubaensis SigG1, an ECF56-family σ factor, was determined revealing σ2, σ4 and the additional carboxi-terminal domain SnoaL_2 tightly packed in a compact conformation. The structure of anti-sigma RsfG was also determined by X-ray crystallography and shows a rare ß-barrel fold. Analysis of the metal binding motifs inside the protein barrel are consistent with Fe(III) binding, which is in agreement with previous findings that the Streptomyces tsukubaensis ECF56 SigG1-RsfG system is involved in metal-ion homeostasis.

SARS-CoV-2 RNA detection in stool samples from acute gastroenteritis cases, Brazil.

We described the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in stool samples from patients presenting only acute gastroenteritis (AGE) symptoms. From January to July 2020, 121 AGE stool samples were screened by quantitative reverse-transcription polymerase chain reaction. We detected SARS-CoV-2 in 27.5% of samples received during the epidemic period. No infectious viruses were observed in Vero E6 cells.

Metal-organic frameworks: a future toolbox for biomedicine?

The present review focuses on the use of Metal-Organic Frameworks, (MOFs) highlighting the most recent developments in the biological field. This review assesses, in the first instance, the cytotoxicity of MOFs (particularly those used for various biological applications described throughout this review), and shows that for standard MOFs based on metals already present in active molecules of the human body, toxicity is not a significant limitation. Here we underline the MIL-, UiO- and ZIF-series of MOFs which remain until now the most used materials in drug delivery of active pharmaceutical ingredients (APIs), such as antitumourals or retroviral drugs (with high loading and slow release time). Porosity remains undoubtedly the most studied key property of MOFs, that allows the protection of active biomolecules such as enzymes or the development of antimicrobial materials. Emphasis is given on the usage of MOFs for the detection of biomarkers in biological fluids such as urine and blood (detection of cystinuria, identification of penicillin anaphylaxis, urea, bilirubin, biomarkers related to human intoxication, tumoural indicators, among several others), for which a number of simple devices (such as paper strips) were developed. Despite the remarkable and promising results presented in recent years, the literature remains scarce (mostly non-existent) in terms of direct comparison of these novel technologies with the solutions presently available in the market. Action on this side may make the difference in the next years concerning research on MOFs, to see if some of these materials may reach the end-user as new and more efficient treatments or detection approaches.

Rotavirus A Infections in Community Childhood Diarrhea in the Brazilian Semiarid Region During Postvaccination Era.

BACKGROUND: Rotavirus A (RVA) is one of the leading causes of acute gastroenteritis worldwide; however, few studies assessed RVA genetics with community surveillance. OBJECTIVES: This study aimed to investigate clinical data, genetic diversity, and coinfection patterns of RVA infections in children from 2 to 36 months old with or without community childhood diarrhea in the Brazilian semiarid region during postvaccination era. METHODS: We enrolled and collected socioeconomic/clinical information using a standardized questionnaire and fecal samples from 291 children. Viral RNA samples were extracted and analyzed using quantitative reverse transcription polymerase chain reaction to establish the diagnosis of RVA. Sequencing of VP7 and VP4 (VP8*) regions and phylogenetic analysis were performed. RESULTS: RVA-negative diagnosis was associated with children 24 to 36 months old with complete vaccination schedule. Genotype G1P[8] was the most prevalent (57%), whereas unusual genotypes including G1P[4], G2P[8], and G3P[9] were also detected. G1- and P[8]-positive samples showed high degrees of similarity with the vaccine strain. RVA coinfections were frequently observed, and enteroaggregative Escherichia coli was the most prevalent copathogen. CONCLUSIONS: These results demonstrate that genotype G1P[8] is the most prevalent strain. VP7 and/or VP8* gene segments arising from RV1 vaccine strain were documented in these children, suggesting shedding or herd vaccination. Moreover, our study indicates full vaccination is important for protection against RVA infections.

Genetic Diversity of Norovirus Infections, Coinfections, and Undernutrition in Children From Brazilian Semiarid Region.

BACKGROUND AND OBJECTIVE: Norovirus (NoV) infections are known to have high-morbidity and mortality rates and are a major health problem globally. The impact of NoV on child development is, however, poorly understood. We evaluated the distribution of NoV genotypes in children from a low-income Brazilian semiarid region, in relation with their clinical symptoms, nutritional status, and co-pathogens. METHODS: The test population included children aged 2 to 36 months from 6 cities of the Brazilian semiarid region. Fecal samples were collected from each child, along with the information regarding their socioeconomic/clinical conditions using a standardized questionnaire. Detection and quantification of NoV were performed by reverse-transcription quantitative polymerase chain reaction, followed by molecular and phylogenetic analyses. RESULTS: The NoV detection rate was 45.2%. Presence of NoV was associated with lower z scores for weight-for-age (P = 0.03), weight-for-height (P = 0.03), and body mass index-for-age (P = 0.03). NoV infection was associated with more frequent respiratory illnesses (P < 0.01). GII.P7 (polymerase) and GII.3 (capsid) were the most frequent NoV genotypes. Analysis of the open reading frame (ORF)1-2 junction identified recombinant NoV strains in 80% of the sequenced samples. Enteroaggregative Escherichia coli coinfection was the major predictor for diarrhea in NoV-positive samples (P < 0.02). Moreover, Shigella spp was also associated with NoV-positive diagnosis (P = 0.02). CONCLUSIONS: This study highlights the genetic variability of NoV and, associated co-infections and undernutrition in infants from low-income Brazilian semiarid region.

Transthyretin monomers: a new plasma biomarker for pre-symptomatic transthyretin-related amyloidosis.

BACKGROUND: Genotyping and amyloid fibril detection in tissues are generally considered the diagnostic gold standard in transthyretin-related amyloidosis. Patients carry less stable TTR homotetramers prone to dissociation into non-native monomers, which rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Thus, the initial event of the amyloid cascade produces the smallest transthyretin species: the monomers. This creates engineering opportunities for diagnosis that remain unexplored. METHODS: We hypothesise that molecular sieving represents a promising method for isolating and concentrating trace TTR monomers from the tetramers present in plasma samples. Subsequently, immunodetection can be utilised to distinguish monomeric TTR from other low molecular weight proteins within the adsorbed fraction. A two-step assay was devised (ImmunoSieve assay), combining molecular sieving and immunodetection for sensing monomeric transthyretin. This assay was employed to analyse plasma microsamples from 10 individuals, including 5 pre-symptomatic carriers of TTR-V30M, the most prevalent amyloidosis-associated TTR variant worldwide, and 5 healthy controls. RESULTS: The ImmunoSieve assay enable sensitive detection of monomeric transthyretin in plasma microsamples. Moreover, the circulating monomeric TTR levels were significantly higher in carriers of amyloidogenic TTR mutation. CONCLUSIONS: Monomeric TTR can function as a biomarker for evaluating disease progression and assessing responses to therapies targeted at stabilising native TTR.

Metal-Organic Frameworks as Sensors for Human Amyloid Diseases.

Metal-organic frameworks (MOFs) are versatile compounds with emergent applications in the fabrication of biosensors for amyloid diseases. They hold great potential in biospecimen protection and unprecedented probing capabilities for optical and redox receptors. In this Review, we summarize the main methodologies employed in the fabrication of MOF-based sensors for amyloid diseases and collect all available data in the literature related to their performance (detection range, limit of detection, recovery, time of analysis, among other parameters). Nowadays, MOF sensors have evolved to a point where they can, in some cases, outperform technologies employed in the detection of several amyloid biomarkers (amyloid ß peptide, α-synuclein, insulin, procalcitonin, and prolactin) present in biological fluids, such as cerebrospinal fluid and blood. A special emphasis has been given by researchers on Alzheimer's disease monitoring to the detriment of other amyloidosis that are underexploited despite their societal relevance (e.g., Parkinson's disease). There are still important obstacles to overcome in order to selectively detect the various peptide isoforms and soluble amyloid species associated with Alzheimer's disease. Furthermore, MOF contrast agents for imaging peptide soluble oligomers in living humans are also scarce (if not nonexistent), and action in this direction is unquestionably required to clarify the contentious link between the amyloidogenic species and the disease, guiding research toward the most promising therapeutic strategies.

Aß31-35 Decreases Neprilysin-Mediated Alzheimer's Amyloid-ß Peptide Degradation.

Alzheimer's disease is associated with the deposition of extracellular senile plaques, made primarily of amyloid-ß (Aß), particularly peptides Aß1-42 and Aß1-40. Neprilysin, or neutral endopeptidase (NEP), catalyzes proteolysis of the amyloid peptides (Aß) and is recognized as one of the major regulators of the levels of these peptides in the brain, preventing Aß accumulation and plaque formation. Here, we used a combination of techniques to elucidate the mechanism of Aß binding and cleavage by NEP. Our findings indicate that the Aß31-X cleavage products remain bound to the neprilysin active site, reducing proteolytic activity. Interestingly, it was already shown that this Aß31-35 sequence is also critical for recognition of Aß peptides by other targets, such as the serpin-enzyme complex receptor in neuronal cells.

Dissection of the key steps of amyloid-ß peptide 1-40 fibrillogenesis.

The aggregation kinetics of Aß1-40 peptide was characterized using a synergistic approach by a combination of nuclear magnetic resonance, thioflavin-T fluorescence, transmission electron microscopy and dynamic light scattering. A major finding is the experimental detection of high molecular weight oligomers (HMWO) that converts into fibrils nuclei. Our observations are consistent with a mechanism of Aß1-40 fibrillogenesis that includes the following key steps: i) slow formation of HMWO (Rh ~ 20 nm); ii) conversion of the HMWO into more compact Rh ~ 10 nm fibrils nuclei; iii) fast formation of additional fibrils nuclei through fibril surface catalysed processes; and iv) growth of fibrils by addition of soluble Aß species. Moreover, NMR diffusion experiments show that at 37 °C soluble Aß1-40 remains intrinsically disordered and mostly in monomeric form despite evidences of the presence of dimers and/or other small oligomers. A mathematical model is proposed to simulate the aggregation kinetics of Aß1-40.

Alzheimer's Aß1-40 peptide degradation by thermolysin: evidence of inhibition by a C-terminal Aß product.

The interaction of the amyloid-ß peptide (Aß) with thermolysin (TLN) was investigated by X-ray crystallography. Structural models of the complexes of TLN with several Aß fragments show that, despite the numerous possible cleavage sites of the Aß sequence, the C-terminal product of Ala30-Ile31 cleavage does not dissociate, thus inhibiting the enzyme. The high similarity between the TLN structural motif and neprilysin (NEP), the most extensively studied peptidase associated with Aß clearance, suggests that NEP should be more efficient against Aß polymorphs where Ala30-Ile31 is inaccessible, which is in agreement with studies in living mice that point to the limited role of NEP in degrading soluble Aß and its higher ability to degrade insoluble and/or oligomeric Aß forms, producing only the Aß10-37 intermediate.

The role of the tyrosine kinase Wzc (Sll0923) and the phosphatase Wzb (Slr0328) in the production of extracellular polymeric substances (EPS) by Synechocystis PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.

Cyanobacterium-Derived Extracellular Carbohydrate Polymer for the Controlled Delivery of Functional Proteins.

The unicellular cyanobacterium Cyanothece sp. CCY 0110 is a highly efficient producer of extracellular polymeric substances (EPS), releasing up to 75% of the polymer to the culture medium. The carbohydrate polymer released to the medium (RPS) was previously isolated and characterized; it is composed of nine different monosaccharides including two uronic acids, and also containing peptides and sulfate groups. Here it is shown that the RPS spontaneously assembles with proteins at high concentrations leading to a phase transition. The proteins are released progressively and structurally intact near physiological conditions, primarily through the swelling of the polymer-protein matrix. The releasing kinetics of the proteins can be modulated through the addition of divalent cations, such as calcium. Notably, the polymer is not toxic to human dermal neonatal fibroblasts in vitro at RPS concentrations bellow 0.1 mg mL-1 . The results show that this polymer is a good candidate for the delivery of therapeutic macromolecules.

Production of microparticles of molinate degrading biocatalysts using the spray drying technique.

Previous studies demonstrated the capability of mixed culture DC1 to mineralize the thiocarbamate herbicide molinate through the activity of molinate hydrolase (MolA). Because liquid suspensions are not compatible with long-term storage and are not easy to handle when bioremediation strategies are envisaged, in this study spray drying was evaluated as a cost-effective method to store and transport these molinate biocatalysts. Microparticles of mixed culture DC1 (DC1) and of cell free crude extracts containing MolA (MA) were obtained without any carrier polymer, and with calcium alginate (CA) or modified chitosan (MCt) as immobilizing agents. All the DC1 microparticles showed high molinate degrading activity upon storage for 6 months, or after 9 additions of ∼0.4 mM molinate over 1 month. The DC1-MCt microparticles were those with the highest survival rate and lowest heterogeneity. For MA microparticles, only MA-MCt degraded molinate. However, its Vmax was only 1.4% of that of the fresh cell free extract (non spray dried). The feasibility of using the DC1-MCt and MA-MCt microparticles in bioaugmentation processes was assessed in river water microcosms, using mass (g):volume (L) ratios of 1:13 and 1:0.25, respectively. Both type of microparticles removed ∼65-75% of the initial 1.5 mg L(-1) molinate, after 7 days of incubation. However, only DC1-MCt microparticles were able to degrade this environmental concentration of molinate without disturbing the native bacterial community. These results suggest that spray drying can be successfully used to produce DC1-MCt microparticles to remediate molinate polluted sites through a bioaugmentation strategy.

Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

Effectiveness of rotavirus vaccine against hospitalized rotavirus diarrhea: A case-control study.

Rotavirus is one of the leading cause of hospitalization and outpatients visits among children under five years. This study evaluated overall and genotype-specific vaccine effectiveness of oral monovalent rotavirus vaccine (G1P[8] strain) in preventing hospital admission of Brazilian children with rotavirus acute diarrhea. A hospital based case-control study was conducted in five Regions of Brazil using the National Rotavirus Acute Diarrhea Surveillance System from July 2008 to August 2011. A total of 215 cases (aged 4-24 months) admitted with confirmed rotavirus diarrhea were recruited and 1961 controls hospitalized without diarrhea were frequency matched by sex and age group to cases. Two-dose adjusted vaccine effectiveness (adjusted by year of birth and the frequency matching variables) was 76% (95%CI: 58-86) lasting for two years. Effectiveness controlled by the available potential confounders was 72% (95%CI: 44-85), suggesting no appreciable confounding by those factors for which adjustment was made. In a half of the cases the rotavirus genotype was G2P[4] and in 15% G1P[8]. Genotype-specific VE (two doses) was 89% (95%CI: 78-95), for G1P[8] and 76% (95%CI: 64-84) for G2P[4]. For all G1, it was 74% (95%CI: 35-90), for all G2, 76% (95%CI: 63-84), and for all non G1/G2 genotypes, 63% (95%CI: -27-99). Effectiveness for one dose was 62% (95%CI: 39-97). Effectiveness of two-dose monovalent rotavirus vaccine in preventing hospital admission with rotavirus diarrhea was high, lasted for two years and it was similar against both G1P[8] and G2P[4]. Based on the findings of the study we recommend the continued use of rotavirus in the Brazilian National Immunization Program and the monitoring of the early emergence of unusual and novel rotavirus genotypes.

Phylogenetic prediction of cis-acting elements: a cre-like sequence in Norovirus genome?

BACKGROUND: Discrete RNA structures such as cis-acting replication elements (cre) in the coding region of RNA virus genomes create characteristic suppression of synonymous site variability (SSSV). Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, for high-resolution thermodynamic scanning and for detecting SSSV. These approaches have been successfully in predicting cis-acting signals in different members of the family Picornaviridae and Caliciviridae. In order to gain insight into the identification of cis-acting signals in viruses whose mechanisms of replication are currently unknown, we performed a phylogenetic analysis of complete genome sequences from 49 Human Norovirus (NoV) strains. FINDINGS: The complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop. CONCLUSION: A new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for Hepatovirus cre stem-loop.

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes.

Most adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 (Aviadenovirus), DAdV-1, and BAdV-4 (Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy.