Neuronal inhibition of the autophagy nucleation complex extends life span in post-reproductive C. elegans.

Autophagy is a ubiquitous catabolic process that causes cellular bulk degradation of cytoplasmic components and is generally associated with positive effects on health and longevity. Inactivation of autophagy has been linked with detrimental effects on cells and organisms. The antagonistic pleiotropy theory postulates that some fitness-promoting genes during youth are harmful during aging. On this basis, we examined genes mediating post-reproductive longevity using an RNAi screen. From this screen, we identified 30 novel regulators of post-reproductive longevity, including pha-4 Through downstream analysis of pha-4, we identified that the inactivation of genes governing the early stages of autophagy up until the stage of vesicle nucleation, such as bec-1, strongly extend both life span and health span. Furthermore, our data demonstrate that the improvements in health and longevity are mediated through the neurons, resulting in reduced neurodegeneration and sarcopenia. We propose that autophagy switches from advantageous to harmful in the context of an age-associated dysfunction.

Antifungal effects of Streptococcus mutans extract on Candida strains susceptible and resistant to fluconazole: An in vivo study.

Previous studies showed that the crude extract obtained from Streptococcus mutans inhibited the growth of Candida albicans reference strains. In this study, we evaluated whether the antifungal effects of S. mutans extract can be extended to clinical Candida isolates, including C. albicans and non-abicans strains with different susceptibilities to fluconazole. We verified that S. mutans extract increased the survival of Galleria mellonella larvae infected with C. albicans and C. glabrata and inhibited the fungal cells in hemolymph. These antifungal effects occurred for both fluconazole-susceptible and fluconazole-resistant strains. However, larvae infected by C. krusei were not affected by S. mutans extract. LAY SUMMARY: Streptococcus mutans crude extract shows antifungal effects on clinical Candida strains susceptible and resistant to fluconazole in Galleria mellonella model.

One hundred twenty years of koala retrovirus evolution determined from museum skins.

Although endogenous retroviruses are common across vertebrate genomes, the koala retrovirus (KoRV) is the only retrovirus known to be currently invading the germ line of its host. KoRV is believed to have first infected koalas in northern Australia less than two centuries ago. We examined KoRV in 28 koala museum skins collected in the late 19th and 20th centuries and deep sequenced the complete proviral envelope region from five northern Australian specimens. Strikingly, KoRV env sequences were conserved among koalas collected over the span of a century, and two functional motifs that affect viral infectivity were fixed across the museum koala specimens. We detected only 20 env polymorphisms among the koalas, likely representing derived mutations subject to purifying selection. Among northern Australian koalas, KoRV was already ubiquitous by the late 19th century, suggesting that KoRV evolved and spread among koala populations more slowly than previously believed. Given that museum and modern koalas share nearly identical KoRV sequences, it is likely that koala populations, for more than a century, have experienced increased susceptibility to diseases caused by viral pathogenesis.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.

N6-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.

Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.

Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing.

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.

The Postbiotic Activity of Lactobacillus paracasei 28.4 Against Candida auris.

Candida auris has emerged as a medically important pathogen with considerable resistance to antifungal agents. The ability to produce biofilms is an important pathogenicity feature of this species that aids escape of host immune responses and antimicrobial agents. The objective of this study was to verify antifungal action using in vitro and in vivo models of the Lactobacillus paracasei 28.4 probiotic cells and postbiotic activity of crude extract (LPCE) and fraction 1 (LPF1), derived from L. paracasei 28.4 supernatant. Both live cells and cells free supernatant of L. paracasei 28.4 inhibited C. auris suggesting probiotic and postbiotic effects. The minimum inhibitory concentration (MIC) for LPCE was 15 mg/mL and ranges from 3.75 to 7.5 mg/mL for LPF1. Killing kinetics determined that after 24 h treatment with LPCE or LPF1 there was a complete reduction of viable C. auris cells compared to fluconazole, which decreased the initial inoculum by 1-logCFU during the same time period. LPCE and LPF1 significantly reduced the biomass (p = 0.0001) and the metabolic activity (p = 0.0001) of C. auris biofilm. There was also a total reduction (~108 CFU/mL) in viability of persister C. auris cells after treatment with postbiotic elements (p < 0.0001). In an in vivo study, injection of LPCE and LPF1 into G. mellonella larvae infected with C. auris prolonged survival of these insects compared to a control group (p < 0.05) and elicited immune responses by increasing the number of circulating hemocytes and gene expression of antimicrobial peptide galiomicin. We concluded that the L. paracasei 28.4 cells and postbiotic elements (LPCE and LPF1) have antifungal activity against planktonic cells, biofilms, and persister cells of C. auris. Postbiotic supplementation derived from L. paracasei 28.4 protected G. mellonella infected with C. auris and enhanced its immune status indicating a dual function in modulating a host immune response.

Streptococcus mutans Secreted Products Inhibit Candida albicans Induced Oral Candidiasis.

In the oral cavity, Candida species form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. In this study, we extracted and fractioned culture filtrate of S. mutans, seeking antifungal agents capable of inhibiting the biofilms, filamentation, and candidiasis by Candida albicans. Active S. mutans UA159 supernatant filtrate components were extracted via liquid-liquid partition and fractionated on a C-18 silica column to resolve S. mutans fraction 1 (SM-F1) and fraction 2 (SM-F2). We found anti-biofilm activity for both SM-F1 and SM-F2 in a dose dependent manner and fungal growth was reduced by 2.59 and 5.98 log for SM-F1 and SM-F2, respectively. The SM-F1 and SM-F2 fractions were also capable of reducing C. albicans filamentation, however statistically significant differences were only observed for the SM-F2 (p = 0.004). SM-F2 efficacy to inhibit C. albicans was confirmed by its capacity to downregulate filamentation genes CPH1, EFG1, HWP1, and UME6. Using Galleria mellonella as an invertebrate infection model, therapeutic treatment with SM-F2 prolonged larvae survival. Examination of the antifungal capacity was extended to a murine model of oral candidiasis that exhibited a reduction in C. albicans colonization (CFU/mL) in the oral cavity when treated with SM-F1 (2.46 log) and SM-F2 (2.34 log) compared to the control (3.25 log). Although both SM-F1 and SM-F2 fractions decreased candidiasis in mice, only SM-F2 exhibited significant quantitative differences compared to the non-treated group for macroscopic lesions, hyphae invasion, tissue lesions, and inflammatory infiltrate. Taken together, these results indicate that the SM-F2 fraction contains antifungal components, providing a promising resource in the discovery of new inhibitors for oral candidiasis.

Amaiouine, a cyclopeptide alkaloid from the leaves of Amaioua guianensis.

Amaiouine, a cyclopeptide alkaloid, was isolated from the ethanolic extract of the leaves of Amaioua guianensis. The structure was elucidated by NMR spectroscopy and confirmed by X-ray crystallography.

Aromatic compounds produced by endophytic fungi isolated from red alga Asparagopsis taxiformis - Falkenbergia stage.

Endophytic fungi were isolated from red alga Asparagopsis taxiformis - Falkenbergia stage, collected from the Brazilian coast, and were identified as Annulohypoxylon stygium (AT-03) and A. yungensis (AT-06) based on their macro/micromorphological and molecular features. Bioassay-guided fractionation of the EtOAc extract from laboratory cultures of both strains yielded known compounds pyrogallol from A. stygium, (3 R)-scytalone and (3 R,4 R)-4-hydroxy-scytalone from A. yungensis. Pyrogallol was active against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli strains. An inactive fraction from A. stygium afforded two additional compounds, (3 R,4 R)-3,4,5-trihydroxy-1-tetralone and tyrosol. Optically active compounds had their stereochemistry determined by circular dichroism (CD) spectroscopy.

Botryane terpenoids produced by Nemania bipapillata, an endophytic fungus isolated from red alga Asparagopsis taxiformis - Falkenbergia stage.

A chemical study of the EtOAc extract of Nemania bipapillata (AT-05), an endophytic fungus isolated from the marine red alga Asparagopsis taxiformis - Falkenbergia stage, led to the isolation of five new botryane sesquiterpenes, including the diastereomeric pair (+)-(2R,4S,5R,8S)-(1) and (+)-(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (2), (+)-(2R,4S,5R,8R)-4-deacetyl-botryenalol (3), one pair of diastereomeric botryane norsesquiterpenes bearing an unprecedented degraded carbon skeleton, (+)-(2R,4R,8R)-(4) and (+)-(2R,4S,8S)-(5), which were named nemenonediol A and nemenonediol B, respectively, in addition to the known 4ß-acetoxy-9ß,10ß,15α-trihydroxyprobotrydial (6). Their structures were elucidated using 1D and 2D NMR, HRESIMS and comparison with literature data of similar known compounds. The absolute configurations of 2, 3 and 4 were deduced by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, while those of 1 and 5 were assigned from vibrational circular dichroism (VCD) data. Compound 4 weakly inhibited acetylcholinesterase, whereas compound 1 inhibited both acetylcholinesterase and butyrylcholinesterase. Compounds 1, 3, 5 and 6 were tested against two carcinoma cell lines (MCF-7 and HCT-116), but showed no significant citotoxicity at tested concentrations (IC50 > 50 µM).

Anti-inflammatory and antiproliferative activities of Ixora brevifolia Benth. (Rubiaceae).

The crude extract and fractions from the branches of Ixora brevifolia, a tree found in the Brazilian Cerrado, were tested for anti-inflammatory and in vitro antiproliferative effects. The crude extract and n-hexane fraction exhibited significant inhibition of ear oedema in mice, while n-hexane-precipitated and chloroform fractions strongly inhibited the myeloperoxidase activity in ear tissue. The n-hexane and n-hexane-precipitated fractions showed strong growth inhibition for glioma cell line and the hydromethanolic fraction inhibited the growth of leukaemia cell line.

Bioprospecting as a strategy for conservation and sustainable use of the Brazilian Flora / Bioprospecção como estratégia para a conservação e uso sustentável da Flora Brasileira

Abstract In Brazil, research with natural products had a strong impulse when FAPESP supported the creation of the Laboratory of Chemistry of Natural Products of the Institute of Chemistry of USP (1966). In 1999, FAPESP launched the Research Program in the Characterization, Conservation, Restoration and Sustainable Use of Biodiversity (BIOTA-FAPESP), which intensified the sustainable exploitation of biodiversity, and which evolved to form the Biota Network for Bioprospection and Bioassays (BIOprospecTA), which integrates groups from all over the country, optimizing the use of the skills already installed for the bioprospecting of microorganisms, plants, invertebrates, vertebrates and marine organisms. Of the 104 projects related to plant sciences, 35 carried out bioprospection of Brazilian flora, belonging to the areas of Chemistry, Botany, Genetics, Plant Physiology, Plant Morphology, Plant (Chemo)taxonomy, Ecosystem Ecology, Plant Genetics. Physical Sciences, Forest Resources, Forestry Engineering, Agronomy, leading to thousands of publications, engagement of hundreds of students and a deeper understanding of natural products in different biological models through macromolecules analysis aided by computational and spectrometric strategies, in addition to pharmacological evaluations. The development of omics approaches led to a more comprehensive view of the chemical profile of an organism, and enabled integrated and concomitant studies of several samples, and faster annotation of known molecules, through the use of hyphenated and chemometric techniques, and molecular networking. This also helped to overcome the lack of information on the safety and efficacy of herbal preparations, in projects dealing with the standardization of herbal products, according to international standards. The BIOTA-FAPESP program has also focused on environmental aspects, in accordance with the principles of Green Chemistry and has had positive effects on international collaboration, on the number and impact of scientific publications and on partnership with companies, a crucial step to add value and expand the production chain of bioproducts. Also, the compilation, systematization and sharing of data were contemplated with the creation of the NUBBEDB database, of free access, and that integrates with international databases (ACD/labs, American Chemical Society - ACS), helping researchers and companies in the development from different areas of science, technology, strengthening the bioeconomy and subsidizing public policies.

Resumo No Brasil, as pesquisas com produtos naturais tiveram um forte impulso quando a FAPESP apoiou a criação do Laboratório de Química de Produtos Naturais do Instituto de Química da USP (1966). Em 1999, a FAPESP lançou o Programa de Pesquisa em Caracterização, Conservação, Restauração e Uso Sustentável da Biodiversidade (BIOTA-FAPESP), que intensificou a exploração sustentável da biodiversidade, e que evoluiu para formar a Rede Biota de Bioprospecção e Bioensaios (BIOprospecTA), que integra grupos de todo o país, otimizando o aproveitamento das competências já instaladas para a bioprospecção de microrganismos, plantas, invertebrados, vertebrados e organismos marinhos. Dos 104 projetos relacionados às ciências vegetais, 35 realizaram a bioprospecção da flora brasileira, em diversas áreas como Química, Botânica, Fisiologia e Morfologia Vegetal, (Quimio)taxonomia Vegetal, Ecologia de Ecossistemas, Genética Vegetal, Recursos Florestais, Engenharia Florestal, dentre outros, levando a milhares de publicações, ao engajamento de centenas de estudantes e ao entendimento mais profundo dos produtos naturais em diferentes modelos biológicos por meio da análise de micromoléculas auxiliada por estratégias computacionais e espectrométricas, além de avaliações farmacológicas. O desenvolvimento de abordagens ômicas ampliou a visão sobre perfil químico dos organismos, possibilitou o estudo integrado e concomitante de várias amostras, e a anotação mais rápida de moléculas conhecidas, por meio do uso de técnicas hifenadas, quimiométricas e redes moleculares. Isso também contribuiu para superar a falta de informação sobre a segurança e eficácia dos fitopreparados, em projetos que tratam da padronização de produtos fitoterápicos, de acordo com normas internacionais. O programa BIOTA-FAPESP também tem focado em aspectos ambientais, de acordo com os princípios da Química Verde e teve reflexos positivos na colaboração internacional, no número e no impacto das publicações científicas e na parceria com empresas, etapa crucial para agregar valor e expandir a cadeia produtiva de bioprodutos. Ainda, a compilação, sistematização e compartilhamento de dados foram contemplados com a criação da base de dados NUBBEDB, de livre acesso, e que se integra com bases internacionais (ACD/labs, American Chemical Society - ACS), auxiliando pesquisadores e empresas no desenvolvimento de diferentes áreas da ciência, tecnologia, fortalecendo a bioeconomia e subsidiando políticas públicas.

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair.

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV-RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV-DDB-CUL4A E3 ligase complex (DDB1-DDB2-CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites.

IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche.

The adult subventricular zone (SVZ) is the main neurogenic niche in normal adult brains of mice and rats. Interferon gamma (IFNγ) has somewhat controversially been associated with SVZ progenitor proliferation and neurogenesis. The in vivo involvement of IFNγ in the physiology of the adult SVZ niche is not fully understood and its intracellular mediators are unknown. Here we show that IFNγ, through activation of its canonical signal transducer and activator of transcription 1 (STAT1) pathway, acts specifically on Nestin+ progenitors by decreasing both progenitor proliferation and the number of cycling cells. In addition, IFNγ increases the number of neuroblasts generated without shifting glial fate determination. The final result is deficient recruitment of newborn neurons to the olfactory bulb (OB), indicating that IFNγ-induced stimulation of neuronal differentiation does not compensate for its antiproliferative effect. We conclude that IFNγ signaling via STAT1 in the SVZ acts dually as an antiproliferative and proneurogenic factor, and thereby regulates neurogenesis in normal adult brains.

Cytotoxic activity of Guettarda pohliana Müll. Arg. (Rubiaceae).

The cytotoxic activity of crude extracts and their fractions from leaves and roots of G. pohliana was assessed against nine human cancer cell lines: melanoma (UACC-62), breast (MCF-7), breast expressing the multidrug resistance phenotype (NCI-ADR), lung (NCI-460), prostate (PCO-3), kidney (786-0), ovarian (OVCAR), colon (HT-29) and leukaemia (K-562). The hexane fraction from leaves (HL) and ethyl acetate (EAR), chloroform (CR) and hydromethanolic (HMR) fractions from roots were the most active fractions against K-562 with GI50 values being lower than 1 µg mL⁻¹. Also, CR and HMR fractions were active against UACC-62 cell line in the same order of magnitude. The phytochemical study of the CR fraction allowed identifying the known iridoids secoxyloganin, sweroside and loganin.

Detection of streptomycin and quinolone resistance in Mycobacterium tuberculosis by a low-density DNA array.

In cases of multidrug-resistant tuberculosis, it is crucial to rule out resistance to second-line antituberculous (anti-TB) agents. In the present study, a low-cost low-density DNA array including four genetic regions (rrs 530 loop, rrs 1400, rpsL and gyrA) was designed for the rapid detection of the most important mutations related to anti-TB injectable drugs (mainly streptomycin) and fluoroquinolone resistance (LD-SQ array). A total of 108 streptomycin- and/or ofloxacin-resistant and 20 streptomycin- and ofloxacin-susceptible Mycobacterium tuberculosis clinical isolates were analysed with the array. The results obtained were compared with sequencing data and phenotypic susceptibility pattern. The LD-SQ array offered a good sensitivity compared to sequencing, especially among resistant strains: 92.5% (37/40) for streptomycin and 87.5% (7/8) for fluoroquinolones. Therefore, this array could be considered a good approach for the rapid detection of mutations related to streptomycin and fluoroquinolone resistance. On the other hand, there were discordant results in 16 resistant strains and six susceptible isolates, mostly concerning the gyrA region, in which the existence of polymorphisms next to informative positions might cause cross-hybridization. These discrepancies were caused by some technical limitations; consequently, the present array should be considered as a first-step prior to a forthcoming optimized version of the array.