Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis.

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically-derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analyses. We produced DRS datasets using modification-free transcripts from in vitro transcription of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analyses of human transcripts.

Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

mRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation.

Pseudouridine (psi) is one of the most abundant human mRNA modifications generated from the isomerization of uridine via psi synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with our recent tool, Mod-p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, among a list of highly conserved sites across cell types, most are TRUB1 substrates and fall within the coding sequence. We find that these conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. We validate these sites by ruling out single-nucleotide variants, analyzing current traces, and performing enzymatic knockdowns of psi synthases. Finally, we characterize sites with multiple psi modifications on the same transcript (hypermodification type II) and found that these can be conserved or cell type specific. Among these, we discovered examples of multiple psi modifications within the same k-mer for the first time and analyzed the effect on current distribution. Our data support the hypothesis that motif sequence and the presence of psi synthase are insufficient to drive modifications, that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.

Angiotensin Receptor-Neprilysin Inhibition (Sacubitril/Valsartan) Reduces Structural Arterial Stiffness in Middle-Aged Mice.

BACKGROUND: Increasing arterial stiffness is a prominent feature of the aging cardiovascular system. Arterial stiffening leads to fundamental alterations in central hemodynamics with widespread detrimental implications for organ function resulting in significant morbidity and death, and specific therapies to address the underlying age-related structural arterial remodeling remain elusive. The present study investigates the potential of the recently clinically available dual angiotensin receptor-neprilysin inhibitor (ARNI) sacubitril/valsartan (LCZ696) to counteract age-related arterial fibrotic remodeling and stiffening in 1-year-old mice. METHODS AND RESULTS: Treatment of in 1-year-old mice with ARNI (sacubitril/valsartan), in contrast to angiotensin receptor blocker monotherapy (valsartan) and vehicle treatment (controls), significantly decreases structural aortic stiffness (as measured by in vivo pulse-wave velocity and ex vivo aortic pressure myography). This phenomenon appears, at least partly, independent of (indirect) blood pressure effects and may be related to a direct antifibrotic interference with aortic smooth muscle cell collagen production. Furthermore, we find aortic remodeling and destiffening due to ARNI treatment to be associated with improved parameters of cardiac diastolic function in aged mice. CONCLUSIONS: This study provides preclinical mechanistic evidence indicating that ARNI-based interventions may counteract age-related arterial stiffening and may therefore be further investigated as a promising strategy to improve cardiovascular outcomes in the elderly.

Light-Driven Tetra- and Octa-ß-substituted Cationic Zinc(II) Phthalocyanines for Eradicating Fusarium oxysporum Conidia.

Photodynamic inactivation (PDI) is an emerging therapeutic approach that can effectively inactivate diverse microbial forms, including vegetative forms and spores, while preserving host tissues and avoiding the development of resistance to the photosensitization procedure. This study evaluates the antifungal and sporicidal photodynamic activity of two water-soluble amphiphilic tetra- and octa-ß-substituted zinc(II) phthalocyanine (ZnPc) dyes with dimethylaminopyridinium groups at the periphery (ZnPcs 1, 2) and their quaternized derivatives (ZnPcs 1a, 2a). Tetra(1, 1a)- and octa(2, 2a)-ß-substituted zinc(II) phthalocyanines were prepared and assessed as photosensitizers (PSs) for their effects on Fusarium oxysporum conidia. Antimicrobial photoinactivation experiments were performed with each PS at 0.1, 1, 10, and 20 µM under white light irradiation at an irradiance of 135 mW·cm-2, for 60 min (light dose of 486 J·cm-2). High PDI efficiency was observed for PSs 1a, 2, and 2a (10 µM), corresponding to inactivation until the method's detection limit. PS 1 (20 µM) also achieved a considerable reduction of >5 log10 in the concentration of viable conidia. The quaternized PSs (1a, 2a) showed better PDI performance than the non-quaternized ones (1, 2), even at the low concentration of 1 µM, and a light dose of 486 J·cm-2. These cationic phthalocyanines are potent photodynamic drugs for antifungal applications due to their ability to effectively inactivate resistant forms, like conidia, with low concentrations and reasonable energy doses.

Astrocytes produce nitric oxide via nitrite reduction in mitochondria to regulate cerebral blood flow during brain hypoxia.

During hypoxia, increases in cerebral blood flow maintain brain oxygen delivery. Here, we describe a mechanism of brain oxygen sensing that mediates the dilation of intraparenchymal cerebral blood vessels in response to reductions in oxygen supply. In vitro and in vivo experiments conducted in rodent models show that during hypoxia, cortical astrocytes produce the potent vasodilator nitric oxide (NO) via nitrite reduction in mitochondria. Inhibition of mitochondrial respiration mimics, but also occludes, the effect of hypoxia on NO production in astrocytes. Astrocytes display high expression of the molybdenum-cofactor-containing mitochondrial enzyme sulfite oxidase, which can catalyze nitrite reduction in hypoxia. Replacement of molybdenum with tungsten or knockdown of sulfite oxidase expression in astrocytes blocks hypoxia-induced NO production by these glial cells and reduces the cerebrovascular response to hypoxia. These data identify astrocyte mitochondria as brain oxygen sensors that regulate cerebral blood flow during hypoxia via release of nitric oxide.

[Syphilis in Argentina]. / Sífilis en Argentina.

Syphilis is a disease preventable through sexual protection barriers, and curable with a simple treatment. Despite this, between 2010 and 2019, its incidence almost tripled in Argentina, reaching 56.1/100 000. The most affected are young people (15-24 years old), with a higher percentage of women, especially among pregnant women, in whom it increased from 2.3% to 5.8%, with a peak of 7.7% between 15 and 24 years old. Cases of secondary or tertiary syphilis, detected by control tests, are on the rise. The increase in syphilis is linked to HIV. For this reason, rapid detection tests must be carried out jointly at the first level of care, which facilitates access to the diagnosis and treatment of positive cases, with the corresponding guidance and control. Treatment with penicillin, for which no resistance has been found, is the key component of control. Doxycillin is also used (not in pregnant women), or ceftriaxone. The resistance of Treponema pallidum to azithromycin is increasing, so its use should be avoided. In 2022, 696 cases of congenital syphilis were reported. The national rate is 1.3 per 1000 live births. The majority of reported cases come from the public sector. Maternal syphilis is, in low-income areas, the most common cause of stillbirths. Strict application of existing regulations, strengthening the primary care system, and prevention during pregnancy can contribute to controlling and eliminating the syphilis problem in Argentina.

La sífilis es una enfermedad prevenible mediante barreras de protección sexual, y curable por un sencillo tratamiento. A pesar de esto, entre 2010 y 2019, su incidencia casi se triplicó en Argentina, alcanzando 56.1/100 000. Los más afectados son los jóvenes (15-24 años), con mayor porcentaje de mujeres, especialmente entre las embarazadas, en que aumentó de 2.3% a 5.8%, con un pico de 7.7% entre 15 y 24 años. Los casos de sífilis secundaria o terciaria, detectados por pruebas de control, están en aumento. El aumento de sífilis va unido al HIV. Por ello las pruebas rápidas de detección deben hacerse conjuntamente en el primer nivel de atención, lo que facilita el acceso al diagnóstico y al tratamiento de los casos positivos, con la orientación y control correspondientes. El tratamiento con penicilina, para la que no se ha hallado resistencia, es el componente clave del control. También se usa doxicilina (no en embarazadas), o ceftriaxona. Está aumentando la resistencia del Treponema pallidum a la azitromicina, por lo que debe evitarse su empleo. En 2022 se notificaron 696 casos de sífilis congénita. La tasa de nacional es 1.3 por 1000 nacidos vivos. La mayoría de los casos notificados provienen del sector público. La sífilis materna es, en áreas de bajos recursos, la causa más común de nacidos muertos. La aplicación estricta de las normas existentes, el fortalecimiento del sistema de atención primaria, y la prevención durante el embarazo, pueden contribuir a controlar y eliminar el problema de la sífilis en Argentina.

Sífilis en Argentina / Syphilis in Argentina

Resumen La sífilis es una enfermedad prevenible mediante barreras de protección sexual, y curable por un sen cillo tratamiento. A pesar de esto, entre 2010 y 2019, su incidencia casi se triplicó en Argentina, alcanzando 56.1/100 000. Los más afectados son los jóvenes (15-24 años), con mayor porcentaje de mujeres, especialmente entre las embarazadas, en que aumentó de 2.3% a 5.8%, con un pico de 7.7% entre 15 y 24 años. Los casos de sífilis secundaria o terciaria, detectados por pruebas de control, están en aumento. El aumento de sífilis va unido al HIV. Por ello las prue bas rápidas de detección deben hacerse conjuntamente en el primer nivel de atención, lo que facilita el acceso al diagnóstico y al tratamiento de los casos positivos, con la orientación y control correspondientes. El tratamiento con penicilina, para la que no se ha hallado resistencia, es el componente clave del control. También se usa doxicilina (no en embarazadas), o cef triaxona. Está aumentando la resistencia del Treponema pallidum a la azitromicina, por lo que debe evitarse su empleo. En 2022 se notificaron 696 casos de sífilis congénita. La tasa de nacional es 1.3 por 1000 nacidos vivos. La mayoría de los casos notificados provienen del sector público. La sífilis materna es, en áreas de bajos recursos, la causa más común de nacidos muertos. La aplicación estricta de las normas existentes, el fortalecimiento del sistema de atención primaria, y la prevención durante el embarazo, pueden contribuir a controlar y eliminar el problema de la sífilis en Argen tina.

Abstract Syphilis is a disease preventable through sexual pro tection barriers, and curable with a simple treatment. Despite this, between 2010 and 2019, its incidence al most tripled in Argentina, reaching 56.1/100 000. The most affected are young people (15-24 years old), with a higher percentage of women, especially among pregnant women, in whom it increased from 2.3% to 5.8%, with a peak of 7.7% between 15 and 24 years old. Cases of secondary or tertiary syphilis, detected by control tests, are on the rise. The increase in syphilis is linked to HIV. For this reason, rapid detection tests must be carried out jointly at the first level of care, which facilitates access to the diagnosis and treatment of positive cases, with the cor responding guidance and control. Treatment with penicillin, for which no resistance has been found, is the key component of control. Doxycillin is also used (not in pregnant women), or ceftriaxone. The resistance of Treponema pallidum to azithromycin is increasing, so its use should be avoided. In 2022, 696 cases of congenital syphilis were re ported. The national rate is 1.3 per 1000 live births. The majority of reported cases come from the public sector. Maternal syphilis is, in low-income areas, the most com mon cause of stillbirths. Strict application of existing regulations, strength ening the primary care system, and prevention during pregnancy can contribute to controlling and eliminating the syphilis problem in Argentina.

Modulatory Properties of Aloe secundiflora's Methanolic Extracts on Targeted Genes in Colorectal Cancer Management.

Colon tumors have a very complicated and poorly understood pathogenesis. Plant-based organic compounds might provide a novel source for cancer treatment with a sufficient novel mode of action. The objective of this study was to analyze and evaluate the efficacy of Aloe secundiflora's (AS) methanolic extracts on the expression of CASPS9, 5-LOX, Bcl2, Bcl-xL, and COX-2 in colorectal cancer (CRC) management. Caco-2 cell lines were used in the experimental study. In the serial exhaustive extraction (SEE) method, methanol was utilized as the extraction solvent. Upon treatment of CASPS9 with the methanolic extracts, the expression of the genes was progressively upregulated, thus, dose-dependently increasing the rate of apoptosis. On the other hand, the expressions of 5-LOX, Bcl2, and Bcl-xL were variably downregulated in a dose-dependent manner. This is a unique novel study that evaluated the effects of AS methanolic extracts in vitro on CRC cell lines using different dosage concentrations. We, therefore, recommend the utilization of AS and the application of methanol as the extraction solvent of choice for maximum modulatory benefits in CRC management. In addition, we suggest research on the specific metabolites in AS involved in the modulatory pathways that suppress the development of CRC and potential metastases.

Biochemical and structural characterization of the RT domain of Leishmania sp. telomerase reverse transcriptase.

BACKGROUND: The Leishmania genus comprises parasites that cause leishmaniasis, a neglected disease spread worldwide. Leishmania sp. telomeres are composed of TTAGGG repeats maintained by telomerase. In most eukaryotes, the enzyme minimal complex contains the TER (telomerase RNA) and the TERT (telomerase reverse transcriptase) components. The TERT holds the enzyme catalytic core and is formed by four structural and functional domains (TEN, Telomerase Essential N-terminal; TRBD, Telomerase RNA Binding Domain; RT, the reverse transcriptase domain and CTE, C-Terminal Extension domain). METHODS AND RESULTS: Amino acid sequence alignments, protein structure prediction analysis, and protein: nucleic acid interaction assays were used to show that the Leishmania major RT domain preserves the canonical structural elements found in higher eukaryotes, including the canonical motifs and the aspartic acid residues that stabilize the Mg2+ ion cofactor. Furthermore, amino acid substitutions specific to the Leishmania genus and partial conservation of the residues involved with nucleic acid interactions are shown. The purified recombinant Leishmania RT protein is biochemically active and interacts with the G-rich telomeric strand and the TER template sequence. CONCLUSION: Our results highlight that the telomerase catalysis mechanism is conserved in a pathogen of medical importance despite the structural peculiarities present in the parasite's RT domain.

First Identification of bla NDM-1 Producing Escherichia coli ST 9499 Isolated from Musca domestica in the Urban Center of Rio de Janeiro, Brazil.

The present study presents phenotypic and molecular characterization of a multidrug-resistant strain of Escherichia coli (Lemef26), belonging to sequence type ST9499 carrying a blaNDM-1 carbapenem resistance gene. The bacterium was isolated from a specimen of Musca domestica, collected in proximity to a hospital in Rio de Janeiro City, Brazil. The strain was identified as E. coli by matrix-assisted laser desorption-ionization time of flight mass spectrometry (Maldi-TOF-MS) and via genotypic analysis (Whole-Genome Sequencing-WGS), followed by phylogenetic analysis, antibiotic resistance profiling (using phenotypic and genotypic methods) and virulence genotyping. Interestingly, the blaNDM-1 was the only resistance determinant detected using a panel of common resistance genes, as evaluated by PCR. In contrast, WGS detected genes conferring resistance to aminoglycosides, fluoroquinolones, quinolones, trimethoprim, beta-lactams, chloramphenicol, macrolides, sulfonamide, tetracycline, lincosamide and streptogramin B. Conjugation experiments demonstrated the transfer of carbapenem resistance, via acquisition of the blaNDM-1 sequence, to a sensitive receptor strain of E. coli, indicating that blaNDM-1 is located on a conjugative plasmid (most likely of the IncA/C incompatibility group, in association with the transposon Tn3000). Phylogenetic analyses placed Lemef26 within a clade of strains exhibiting allelic and environment diversity, with the greatest level of relatedness recorded with a strain isolated from a human source suggesting a possible anthropogenic origin. Analysis of the virulome revealed the presence of fimbrial and pilus genes, including a CFA/I fimbriae (cfaABCDE), common pilus (ecpABCDER), laminin-bind fimbrae (elfADG), hemorrhagic pilus (hcpABC) and fimbrial adherence determinants (stjC) indicates the ability of strain Lemef26 to colonize animal hosts. To the best of our knowledge, this study represents the first report of blaNDM-1 carbapenemase gene in an E. coli strain isolated from M. domestica. In concordance with the findings of previous studies on the carriage of MDR bacteria by flies, the data presented herein provide support to the idea that flies may represent a convenient means (as sentinel animals) for the monitoring of environmental contamination with multidrug-resistant bacteria.

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Career flexibility and its relation to time perspective: a study with college students in the Portuguese context.

The volatility of the labor market resulting from globalization, rapid technology changes, economic competition, and the impact of events such as the Covid-2019 Pandemic, demand from vocational psychology a more refined understanding of the processes individuals face while dealing with those new challenges and opportunities, especially in uncertain contexts. Theories such as Planned Happenstance address constructs such as career flexibility, an essential skill to recognize, create and use chance events as career opportunities. Furthermore, when fortuitous events and uncertain contexts are considered for career development, it becomes relevant to understand how subjective time perspective evolves, that is how the life events and career goals are projected, accessed, valued, and organized. Given this context, the objectives of the present study are to adapt and validate a Portuguese version of the Career Flexibility Inventory and to explore the possible relationships between career flexibility, time perspective, and variables inherent to the educational context. The Portuguese version of the Career Flexibility Inventory, the Time Perspective Inventory and a sociodemographic form were answered by 1,380 students from Portuguese higher education institutions. The results indicated that the Portuguese version of the CFI has an adequate three-factor structure with good reliability indices. Some limitations regarding psychometric validity show the importance of further research to improve the measure. However, the findings contribute to theoretically and operationally deepening discussions on the multidimensionality of Career Flexibility. The results regarding the relationships between time perspective and career flexibility seem to be in line with the theoretical indicators of the variables and support the formulated hypotheses, specifically, that future orientation presents a positive correlation with active adaptation; a negative correlation with wavering, and wavering presents a positive correlation with the negative future orientation. The results partially support the hypothesis of differences on time perspective and career flexibility among students with different academic grade averages and from diverse scientific areas of study. Finally, the study advances a theoretical reflection on the different nature of career flexibility dimensions and contributes to broadening and promoting theoretical and operational discussions on the relationships between time perspective and career flexibility, which are still incipient.

Abdominal Aortic Endograft Implantation Immediately Induces Vascular Stiffness Gradients That May Promote Adverse Aortic Neck Dilatation: Results of A Porcine Ex Vivo Study.

PURPOSE: Endovascular aortic repair (EVAR) is the method of choice for most abdominal aortic aneurysm (AAA) patients requiring intervention. However, chronic aortic neck dilatation (AND) following EVAR progressively weakens the structural seal between vessel and endograft and compromises long-term results of the therapy. This experimental ex vivo study seeks to investigate mechanisms of AND. MATERIALS AND METHODS: Porcine abdominal aortas (n=20) were harvested from slaughterhouse pigs and connected to a mock circulation. A commercially available endograft was implanted (n=10) or aortas were left untreated as controls (n=10). Vascular circumferential strain was assessed via ultrasound in defined aortic segments as a parameter of aortic stiffness. Histology and aortic gene expression analysis were performed to investigate potential changes of aortic wall structure and molecular differences due to endograft implantation. RESULTS: We found that endograft implantation acutely induces a significant stiffness gradient directly at the interface between stented and unstented aortic segments under pulsatile pressure. Comparing stented aortas with unstented controls, we detected increased aortic expression levels of inflammatory cytokines (Il6 and Ccl2) and matrix metalloproteinases (Mmp2 and Mmp9) after 6 hours of pulsatile pressurization. This effect, however, was abolished when repeating the same experiment under 6 hours of static pressure. CONCLUSIONS: We identified endograft-induced aortic stiffness gradients as an early trigger of inflammatory aortic remodeling processes that might promote AND. These results highlight the importance of adequate endograft designs to minimize vascular stiffness gradients and forestall late complications, such as AND. CLINICAL IMPACT: AND may compromise the long-term results following endovascular aortic repair. However, the mechanisms behind the underlying detrimental aortic remodeling are still unclear. In this study we find that endograft-induced aortic stiffness gradients induce an inflammatory aortic remodeling response consistent with AND. This novel pathomechanistic insight may guide the design of new aortic endografts that minimize vascular stiffness gradients and forestall late complications such as AND.

Multicellular, IVT-derived, unmodified human transcriptome for nanopore direct RNA analysis.

Nanopore direct RNA sequencing (DRS) enables measurements of native RNA modifications. Modification-free transcripts are an important control for DRS. Additionally, it is advantageous to have canonical transcripts from multiple cell lines to better account for human transcriptome variation. Here we generated and analyzed Nanopore DRS datasets for five human cell lines using in vitro transcribed (IVT) RNA. We compared performance statistics amongst biological replicates. We also documented nucleotide and ionic current level variation across cell lines. These data will serve as a resource to the community for RNA modification analysis.

Formation of lanthanide luminescent di-metallic helicates in solution using a bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) ligand.

The chiral bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) ligand 1 was used in the formation of lanthanide di- and triple stranded di-metallic helicates in acetonitrile solution, where the changes in the ground and the Tb(III) excited state properties were used to monitor the formation of these supramolecular structures in situ under kinetic control.