Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia.

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

Spinocerebellar ataxia type 11 (SCA11): TTBK2 variants, functions and associated disease mechanisms.

Spinocerebellar ataxia type 11 (SCA11) is a rare type of autosomal dominant cerebellar ataxia, mainly characterized by progressive cerebellar ataxia, abnormal eye signs and dysarthria. SCA11 is caused by variants in TTBK2, which encodes tau tubulin kinase 2 (TTBK2) protein. Only a few families with SCA11 were described to date, all harbouring small deletions or insertions that result in frameshifts and truncated TTBK2 proteins. In addition, TTBK2 missense variants were also reported but they were either benign or still needed functional validation to ascertain their pathogenic potential in SCA11. The mechanisms behind cerebellar neurodegeneration mediated by TTBK2 pathogenic alleles are not clearly established. There is only one neuropathological report and a few functional studies in cell or animal models published to date. Moreover, it is still unclear whether the disease is caused by TTBK2 haploinsufficiency of by a dominant negative effect of TTBK2 truncated forms on the normal allele. Some studies point to a lack of kinase activity and mislocalization of mutated TTBK2, while others reported a disruption of normal TTBK2 function caused by SCA11 alleles, particularly during ciliogenesis. Although TTBK2 has a proven function in cilia formation, the phenotype caused by heterozygous TTBK2 truncating variants are not clearly typical of ciliopathies. Thus, other cellular mechanisms may explain the phenotype seen in SCA11. Neurotoxicity caused by impaired TTBK2 kinase activity against known neuronal targets, such as tau, TDP-43, neurotransmitter receptors or transporters, may contribute to neurodegeneration in SCA11.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Long-Term Mortality and Health-Related Quality of Life After Continuous Versus Intermittent Renal Replacement Therapy in ICU Survivors: A Secondary Analysis of the Quality of Life After ICU Study.

Purpose: We assessed long-term outcomes in intensive care unit (ICU) survivors with acute kidney injury (AKI) submitted to intermittent or continuous renal replacement therapy (RRT) for comparisons between groups. Methods: The multicenter prospective cohort study included 195 adult ICU survivors with an ICU stay >72 h in 10 ICUs that had at least one episode of AKI treated with intermittent RRT (IRRT) or continuous RRT (CRRT) during ICU stay. The main outcomes were mortality and health-related quality of life (HRQoL). Hospital readmissions and physical dependence were also assessed. Results: Regarding RRT, 83 (42.6%) patients received IRRT and 112 (57.4%) received CRRT. Despite the similarity regarding sociodemographic characteristics, pre-ICU state of health and type of admission between groups, the risk of death (23.5% vs 42.7%; P < .001), the prevalence of sepsis (60.7%) and acute respiratory distress syndrome (17%) were higher at ICU admission among CRRT patients. The severity of critical illness was higher among CRRT patients, regarding the need for mechanical ventilation (75.0% vs 50.6%, P = .002) and vasopressors (91.1% vs 63.9%, P < .001). One year after ICU discharge, 67 of 195 ICU survivors died (34.4%) and, after adjustment for confounders, there were no significant differences in mortality when comparing IRRT and CRTT patients (34.9% vs 33.9%; P = .590), on HRQoL in both physical (41.9% vs 42.2%; P = .926) and mental dimensions (57.6% vs 56.6%; P = .340), and on the number of hospital readmissions and physical dependence. Conclusions: Our study suggests that among ICU survivors RRT modality (IRRT vs CRRT) in the ICU does not impact long-term outcomes after ICU discharge.

Hippocampal acetylcholinesterase activation induced by streptozotocin in mice is protected by an organotellurium compound without evidence of toxicity.

The cognitive deficit, which is like Alzheimer's disease and is associated with oxidative damage, may be induced by exposure to streptozotocin. This study aimed to evaluate if the tellurium-containing organocompound, 3j, 5'-arylchalcogeno-3-aminothymidine derivative, interferes with the effects of streptozotocin, as well as to investigate its toxicity in adult mice. Cognitive deficit was induced by two doses of streptozotocin (2.25 mg/kg/day, 48 h interval) intracerebroventricularly. After, the mice were subcutaneously treated with 3j (8.62 mg/kg/day) for 25 days. The effects were assessed by evaluating hippocampal and cortical acetylcholinesterase and behavioral tasks. 3j toxicity was investigated for 10 (0, 21.55, or 43.10 mg/kg/day) and 37 (0, 4.31, or 8.62 mg/kg/day) days by assessing biometric parameters and glucose and urea levels, and alanine aminotransferase activity in blood plasma. 3j exposure did not alter the behavioral alterations induced by streptozotocin exposure. On the other hand, 3j exposure normalized hippocampus acetylcholinesterase activity, which is enhanced by streptozotocin exposure. Toxicity evaluation showed that the administration of 3j for either 10 or 37 days did not cause harmful effects on the biometric and biochemical parameters analyzed. Therefore, 3j does not present any apparent toxicity and reverts acetylcholinesterase activity increase induced by streptozotocin in young adult mice.

Effectiveness of a home-based pulmonary rehabilitation maintenance programme: the Rehab2Life study protocol.

Pulmonary rehabilitation (PR) is the bedrock of non-pharmacological treatment for people with COPD. Nonetheless, it is well described in the literature that unless the patient changes his behaviour, the benefits of PR programmes will decline in six to twelve months after finishing the programme. Therefore, maintenance programmes can address the problem of PR programmes' effect loss over time.Community care units can provide multidisciplinary care in the current Portuguese primary health care context. These units have an interdisciplinary team that aims to develop competencies in COPD patients to self-manage the disease.This study aims to test the effectiveness of a 12-month home-based PR programme (Rehab2Life) compared to usual care through a single-blind randomised controlled trial with two parallel groups. The Rehab2Life programme includes two distinct phases. The first is an 8-week PR programme delivered to both groups, and the second is a PR maintenance programme delivered to the intervention group after the initial eight weeks. The control group receive the usual care and regular appointments. The primary outcome is functional capacity, and secondary outcomes are dyspnea, Health-Related Quality of Life (HRQoL), number of exacerbations, symptoms burden, anxiety and depression symptoms, and physical activity.We expect to observe that the home-based PR programme brings clinically relevant benefits to the participants at the end of the first eight weeks and that, at 12 months after the maintenance phase of the programme, benefits are less dissipated than in the control group. We expect to identify the characteristics of the patients who benefit the most from home-based programmes.The trial was registered on 7 April 2022 at ClinicalTrials.gov (NCT05315505).

Targeting phosphatidylinositol-3-kinase for inhibiting maxillary bone resorption.

Previous studies have suggested a role of phosphatidylinositol-3-kinase gamma (PI3Kγ) in bone remodeling, but the mechanism remains undefined. Here, we explored the contribution of PI3Kγ in the resorption of maxillary bone and dental roots using models of orthodontic tooth movement (OTM), orthodontic-induced inflammatory root resorption, and rapid maxillary expansion (RME). PI3Kγ-deficient mice (PI3Kγ-/- ), mice with loss of PI3Kγ kinase activity (PI3KγKD/KD ) and C57BL/6 mice treated with a PI3Kγ inhibitor (AS605240) and respective controls were used. The maxillary bones of PI3Kγ-/- , PI3KγKD/KD , and C57BL/6 mice treated with AS605240 showed an improvement of bone quality compared to their controls, resulting in reduction of the OTM and RME in all experimental groups. PI3Kγ-/- mice exhibited increased root volume and decreased odontoclasts counts. Consistently, the pharmacological blockade or genetic deletion of PI3K resulted in increased numbers of osteoblasts and reduction in osteoclasts during OTM. There was an augmented expression of Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (Alp), a reduction of interleukin-6 (Il-6), as well as a lack of responsiveness of receptor activator of nuclear factor kappa-Β (Rank) in PI3Kγ-/- and PI3KγKD/KD mice compared to control mice. The maxillary bones of PI3Kγ-/- animals showed reduced p-Akt expression. In vitro, bone marrow cells treated with AS605240 and cells from PI3Kγ-/- mice exhibited significant augment of osteoblast mineralization and less osteoclast differentiation. The PI3Kγ/Akt axis is pivotal for bone remodeling by providing negative and positive signals for the differentiation of osteoclasts and osteoblasts, respectively.

Aberrant Splicing Caused by a Novel VPS16 Variant Linked to Dystonia Type 30.

Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent involuntary muscle contractions, causing abnormal postures and/or repetitive movements. In this report, we identified a novel heterozygous splice-site variant in VPS16 (NM_022575.4:c.240+3G>C) in a patient with cervical and upper limb dystonia without other neurological or extra-neurological features. Analysis of patient's blood mRNA showed disruption of exon 3/intron 3 donor splice-site, leading to exon 3 skipping, which predictably results in a frameshift [p.(Ala48Valfs*14)]. Despite the scarcity of splice-affecting variants described in VPS16-related dystonia, our report contributes with the first fully characterized variant at the mRNA level.

Functional characterization of a novel PRRT2 variant found in a Portuguese patient with hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare autosomal-dominant form of migraine with aura. Three disease-causing genes have been identified for FHM: CACNA1A, ATP1A2 and SCN1A. However, not all families are linked to one of these three genes.PRRT2 variants were also commonly associated with HM symptoms; therefore, PRRT2 is hypothesized as the fourth gene causing FHM. PRRT2 plays an important role in neuronal migration, spinogenesis, and synapse mechanisms during development and calcium-dependent neurotransmitter release. We performed exome sequencing to unravel the genetic cause of migraine in one family, and a novel PRRT2 variant (c.938C > T;p.Ala313Val) was identified with further functional studies to confirm its pathogenicity. PRRT2-A313V reduced protein stability, led to protein premature degradation by the proteasome and altered the subcellular localization of PRRT2 from the plasma membrane (PM) to the cytoplasm. We identified and characterized for the first time in a Portuguese patient, a novel heterozygous missense variant in PRRT2 associated with HM symptoms. We suggest that PRRT2 should be included in the diagnosis of HM.

SOCS2 regulates alveolar bone loss in Aggregatibacter actinomycetemcomitans-induced periodontal disease.

INTRODUCTION: The role of suppressor of cytokine signaling 2 (SOCS2) in Aggregatibacter actinomycetemcomitans (Aa)-induced alveolar bone loss is unknown; thus, it was investigated in this study. METHODS: Alveolar bone loss was induced by infecting C57BL/6 wild-type (WT) and Socs2-knockout (Socs2-/-) mice with Aa. Bone parameters, bone loss, bone cell counts, the expression of bone remodeling markers, and cytokine profile were evaluated by microtomography, histology, qPCR, and/or ELISA. Bone marrow cells (BMC) from WT and Socs2-/- mice were differentiated in osteoblasts or osteoclasts for analysis of the expression of specific markers. RESULTS: Socs2-/- mice intrinsically exhibited irregular phenotypes in the maxillary bone and an increased number of osteoclasts. Upon Aa infection, SOCS2 deficiency resulted in the increased alveolar bone loss, despite decreased proinflammatory cytokine production, in comparison to the WT mice. In vitro, SOCS2 deficiency resulted in the increased osteoclasts formation, decreased expression of bone remodeling markers, and proinflammatory cytokines after Aa-LPS stimulus. CONCLUSIONS: Collectively, data suggest that SOCS2 is a regulator of Aa-induced alveolar bone loss by controlling the differentiation and activity of bone cells, and proinflammatory cytokines availability in the periodontal microenvironment and an important target for new therapeutic strategies. Thus, it can be helpful in preventing alveolar bone loss in periodontal inflammatory conditions.

Effect of increasing doses of chitosan to grazing beef steers on the relative population and transcript abundance of Archaea and cellulolytic and amylolytic bacterias.

This paper aims to investigate the influence of increasing chitosan doses on the relative proportion and abundance of cellulotytic, amylolytic bacteria, and Archaea transcripts for grazing cattle. Five rumen cannulated crossbread steers [3.6 months and 300 ± 25 kg body LW (live weight), mean ± standard deviation] were used in a 5 × 5 latin square design, randomly assigned to treatment sequence containing chitosan added to 0, 400, 800, 1200, or 1600 mg/kg concentrate. There was the effect of chitosan on the population of Fibrobacter succinogenes, Ruminococcus albus, and Archaea. The lowest population of these bacteria of 576.60 mg/kg DM (dry matter), 1010.40 mg/kg DM, and 634.80 mg/kg DM were noted when chitosan was added at levels of 3.87, 4.16, and 3.52. Except for Ruminococcus albus, which was not affected by increasing chitosan doses, supplementation of this additive in the concentrate quadratically increased the relative abundance of Fibrobacter succinogenes and Archaea Supplemental 740 mg CHI/kg concentrate for grazing steers receiving concentrate at 150 grams/100 kg LW is recommended to promote minimal effect on the relative population and abundance of cellulolytics and amylomatics and to restrict Archaea growth.

Evaluation of Single-Shade Composite Resin Color Matching on Extracted Human Teeth.

Introduction: Universal single-shade composite resins are characterized by a property that enables the creation of restorations that mimic tooth structure to the extent possible with fewer shades of color. Objectives: This study aimed to instrumentally and visually evaluate the color correspondence of two single-shade composite resins in extracted human teeth multishade composite resins. Methods: Upper central incisors and upper and/or lower molars with intact buccal surfaces were selected. The study consisted of a control group (n = 20): Z250 XT (3M ESPE) (G1) multishade composite resin in colors A1 to A4, and a test group (n = 20) divided further into two equal groups, consisting of single-shade composite resin Omnichroma (Tokuyama Dental) (G2) and single-shade composite resin Vittra APS Unique from (FGM) (G3). Instrumental evaluation was performed using a spectrophotometer, and visual evaluation was performed by three observers. Descriptive measurements related to the differences in color obtained through instrumental means were analyzed using mean and standard deviation, wherein the means were compared using ANOVA, applying the Bonferroni post hoc test. Results: A statistically significant difference was observed among the groups (G1, G2, and G3) (ANOVA: p < 0.001). For the visual assessment, regardless of the assessment group, 77.49% of the teeth were within the acceptable color-match classification, with the single-shade resins showing better correspondence than the multishade resins. Conclusion: Single-shade composite resins showed different color-matching results when compared to multishade resins, both in spectrophotometry and visual evaluations. Clinical Significance. Single-shade composite resins simplify the shade-selection process and are promising materials for use in dental practice.

[Schistosomiasis, soil-transmitted helminthiases and sanitation in Latin America and the Caribbean: a systematic reviewRelación entre la prevalencia de esquistosomiasis y geohelmintiasis y las condiciones sanitarias en América Latina y el Caribe: una revisión sistemática]. / Esquistossomose, geo-helmintíases e condições sanitárias na América Latina e Caribe: uma revisão sistemática.

Objective: To investigate the relationship between the prevalence of schistosomiasis and soil-transmitted helminthiasis with variables related to access to water, sanitation and solid waste in Latin American and Caribbean (LAC) countries. Method: A systematic review was performed in the LILACS, PubMed, Web of Science, and SciELO databases. Studies published between 1950 and August 2021, with an ecological design and a focus on population groups (states, municipalities and/or districts), having the prevalence of infection by Schistosoma mansoni, Ancylostoma sp., Necator americanus, Ascaris lumbricoides or Trichuris trichiura as primary variable and access to water, sewage and/or solid waste as explanatory variables were included. Open access articles with full text available in English, Spanish, or Portuguese were considered. The risk of bias and the quality of the studies were assessed according to the Joanna Briggs Institute manual. Results: Of 2 714 articles, nine were eligible, published between 1994 and 2021 and covering 22 LAC countries and 14 350 municipalities. All articles had moderate methodological quality. Environmental variables indicated an association between water supply and solid waste collection with schistosomiasis; water supply with ascariasis, trichuriasis and hookworm; and sewage with ascariasis and hookworm. Except for one article, which had regional coverage for LAC, all the others were developed in Brazil. Conclusion: There is a clear need to expand research on the association between household and collective health conditions and parasitic diseases for all endemic countries in LAC to support environmental strategies to control these diseases.

Objetivo: Investigar la relación entre la prevalencia de esquistosomiasis y geohelmintiasis y las variables de acceso al agua, el saneamiento y el manejo de residuos sólidos en los países de América Latina y el Caribe. Métodos: Se realizó una revisión sistemática en las bases de datos LILACS, PubMed, Web of Science y SciELO. Todos los artículos fueron de calidad metodológica moderada. Se incluyeron estudios publicados entre 1950 y agosto del 2021, con diseño ecológico y atención en agregados demográficos (estados, municipios o distritos), que tuvieran como resultado principal la prevalencia de infección por Schistosoma mansoni, Ancylostoma spp., Necator americanus, Ascaris lumbricoides o Trichuris trichiura y como variable explicativa el acceso al agua, el saneamiento y el manejo de residuos sólidos. Se analizaron artículos de texto completo y acceso libre en español, inglés o portugués. El riesgo de sesgo y la calidad de los estudios se evaluaron según las normas del manual del Instituto Joanna Briggs. Resultados: De los 2 714 artículos, hubo 9 que cumplieron con los requisitos establecidos; estos se publicaron entre 1994 y el 2021 y abarcaron 22 países y 14 350 municipios de América Latina y el Caribe. Las variables ambientales indicaron una relación del abastecimiento de agua y la recolección de residuos sólidos con la esquistosomiasis; del abastecimiento de agua con la ascariasis, la tricuriasis y la anquilostomiasis; y del saneamiento con la ascariasis y la anquilostomiasis. Con excepción de un artículo que abarcó la Región de América Latina y el Caribe, todos los demás se realizaron en Brasil. Conclusiones: Es evidente la necesidad de ampliar las investigaciones sobre la relación entre las condiciones sanitarias domésticas y colectivas y las enfermedades parasitarias en todos los países de América Latina y el Caribe donde son endémicas, con el fin de formular estrategias centradas en el medio ambiente para controlar esas enfermedades.

The management of osteoarthritis symptomatology through nanotechnology: a patent review.

Osteoarthritis is considered a degenerative joint disease that is characterised by inflammation, chronic pain, and functional limitation. The increasing development of nanotechnology in drug delivery systems has provided new ideas and methods for osteoarthritis therapy. This review aimed to evaluate patents that have developed innovations, therapeutic strategies, and alternatives using nanotechnology in osteoarthritis treatment. The results show patents deposited from 2015 to November 2021 in the online databases European Patent Office and World Intellectual Property Organisation. A total of 651 patents were identified for preliminary assessment and 16 were selected for full reading and discussion. The evaluated patents are focused on the intraarticular route, oral route, and topical route for osteoarthritis treatment. The intraarticular route presented a higher patent number, followed by the oral and topical routes, respectively. The development of new technologies allows us to envision a promising and positive future in osteoarthritis treatment.

Non-coding variants in VAMP2 and SNAP25 affect gene expression: potential implications in migraine susceptibility.

Migraine is a common and complex neurological disease potentially caused by a polygenic interaction of multiple gene variants. Many genes associated with migraine are involved in pathways controlling the synaptic function and neurotransmitters release. However, the molecular mechanisms underpinning migraine need to be further explored.Recent studies raised the possibility that migraine may arise from the effect of regulatory non-coding variants. In this study, we explored the effect of candidate non-coding variants potentially associated with migraine and predicted to lie within regulatory elements: VAMP2_rs1150, SNAP25_rs2327264, and STX1A_rs6951030. The involvement of these genes, which are constituents of the SNARE complex involved in membrane fusion and neurotransmitter release, underscores their significance in migraine pathogenesis. Our reporter gene assays confirmed the impact of at least two of these non-coding variants. VAMP2 and SNAP25 risk alleles were associated with a decrease and increase in gene expression, respectively, while STX1A risk allele showed a tendency to reduce luciferase activity in neuronal-like cells. Therefore, the VAMP2_rs1150 and SNAP25_rs2327264 non-coding variants affect gene expression, which may have implications in migraine susceptibility. Based on previous in silico analysis, it is plausible that these variants influence the binding of regulators, such as transcription factors and micro-RNAs. Still, further studies exploring these mechanisms would be important to shed light on the association between SNAREs dysregulation and migraine susceptibility.

Early Intervention Involving Specific Task-Environment-Participation (STEP) Protocol for Infants at Risk: A Feasibility Study.

Aims: To verify the feasibility and preliminary effects of the STEP protocol, an intervention based on specific motor skills, environmental factors and participation, in infants at biological risk.Methods: Twenty-eight at-risk infants (STEP Protocol = 14; Standard Intervention = 14), aged 3-9 months and at risk for developmental delay. The following outcomes were assessed: motor skills (Alberta Infant Motor Scale-AIMS); frequency and involvement of participation (Young Children's Participation and Environment Measure-YC-PEM), and home environment opportunities (Affordances in the Home Environment for Motor Development-AHEMD-IS). For both groups, interventions were provided by parents. The intervention for group was based on the following principles: (1) standard intervention: stimulation of motor skills; (2) STEP: stimulation of motor skills, participation, mother-infant interaction, environmental enrichment. A mean comparison test was applied to verify difference between groups after the intervention.Results: The protocol showed good retention and recruitment rates. The STEP group had significantly higher outcomes after intervention on the AIMS (p = 0.014); frequency (p = 0.02) and engagement (p = 0.03) in participation, when compared to standard intervention.Conclusions: The results showed that the STEP protocol is feasible, and presents better results compared to the standard intervention, which reinforces the importance of promoting participation, specific motor skills and family involvement.

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays.

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

Role of Suppressor of cytokine signaling 2 during the development and resolution of an experimental arthritis.

Rheumatoid arthritis(RA) is a debilitating chronic inflammatory disease. Suppressors of Cytokine Signaling(SOCS) proteins regulate homeostasis and pathogenesis in several diseases. The intersection between RA pathophysiology and SOCS2 is unclear. Herein, we investigated the roles of SOCS2 during the development of an experimental antigen-induced arthritis(AIA). In wild type mice, joint SOCS2 expression was reduced during AIA development. At the peak of inflammation, SOCS2-/- mice presented with reduced numbers of infiltrated cells in their joints. At the late phase of AIA, however, exhibited increased adhesion/infiltration of neutrophils, macrophages, CD4+-T cells, CD4+CD8+-T cells, and CD4-CD8--T cells associated with elevated IL-17 and IFN-γ levels, joint damage, proteoglycan loss, and nociception. SOCS2 deficiency resulted in lower numbers of apoptotic neutrophils and reduced efferocytosis. The present study demonstrated the vital role of SOCS2 during the development and resolution of an experimental RA model. Hence, this protein may be a novel therapeutic target for this disorder.

Clinical and Genetic Characterization of Brazilian Patients with Ataxia and Oculomotor Apraxia.

BACKGROUND: Ataxia with oculomotor apraxia (AOA) is characterized by early-onset cerebellar ataxia associated with oculomotor apraxia. AOA1, AOA2, AOA3, and AOA4 subtypes may present pathogenic variants in APTX, SETX, PIK3R5, and PNKP genes, respectively. Mutations in XRCC1 have been found to cause autosomal recessive spinocerebellar ataxia-26 (SCAR26) now considered AOA5. OBJECTIVES: To examine a cohort of Brazilians with autosomal recessive cerebellar ataxia plus oculomotor apraxia and determine the frequencies of AOA subtypes through genetic investigation. METHODS: We evaluated clinical, biomarkers, electrophysiological, and radiological findings of 52 patients with AOA phenotype and performed a genetic panel including APTX, SETX, PIK3R5, PNKP, and XRCC1. RESULTS: We found pathogenic variants in SETX (15 patients), PNKP (12), and APTX (5). No mutations in PIK3R5 or XRCC1 were identified. CONCLUSIONS: AOA2 and AOA4 were the most common forms of AOA in Brazil. Mutations in PIK3R5 and XRCC1 were not part of this genetic spectrum. © 2022 International Parkinson and Movement Disorder Society.