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Life Science Alliance Aug 2024In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is...
In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.
Topics: Dynamins; Mitochondrial Dynamics; Humans; Single-Domain Antibodies; Mitochondria; Proteomics; Animals; Protein Binding; HeLa Cells; Mitochondrial Proteins
PubMed: 38816213
DOI: 10.26508/lsa.202402608 -
RMD Open May 2024To compare the effectiveness of a strategy administering baricitinib versus one using TNF-inhibitors (TNFi) in patients with rheumatoid arthritis (RA) after conventional... (Randomized Controlled Trial)
Randomized Controlled Trial Comparative Study
PERFECTRA: a pragmatic, multicentre, real-life study comparing treat-to-target strategies with baricitinib versus TNF inhibitors in patients with active rheumatoid arthritis after failure on csDMARDs.
OBJECTIVE
To compare the effectiveness of a strategy administering baricitinib versus one using TNF-inhibitors (TNFi) in patients with rheumatoid arthritis (RA) after conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) failure in a real-life treat-to-target (T2T) setting.
METHODS
Patients with biological and targeted synthetic DMARD (b/tsDMARD) naïve RA with disease duration ≤5 years without contraindications to b/tsDMARD were randomised to either TNFi or baricitinib when csDMARD failed to achieve disease control in a T2T setting. Changes in clinical and patient-reported outcome measures (PROMs) were assessed at 12-week intervals for 48 weeks. The primary endpoint was non-inferiority, with testing for superiority if non-inferiority is demonstrated, of baricitinib strategy in the number of patients achieving American College of Rheumatology 50 (ACR50) response at 12 weeks. Secondary endpoints included 28-joint count Disease Activity Score with C reactive protein (DAS28-CRP) <2.6, changes in PROMs and radiographic progression.
RESULTS
A total of 199 patients (TNFi, n=102; baricitinib, n=97) were studied. Both study groups were similar. Baricitinib was both non-inferior and superior in achieving ACR50 response at week 12 (42% vs 20%). Moreover, 75% of baricitinib patients achieved DAS28-CRP <2.6 at week 12 compared with 46% of TNFi patients. On secondary outcomes throughout the duration of the study, the baricitinib strategy demonstrated comparable or better outcomes than TNFi strategy. Although not powered for safety, no unexpected safety signals were seen in this relatively small group of patients.
CONCLUSION
Up to present, in a T2T setting, patients with RA failing csDMARDs have two main strategies to consider, Janus Kinases inhibitor versus bDMARDs (in clinical practice, predominantly TNFi). The PERFECTRA study suggested that starting with baricitinib was superior over TNFi in achieving response at 12 weeks and resulted in improved outcomes across all studied clinical measures and PROMs throughout the study duration in these patients.
Topics: Humans; Purines; Sulfonamides; Arthritis, Rheumatoid; Pyrazoles; Azetidines; Male; Female; Middle Aged; Antirheumatic Agents; Aged; Treatment Outcome; Tumor Necrosis Factor Inhibitors; Treatment Failure; Adult; Patient Reported Outcome Measures; Severity of Illness Index
PubMed: 38816210
DOI: 10.1136/rmdopen-2024-004291 -
The Journal of Biological Chemistry May 2024ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice...
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
PubMed: 38815867
DOI: 10.1016/j.jbc.2024.107418 -
The Journal of Biological Chemistry May 2024Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of...
Infiltration of monocyte-derived cells to sites of infection and injury is greater in males than in females, due in part, to increased chemotaxis, the process of directed cell movement toward a chemical signal. The mechanisms governing sexual dimorphism in chemotaxis are not known. We hypothesized a role for the store-operated calcium entry (SOCE) pathway in regulating chemotaxis by modulating leading and trailing edge membrane dynamics. We measured the chemotactic response of bone marrow derived macrophages (BMDMs) migrating towards complement component 5a (C5a). Chemotactic ability was dependent on sex and inflammatory phenotype (M0, M1, M2), and correlated with SOCE. Notably, females exhibited a significantly lower magnitude of SOCE than males. When we knocked out the SOCE gene, stromal interaction molecule 1 (STIM1), it eliminated SOCE and equalized chemotaxis across both sexes. Analysis of membrane dynamics at the leading and trailing edges showed that STIM1 influences chemotaxis by facilitating retraction of the trailing edge. Using BTP2 to pharmacologically inhibit SOCE mirrored the effects of STIM1 knockout, demonstrating a central role of STIM/Orai-mediated calcium signaling. Importantly, by monitoring the recruitment of adoptively transferred monocytes in an in vivo model of peritonitis, we show that increased infiltration of male monocytes during infection is dependent on STIM1. These data support a model in which STIM1-dependent SOCE is necessary and sufficient for mediating the sex difference in monocyte recruitment and macrophage chemotactic ability by regulating trailing edge dynamics.
PubMed: 38815866
DOI: 10.1016/j.jbc.2024.107422 -
JCI Insight May 2024The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells...
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
PubMed: 38815134
DOI: 10.1172/jci.insight.174329 -
ELife May 2024Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.
Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.
Topics: Eye Proteins; Humans; Wnt Signaling Pathway; Nerve Tissue Proteins; Retina; Animals; Protein Binding
PubMed: 38814685
DOI: 10.7554/eLife.98933 -
ELife May 2024Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we...
Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.
Topics: Humans; SARS-CoV-2; tRNA Methyltransferases; Proteolysis; RNA, Transfer; COVID-19; Coronavirus 3C Proteases; HEK293 Cells; Virus Replication; Viral Nonstructural Proteins
PubMed: 38814682
DOI: 10.7554/eLife.90316 -
Canadian Association of Radiologists... May 2024Positron emission tomography/magnetic resonance (PET/MR) imaging has gone through major hardware improvements in recent years, making it a reliable state-of-the-art... (Review)
Review
Positron emission tomography/magnetic resonance (PET/MR) imaging has gone through major hardware improvements in recent years, making it a reliable state-of-the-art hybrid modality in clinical practice. At the same time, image reconstruction, attenuation correction, and motion correction algorithms have significantly evolved to provide high-quality images. Part I of the current review discusses technical basics, pre-clinical applications, and clinical applications of PET/MR in radiation oncology and head and neck imaging. PET/MR offers a broad range of advantages in preclinical and clinical imaging. In the preclinic, small and large animal-dedicated devices were developed, making PET/MR capable of delivering new insight into animal models in diseases and facilitating the development of methods that inform clinical PET/MR. Regarding PET/MR's clinical applications in radiation medicine, PET and MR already play crucial roles in the radiotherapy process. Their combination is particularly significant as it can provide molecular and morphological characteristics that are not achievable with other modalities. In addition, the integration of PET/MR information for therapy planning with linear accelerators is expected to provide potentially unique biomarkers for treatment guidance. Furthermore, in clinical applications in the head and neck region, it has been shown that PET/MR can be an accurate modality in head and neck malignancies for staging and resectability assessment. Also, it can play a crucial role in diagnosing residual or recurrent diseases, reliably distinguishing from oedema and fibrosis. PET/MR can furthermore help with tumour characterization and patient prognostication. Lastly, in head and neck carcinoma of unknown origin, PET/MR, with its diagnostic potential, may obviate multiple imaging sessions in the near future.
PubMed: 38813998
DOI: 10.1177/08465371241255903 -
Turkish Journal of Medical Sciences 2023Neuropathic pain (NP) is a type of chronic pain usually caused by damage to the somatosensory system. Bioactive antioxidant compounds, such as curcumin and ginger, are...
BACKGROUND/AIM
Neuropathic pain (NP) is a type of chronic pain usually caused by damage to the somatosensory system. Bioactive antioxidant compounds, such as curcumin and ginger, are widely preferred in the treatment of NP. However, the ingredient-based mechanism that underlies their pain-relieving activity remains unknown. The aim of this study was to investigate the therapeutic effects of trans-[6]-Shogaol and [6]-Gingerol active ingredients of the Roscoe extract on the spinal cord and cortex in the neuroinflammatory pathway in rats with experimental sciatic nerve injury.
MATERIALS AND METHODS
Forty-six volatile phenolic components were identified in ginger samples using gas chromatography-mass spectrometry analysis. Thirty 3-month-old male 250-300 g Wistar Albino rats were divided into three groups as (i) sham, (ii) chronic constriction injury (CCI), and (iii) CCI+ginger. NP was induced using the CCI model. A ginger extract treatment enriched with trans-[6]-shogaol and [6]-gingerol active ingredients was administered by gavage at 200 mg/kg/day for 7 days. On the 14th day of the experiment, locomotor activity was evaluated in open field and hyperalgesia in tail flick tests.
RESULTS
In behavioural experiments, a significant decrease was observed in the CCI group compared to the sham group, while a significant increase was observed in the CCI+ginger group compared to the CCI group (p < 0.05). In the spinal cord and cortex tissues, there was a significant increase in the TNF-α, IL-1β, and IL-18 neuroinflammation results of the CCI group compared to the sham group, while there was a significant decrease in the CCI+ginger group compared to the CCI group.
CONCLUSION
In this study, ginger treatment was shown to have a therapeutic effect on neuroinflammation against sciatic nerve damage.
Topics: Animals; Fatty Alcohols; Catechols; Neuralgia; Rats; Male; Rats, Wistar; Zingiber officinale; Disease Models, Animal; Cytokines; Plant Extracts; Sciatic Nerve; Spinal Cord
PubMed: 38813490
DOI: 10.55730/1300-0144.5728 -
Central-European Journal of Immunology 2024The flow cytometry method could support physicians' decisions in the diagnosis and treatment monitoring of immunodeficient patients. Most clinical recommendations are...
The flow cytometry method could support physicians' decisions in the diagnosis and treatment monitoring of immunodeficient patients. Most clinical recommendations are focused on the search for alterations in T- and B-lymphocyte subsets, less commonly natural killer (NK) cells and granulocytes. While reference values for clinically meaningful lymphocyte subsets have been published ubiquitously among numerous countries, we have not found significant data for a population of adult Polish habitats; thus we determined reference values for T, B, and NK subsets according to sex and age. The female group showed a higher percentage of lymphocytes (CD45), T helper lymphocytes with a higher absolute count, as well as CD4/CD8 ratio, marginal zone-like B cells, class-switched B cells, and CD21 B cells than the male group. The male group was found to have elevated percentages of naïve B lymphocytes, transitional B cells, and plasmablasts. A weak positive correlation with age was found among double positive T lymphocytes, natural killer T cells (NKT) lymphocytes, and CD21 B cells. A negative correlation with age for double negative T lymphocytes, marginal zone-like B cells, and plasmablasts was noted. The results indicated the importance of creating distinct reference ranges regarding sex and age concerning immunophenotype.
PubMed: 38812608
DOI: 10.5114/ceji.2024.136371