Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation.

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Bacterial and fungal superinfections are detected at higher frequency in critically ill patients affected by SARS CoV-2 infection than negative patients and are associated to a worse outcome.

Patients with viral infections are at higher risk to acquire bacterial and fungal superinfections associated with a worse prognosis. We explored this critical point in the setting of patients with severe COVID-19 disease. The study included 1911 patients admitted to intensive care unit (ICU) during a 2-year study period (March 2020-March 2022). Of them, 713 (37.3%) were infected with SARS-CoV-2 and 1198 were negative (62.7%). Regression analysis was performed to determine risk factors associated with the presence of bacterial and/or fungal superinfections in SARS-CoV-2 patients and to evaluate predictors of ICU mortality. Of the 713 patients with SARS-CoV-2 infection, 473 (66.3%) had respiratory and/or bloodstream bacterial and/or fungal superinfections, while of the 1198 COVID-19-negative patients, only 369 (30%) showed respiratory and/or bloodstream bacterial and/or fungal superinfections (p < 0.0001). Baseline characteristics of COVID-19 patients included a median age of 66 (interquartile range [IQR], 58-73), a predominance of males (72.7%), and the presence of a BMI higher than 24 (median 26; IQR, 24.5-30.4). Seventy-four percent (527, 73.9%) had one or more comorbidities and 135 (18.9%) of them had received previous antibiotic therapy. Furthermore, most of them (473, 66.3%) exhibited severe radiological pictures and needed invasive mechanical ventilation. Multivariate logistic regression analysis showed that 1 unit increment in BMI rises the risk of bacterial and/or fungal superinfections acquisition by 3% and 1-day increment in ICU stays rises the risk of bacterial and/or fungal superinfections acquisition by 11%. Furthermore, 1-day increment in mechanical ventilation rises the risk of bacterial and/or fungal superinfection acquisition by 2.7 times. Furthermore, patients with both bacterial and fungal infections had a significantly higher mortality rate than patients without superinfections (45.8% vs. 26.2%, p < 0.0001). Therefore, bacterial and fungal superinfections are frequent in COVID-19 patients admitted to ICU and their presence is associated with a worse outcome. This is an important consideration for targeted therapies in critically ill SARS-CoV-2 infected patients to improve their clinical course.

Cystatin B-deficiency triggers ectopic histone H3 tail cleavage during neurogenesis.

Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency.

The Feasibility and Diagnostic Adequacy of PD-L1 Expression Analysis Using the Cytoinclusion Technique in Bladder Cancer: A Prospective Single-Center Study.

Background: Programmed death-ligand 1 (PD-L1) expression has been recognized as a potential biomarker for various cancers, yet its diagnostic and prognostic significance in urothelial bladder cancer (BCa) requires further investigation. Methods: In this prospective single-center study, we aimed to assess the feasibility and diagnostic adequacy of PD-L1 expression analysis using cytoinclusion in BCa patients. We enrolled consecutive patients undergoing endoscopic transurethral resection of bladder tumor (TURBT), repeat TURBT, or robot-assisted radical cystectomy. Urinary and tissue specimens were collected from these patients for cytoinclusion and histopathological analysis to evaluate PD-L1 expression. Results: Out of 29 patients, PD-L1 expression was detected from cytoinclusion in 42.8% (3 out of 7), 10% (1 out of 10), and 66.8% (8 out of 12) of patients with negative/papilloma, low-grade, and high-grade tumors, respectively. Conversely, histopathological analysis identified PD-L1 expression in 57.2% (4 out of 7), 30% (3 out of 10), and 83.3% (10 out of 12) of patients with negative/papilloma, low-grade, and high-grade tumors, respectively. The diagnostic concordance between cytoinclusion and histopathology was 85.7%, 80%, and 83.3% in patients with negative/papilloma, low-grade, and high-grade tumors, respectively. Conclusions: Our study underscores the promise of cytoinclusion as a minimally invasive method for quantifying urinary PD-L1 percentages. This approach could serve as both a potential prognostic and diagnostic indicator, easily obtainable from urine samples. Standardizing this technique could facilitate its widespread use as a valuable tool.

Placenta Accreta Spectrum Disorders: How to reduce maternal transfusion? A center experience on extraperitoneal retrograde hysterectomy.

OBJECTIVE: Placenta Accreta Spectrum disorders (PASd) refer to the range of pathologic adherence of placenta associated with high maternal morbidity and mortality due to severe and sometimes life-threatening hemorrhage at the time of delivery. The aim of this study is to describe the surgical technique of extraperitoneal retrograde hysterectomy, which has allowed a reduction of blood transfusions compared to patients who underwent classical post-partum hysterectomy. STUDY DESIGN: We collected data from twelve patients with antenatal diagnosis of PASd treated between 2018 and 2021 with an extra-peritoneal hysterectomy using a posterior retrograde approach and we compared them to patients who underwent classical hysterectomy for suspected PASd, treated between 2007 and 2017. RESULTS: The classical hysterectomy group presented a higher frequency of blood and plasma transfusion compared to the extraperitoneal hysterectomy group. In particular, classical hysterectomy resulted in an independent risk factor for transfusion, with an increment of 6.6 times of risk. CONCLUSION: Even if future studies are required, we think that extraperitoneal hysterectomy could be a safe option in case of PASd, considering that classical hysterectomy compared to this approach increases, in our population, the risk of blood and plasma transfusion.

Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo.

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1ß, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.

The Role of Checkpoint Inhibitor Expression Directly on Exfoliated Cells from Bladder Cancer: A Narrative Review.

Bladder cancer (BCa) is a common type of cancer that affects the urinary bladder. The early detection and management of BCa is critical for successful treatment and patient outcomes. In recent years, researchers have been exploring the use of biomarkers as a non-invasive and effective tool for the detection and monitoring of BCa. One such biomarker is programmed death-ligand 1 (PD-L1), which is expressed on the surface of cancer cells and plays a crucial role in the evasion of the immune system. Studies have shown that the PD-L1 expression is higher in BCa tumors than in healthy bladder tissue. Additionally, PD-L1 expression might even be detected in urine samples in BCa patients, in addition to the examination of a histological sample. The technique is being standardized and optimized. We reported how BCa patients had higher urinary PD-L1 levels than controls by considering BCa tumors expressing PD-L1 in the tissue specimen. The expression of PD-L1 in urinary BCa cells might represent both a diagnostic and a prognostic tool, with the perspective that the PD-L1 expression of exfoliate urinary cells might reveal and anticipate eventual BCa recurrence or progression. Further prospective and longitudinal studies are needed to assess the expression of PD-L1 as a biomarker for the monitoring of BCa patients. The use of PD-L1 as a biomarker for the detection and monitoring of BCa has the potential to significantly improve patient outcomes by allowing for earlier detection and more effective management of the disease.

Schmidt camera correctors for large telescopes.

Designing large Schmidt cameras using the polynomial expansion may yield a not-perfect spherical aberration correction. We indicate an alternative method to design the corrector, whose integration in a software package is in progress. The shape of the corrector can be determined with a simple recursive method. The incidence point can also be calculated for any ray, as well as the corresponding unitary vector normal to the surface.

Nuclear Import Receptors Directly Bind to Arginine-Rich Dipeptide Repeat Proteins and Suppress Their Pathological Interactions.

Nuclear import receptors, also called importins, mediate nuclear import of proteins and chaperone aggregation-prone cargoes (e.g., neurodegeneration-linked RNA-binding proteins [RBPs]) in the cytoplasm. Importins were identified as modulators of cellular toxicity elicited by arginine-rich dipeptide repeat proteins (DPRs), an aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanistically, the link between importins and arginine-rich DPRs remains unclear. Here, we show that arginine-rich DPRs (poly-GR and poly-PR) bind directly to multiple importins and, in excess, promote their insolubility and condensation. In cells, poly-GR impairs Impα/ß-mediated nuclear import, including import of TDP-43, an RBP that aggregates in C9orf72-ALS/FTD patients. Arginine-rich DPRs promote phase separation and insolubility of TDP-43 in vitro and in cells, and this pathological interaction is suppressed by elevating importin concentrations. Our findings suggest that importins can decrease toxicity of arginine-rich DPRs by suppressing their pathological interactions.

Human Ovarian Cancer Tissue Exhibits Increase of Mitochondrial Biogenesis and Cristae Remodeling.

Ovarian cancer (OC) is the most lethal gynecologic cancer characterized by an elevated apoptosis resistance that, potentially, leads to chemo-resistance in the recurrent disease. Mitochondrial oxidative phosphorylation was found altered in OC, and mitochondria were proposed as a target for therapy. Molecular evidence suggests that the deregulation of mitochondrial biogenesis, morphology, dynamics, and apoptosis is involved in carcinogenesis. However, these mitochondrial processes remain to be investigated in OC. Eighteen controls and 16 OC tissues (serous and mucinous) were collected. Enzymatic activities were performed spectrophotometrically, mitochondrial DNA (mtDNA) content was measured by real-time-PCR, protein levels were determined by Western blotting, and mitochondrial number and structure were measured by electron microscopy. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, and principal component analysis (PCA). We found, in OC, that increased mitochondrial number associated with increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mitochondrial transcription factor A (TFAM) protein levels, as well as mtDNA content. The OC mitochondria presented an increased maximum length, as well as reduced cristae width and junction diameter, associated with increased optic atrophy 1 protein (OPA1) and prohibitin 2 (PHB2) protein levels. In addition, in OC tissues, augmented cAMP and sirtuin 3 (SIRT3) protein levels were observed. PCA of the 25 analyzed biochemical parameters classified OC patients in a distinct group from controls. We highlight a "mitochondrial signature" in OC that could result from cooperation of the cAMP pathway with the SIRT3, OPA1, and PHB2 proteins.