Novel prognostic nomogram for predicting recurrence-free survival in medullary thyroid carcinoma.

AIMS: Recently, there have been attempts to improve prognostication and therefore better guide treatment for patients with medullary thyroid carcinoma (MTC). In 2022, the International MTC Grading System (IMTCGS) was developed and validated using a multi-institutional cohort of 327 patients. The aim of the current study was to build upon the findings of the IMTCGS to develop and validate a prognostic nomogram to predict recurrence-free survival (RFS) in MTC. METHODS AND RESULTS: Data from 300 patients with MTC from five centres across the USA, Europe, and Australia were used to develop a prognostic nomogram that included the following variables: age, sex, AJCC stage, tumour size, mitotic count, necrosis, Ki67 index, lymphovascular invasion, microscopic extrathyroidal extension, and margin status. A process of 10-fold cross-validation was used to optimize the model's performance. To assess discrimination and calibration, the area-under-the-curve (AUC) of a receiver operating characteristic (ROC) curve, concordance-index (C-index), and dissimilarity index (D-index) were calculated. Finally, the model was externally validated using a separate cohort of 87 MTC patients. The model demonstrated very strong performance, with an AUC of 0.94, a C-index of 0.876, and a D-index of 19.06. When applied to the external validation cohort, the model had an AUC of 0.9. CONCLUSIONS: Using well-established clinicopathological prognostic variables, we developed and externally validated a robust multivariate prediction model for RFS in patients with resected MTC. The model demonstrates excellent predictive capability and may help guide decisions on patient management. The nomogram is freely available online at https://nomograms.shinyapps.io/MTC_ML_DFS/.

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing.

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

Zebrafish CCNF and FUS Mediate Stress-Specific Motor Responses.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the cyclin F (CCNF) and fused in sarcoma (FUS) genes have been associated with ALS pathology. In this study, we aimed to investigate the functional role of CCNF and FUS in ALS by using genome editing techniques to generate zebrafish models with genetic disruptions in these genes. Sequence comparisons showed significant homology between human and zebrafish CCNF and FUS proteins. We used CRISPR/Cas9 and TALEN-mediated genome editing to generate targeted disruptions in the zebrafish ccnf and fus genes. Ccnf-deficient zebrafish exhibited abnormal motor neuron development and axonal outgrowth, whereas Fus-deficient zebrafish did not exhibit developmental abnormalities or axonopathies in primary motor neurons. However, Fus-deficient zebrafish displayed motor impairments in response to oxidative and endoplasmic reticulum stress. The Ccnf-deficient zebrafish were only sensitized to endoplasmic reticulum stress, indicating that ALS genes have overlapping as well as unique cellular functions. These zebrafish models provide valuable platforms for studying the functional consequences of CCNF and FUS mutations in ALS pathogenesis. Furthermore, these zebrafish models expand the drug screening toolkit used to evaluate possible ALS treatments.

Lipid-Based Nanoparticles for Drug/Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development.

Over the past decade, the therapeutic potential of nanomaterials as novel drug delivery systems complementing conventional pharmacology has been widely acknowledged. Among these nanomaterials, lipid-based nanoparticles (LNPs) have shown remarkable pharmacological performance and promising therapeutic outcomes, thus gaining substantial interest in preclinical and clinical research. In this review, we introduce the main types of LNPs used in drug formulations such as liposomes, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers, and lipid polymer hybrid nanoparticles, focusing on their main physicochemical properties and therapeutic potential. We discuss computational studies and modeling techniques to enhance the understanding of how LNPs interact with therapeutic cargo and to predict the potential effectiveness of such interactions in therapeutic applications. We also analyze the benefits and drawbacks of various LNP production techniques such as nanoprecipitation, emulsification, evaporation, thin film hydration, microfluidic-based methods, and an impingement jet mixer. Additionally, we discuss the major challenges associated with industrial development, including stability and sterilization, storage, regulatory compliance, reproducibility, and quality control. Overcoming these challenges and facilitating regulatory compliance represent the key steps toward LNP's successful commercialization and translation into clinical settings.

A novel prognostic nomogram for predicting survival in diffuse pleural mesothelioma.

Recent advances in the management of diffuse pleural mesothelioma (DPM) have increased interest in prognostication and risk stratification on the basis that maximum benefit of combination immunotherapy appears to be seen in patients who otherwise would have the worst prognosis. Various grading schemes have been proposed, including the recently published Mesothelioma Weighted Grading Scheme (MWGS). However, predictive modelling using deep learning algorithms is increasingly regarded as the gold standard in prognostication. We therefore sought to develop and validate a prognostic nomogram for DPM. Data from 369 consecutive patients with DPM were used as independent training and validation cohorts to develop a prognostic tool that included the following variables: age, sex, histological type, nuclear atypia, mitotic count, necrosis, and BAP1 immunohistochemistry. Patients were stratified into four risk groups to assess model discrimination and calibration. To assess discrimination, the area-under-the-curve (AUC) of a receiver-operator-curve (ROC), concordance-index (C-index), and dissimilarity index (D-index) were calculated. Based on the 5-year ROC analysis, the AUC for our model was 0.75. Our model had a C-index of 0.67 (95% CI 0.53-0.79) and a D-index of 2.40 (95% CI 1.69-3.43). Our prognostic nomogram for DPM is the first of its kind, incorporates well established prognostic markers, and demonstrates excellent predictive capability. As these factors are routinely assessed in most pathology laboratories, it is hoped that this model will help inform prognostication and difficult management decisions, such as patient selection for novel therapies. This nomogram is now freely available online at: https://nomograms.shinyapps.io/Meso_Cox_ML/.

A Critical Assessment of Current Grading Schemes for Diffuse Pleural Mesothelioma With a Proposal for a Novel Mesothelioma Weighted Grading Scheme (MWGS).

Although there is early support for schemes based on nuclear grade, necrosis and mitotic rate, there is currently no widely implemented grading system for diffuse pleural mesothelioma (DPM). We investigated current systems and propose a novel Mesothelioma Weighted Grading Scheme (MWGS). The MWGS assigns weighted scores from 0 to 10 based on age (≤74, >74 yrs: 0,1); histologic type (epithelioid, biphasic, sarcomatoid: 0,1,2); necrosis (absent, present: 0,2); mitotic count per 2 mm2 (≤1, 2 to 4, ≥5: 0,1,2); nuclear atypia (mild, moderate, severe: 0,1,2); and BRCA1-associated protein 1 (BAP1) expression (lost, retained: 0,1). A score of 0 to 3 is low grade, 4 to 6 intermediate grade, and 7 to 10 high grade. In 369 consecutive DPMs, median survival was 17.1, 10.1, and 4.1 months for low, intermediate, and high grades (P<0.0001). A progressive increase in score correlated with worsening overall survival (P<0.0001). Interobserver concordance was substantial (κ=0.588), with assessment of nuclear grade being the most subjective parameter (κ=0.195). We compared the MWGS to the 2-tiered system discussed in the World Health Organization (WHO) fifth edition. The WHO system predicted median survival in epithelioid (median 18.0 vs. 11.3 mo, P=0.003) and biphasic (16.2 vs. 4.2 mo, P=0.002), but not sarcomatoid DPM (5.4 vs. 4.7 mo, P=0.407). Interestingly, the WHO grading system was prognostic in cases with BAP1 loss (median survival 18.7 vs. 10.4 mo, P<0.0001), but not retained BAP1 expression (8.9 vs. 6.2 mo, P=0.061). In conclusion, the WHO scheme has merit in epithelioid/biphasic and BAP1-deficient DPM, however, the MWGS can be used for risk stratification of all DPMs, regardless of histologic subtype and BAP1 status.

"STRESSED OUT": The role of FUS and TDP-43 in amyotrophic lateral sclerosis.

Mutations in fused-in-sarcoma (FUS) and TAR DNA binding protein-43 (TDP-43; TARDBP) are known to cause the severe adult-onset neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Proteinopathy caused by cellular stresses such as endoplasmic reticulum (ER) stress, oxidative stress, mitochondrial stress and proteasomal stress and the formation of stress granules (SGs), cytoplasmic aggregates and inclusions is a hallmark of ALS. FUS and TDP-43, which are DNA/RNA binding proteins that regulate transcription, RNA homeostasis and protein translation are implicated in ALS proteinopathy. Disease-causing mutations in FUS and TDP-43 cause sequestration of these proteins and their interacting partners in the cytoplasm, which leads to aggregation. This mislocalization and formation of aggregates and SGs is cytotoxic and a contributor to neuronal death. We explore how loss-of-nuclear-function and gain-of-cytoplasmic function mechanisms that affect FUS and TPD-43 localization can generate a 'stressed out' neuronal pathology and proteinopathy that drives ALS progression.

Spatial and Temporal Control of CRISPR-Cas9-Mediated Gene Editing Delivered via a Light-Triggered Liposome System.

The CRISPR-Cas9 and related systems offer a unique genome-editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with the CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with the CRISPR-Cas9 system. We demonstrated its efficient protein release by respectively assessing the targeted knockout of the eGFP gene in human HEK293/GFP cells and the TNFAIP3 gene in TNFα-induced HEK293 cells. We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). These findings indicate that light-triggered liposomes may have new options for precise control of CRISPR-Cas9 release and editing.

Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.

Impact of transrectal prostate needle biopsy on erectile function: results of power Doppler ultrasonography of the prostate.

We evaluated the impact of transrectal prostate needle biopsy (TPNB) on erectile function and on the prostate and bilateral neurovascular bundles using power Doppler ultrasonography imaging of the prostate. The study consisted of 42 patients who had undergone TPNB. Erectile function was evaluated prior to the biopsy, and in the 3rd month after the biopsy using the first five-item version of the International Index of Erectile Function (IIEF-5). Prior to and 3 months after the biopsy, the resistivity index of the prostate parenchyma and both neurovascular bundles was measured. The mean age of the men was 64.2 (47-78) years. Prior to TPNB, 10 (23.8%) patients did not have erectile dysfunction (ED) and 32 (76.2%) patients had ED. The mean IIEF-5 score was 20.8 (range: 2-25) prior to the biopsies, and the mean IIEF-5 score was 17.4 (range: 5-25; p < 0.001) after 3 months. For patients who were previously potent in the pre-biopsy period, the ED rate was 40% (n = 4/10) at the 3rd month evaluation. In these patients, all the resistivity index values were significantly decreased. Our results showed that TPNB may lead to an increased risk of ED. The presence of ED in men after TPNB might have an organic basis.

TrpA1 regulates thermal nociception in Drosophila.

Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca²âº channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca²âº signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide "pain" gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.