In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

Fazirsiran for Adults with Alpha-1 Antitrypsin Deficiency Liver Disease: A Phase 2 Placebo Controlled Trial (SEQUOIA).

BACKGROUND & AIMS: Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT mRNA, reducing deleterious protein synthesis. METHODS: This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25/100/200 mg. The primary endpoint was percentage change in serum Z-AAT concentration from baseline to Week 16. Patients with fibrosis on baseline liver biopsy received treatment on Day 1, Week 4, and then every 12 weeks, and had a second liver biopsy at or after Weeks 48, 72, or 96. Patients without fibrosis received two doses on Day 1 and Week 4. RESULTS: At Week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83% and -94% with fazirsiran 25/100/200 mg, respectively, versus placebo (all P< .0001). Efficacy was sustained through Week 52. At post-dose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histological reduction from baseline in hepatic globule burden. Portal inflammation improved in 5/12 and 0/8 patients with baseline score >0 in the fazirsiran and placebo groups, respectively. Histological METAVIR score improved by >1 point in 7/14 and 3/8 patients with fibrosis >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation and pulmonary function tests remained stable. CONCLUSIONS: Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose dependent manner and reduced hepatic globule burden (NCT03945292).

Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.

Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms.

Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present DeepFinder, a computational procedure that uses artificial neural networks to simultaneously localize multiple classes of macromolecules. Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase-oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms.

Assessing the rebound phenomenon in different myopia control treatments: A systematic review.

PURPOSE: To review the rebound effect after cessation of different myopia control treatments. METHODS: A systematic review that included full-length randomised controlled studies (RCTs), as well as post-hoc analyses of RCTs reporting new findings on myopia control treatments rebound effect in two databases, PubMed and Web of Science, was performed according to the PRISMA statement. The search period was between 15 June 2023 and 30 June 2023. The Cochrane risk of bias tool was used to analyse the quality of the selected studies. RESULTS: A total of 11 studies were included in this systematic review. Unifying the rebound effects of all myopia control treatments, the mean rebound effect for axial length (AL) and spherical equivalent refraction (SER) were 0.10 ± 0.07 mm [-0.02 to 0.22] and -0.27 ± 0.2 D [-0.71 to -0.03] after 10.2 ± 7.4 months of washout, respectively. In addition, spectacles with highly aspherical lenslets or defocus incorporated multiple segments technology, soft multifocal contact lenses and orthokeratology showed lower rebound effects compared with atropine and low-level light therapy, with a mean rebound effect for AL and SER of 0.04 ± 0.04 mm [0 to 0.08] and -0.13 ± 0.07 D [-0.05 to -0.2], respectively. CONCLUSIONS: It appears that the different treatments for myopia control produce a rebound effect after their cessation. Specifically, optical treatments seem to produce less rebound effect than pharmacological or light therapies. However, more studies are required to confirm these results.

Resilience and psychological factors among dentistry students who received face-to-face lectures during the COVID-19 pandemic.

BACKGROUND: This research evaluated whether the relationships between factors of resilience, self-esteem, depression, and anxiety in dental students with changes in teaching and learning methods. We also studied the psychological impact of face-to-face lectures during the COVID-19 pandemic. METHODS: This cross-sectional descriptive study used Google Forms to collect data with the Rosenberg Self-Esteem Scale (RSE), Connor-Davidson Risk Resilience Scale (CD-RISC), Beck Anxiety Inventory (BAI), and Beck Depression Inventory (BDI and BDI-II). An open-ended question was also asked about important learning difficulties. RESULTS: The analysis revealed very high levels of resilience (30.23 ± 5.84), self-esteem in the normal range (29.08 ± 4.03), minimal depression levels (12.32 ± 8.05), and low anxiety levels (17.20 ± 12.41). There were no significant differences between sociodemographic variables ranges in regard to all psychological questionnaires. No high levels of depression and anxiety were found. CONCLUSIONS: The levels were low compared to other studies in which online teaching was used, which is explained by the fact that the students retained adequate resilience and self-esteem thanks to being able to contact teachers and, above all, their own peers.

Acute complications in open/miss primary and revision thoracolumbar spine surgery: a descriptive study of the most common complications and treatment of choice.

PURPOSE: Main question The aim of this study is to describe and analyze the frequency of acute perioperative (intraoperatively and 30 days after) complications of open/MISS thoracolumbar spine surgery. Secondary questions A) Describe the treatment of choice for every kind of complication mentioned. B) Perform a bibliographic search and compare the complications described and their frequency with those studied in the manuscript. METHODS: A retrospective cohort of 816 patients undergoing spinal surgery over a two year period was analyzed. Acute complications of 59 patients are described whether those with a greater number of levels required longer periods of hospitalization. RESULTS: The frequency of acute complications was 7.2%. The most common was infection (2.7%), followed by dural tear (1.7%), and screw malpositioning (1%), which is consistent with the current literature. No statistically significant results were observed when comparing the mean length of hospital stay among patients operated on a greater number of levels compared to the rest (P: 0.344; 95% CI: -3.88-10.93). CONCLUSIONS: The subsidiary patient of spinal surgery is getting older and has more comorbidities, and therefore, has a higher risk of complications. Although there are models predicting the risk of complications, they are not used in routine clinical practice. It would be necessary to unify the main criteria and establish guidelines for risk detection and therapeutic algorithms based on new high-quality studies.

Enhancing Structural Diversity of Lathyrane Derivatives through Biotransformation by the Marine-Derived Actinomycete Streptomyces puniceus BC-5GB.11.

Lathyrane-type diterpenes have a wide range of biological activities. Among them, euphoboetirane A (1) exerts neurogenesis-promoting activity. In order to increase the structural diversity of this type of lathyrane and explore its potential use in neurodegenerative disorders, the biotransformation of 1 by Streptomyces puniceus BC-5GB.11 has been investigated. The strain BC-5GB.11, isolated from surface sediments collected from the intertidal zone of the inner Bay of Cadiz, was identified as Streptomyces puniceus, as determined by phylogenetic analysis using 16S rRNA gene sequence. Biotransformation of 1 by BC-5GB.11 afforded five products (3-7), all of which were reported here for the first time. The main biotransformation pathways involved regioselective oxidation at non-activated carbons (3-5) and isomerization of the ∆12,13 double bond (6). In addition, a cyclopropane-rearranged compound was found (7). The structures of all compounds were elucidated on the basis of extensive NMR and HRESIMS spectroscopic studies.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.

Microbial community and volatilome changes in brines along the spontaneous fermentation of Spanish-style and natural-style green table olives (Manzanilla cultivar).

Microbial community and volatilome of brines were monitored during the spontaneous fermentations of Spanish-style and Natural-style green table olives from Manzanilla cultivar. Fermentation of olives in the Spanish style was carried out by lactic acid bacteria (LAB) and yeasts, whereas halophilic Gram-negative bacteria and archaea, along with yeasts, drove the fermentation in the Natural style. Clear differences between the two olive fermentations regarding physicochemical and biochemical features were found. Lactobacillus, Pichia, and Saccharomyces were the dominant microbial communities in the Spanish style, whereas Allidiomarina, Halomonas, Saccharomyces, Pichia, and Nakazawaea predominated in the Natural style. Numerous qualitative and quantitative differences in individual volatiles between both fermentations were found. The final products mainly differed in total amounts of volatile acids and carbonyl compounds. In addition, in each olive style, strong positive correlations were found between the dominant microbial communities and various volatile compounds, some of them previously reported as aroma-active compounds in table olives. The findings from this study provide a better understanding of each fermentation process and may help the development of controlled fermentations using starter cultures of bacteria and/or yeasts for the production of high-quality green table olives from Manzanilla cultivar.

Value of Prostate-Specific Antigen Kinetics in Patients with Low-Risk Prostate Cancer under Active Surveillance.

INTRODUCTION: This study analyzes the value of PSA kinetics, PSA speed (vPSA), and PSA doubling time (PSAdt), in patients with low-risk prostate cancer who are in an active surveillance (AS) program. METHODS: An observational, retrospective, and longitudinal study of a sample of 86 patients included in AS program between January 2014 and October 2021 was conducted. A review of their medical records was performed, and PSA kinetics were calculated, analyzing the causes of discontinuation of the AS program and its relationship with PSA kinetics. RESULTS: The mean age was 63.39 years, and the median follow-up was 62.55 months. The mean PSA at diagnosis was 8.27 ng/mL. A median of PSAdt of 62.55 months and 1.3 ng/mL/year for vPSA was obtained. 35 patients left the program, with a higher percentage of patients leaving with a PSAdt less than 36 months (73.7 vs. 31.1%) and a vPSA greater than 2 ng/mL/year (68.2 vs. 31.3%). The probability of permanence and the time of permanence in AS were statistically significantly higher for those patients with favorable kinetic parameters. CONCLUSION: PSA kinetics is a parameter to take into account when making decisions to keep patients in an AS program.

Complete mitochondrial genome of Metathelazia capsulata (Pneumospiruridae) and comparison with other Spiruromorpha species.

Metathelazia capsulata (family Pneumospiruridae) is a lungworm parasitizing the bronchi and bronchioles, described in four species of wild carnivores. Very little molecular data are available on this nematode and none on other species of the Pneumospiruridae family. In this work, we describe for the first time the complete mitogenome (mitochondrial genome) of M. capsulata, being the first described of the family Pneumospiruridae. The mitogenome of M. capsulata has 13,659 bp in length, an A + T content of 79.2%. The mitogenome included 12 protein-coding genes (PCGs) (lacking the atp8 gene), 22 tRNA genes, 2 rRNA genes (all the genes are coded by the heavy strand), and an AT-rich region. The PCGs varied in size (232 bp-1645 bp). Only the tRNA-Trp has the standard cloverleaf secondary structure, while the other 21 do not. The AT-rich region, with a 90.5% A + T content and a length of 389 bp, is located between the cox3 and tRNA-Ala genes. Comparison with the mitogenomes of 29 species of Spiruromorpha infraorder, belonging to different families, demonstrates that M. capsulata mitogenome shared the common characteristics of most of them. The phylogeny constructions yielded phylogenies that were in agreement with the obtained previously by using sequences and gene order data of mitogenomes.

Intraobserver Repeatability Assessment of the S390L Firefly WDR Slitlamp in Patients With Dry Eye Disease: Objective, Automated, and Noninvasive Measures.

OBJECTIVES: To assess the intraobserver repeatability of automated, objective, and noninvasive measures obtained with the S390L Firefly WDR slitlamp. METHODS: This cross-sectional study included 50 eyes of patients with dry eye disease with a mean age of 55.06±12.96 years. Three consecutively repeated measures of the following variables were obtained: first noninvasive break-up time (F-NIBUT), average noninvasive break-up time (A-NIBUT), tear meniscus height, tear meniscus area (TMA), nasal ciliary hyperemia (NCIH), temporal ciliary hyperemia (TCIH), nasal conjunctival hyperemia (NCOH), temporal conjunctival hyperemia (TCOH), upper loss area meibomian gland (U-LAMG), lower loss area meibomian gland (L-LAMG), upper meibomian gland dysfunction grade (U-MGD grade), and lower meibomian gland dysfunction grade (L-MGD grade). Intraobserver repeatability was estimated with coefficient of variation (CoV), intrasubject standard deviation (SD) (S w ), and Bland-Altman plots. RESULTS: All variables showed no statistically significant differences in the repeated-measures analysis except for L-MGD grade ( P =0.045). F-NIBUT and A-NIBUT obtained the highest CoV with an average value of 0.48±0.41 [0.02-1.00] and 0.34±0.25 [0.02-1.00], respectively. The remaining variables showed CoVs between 0.04±0.11 [0.00-0.43] and 0.18±0.16 [0.00-0.75]. A-NIBUT, TMA, NCOH, and L-LAMG obtained an S w of 2.78s, 0.21 mm 2 , <0.001, and 4.11%, respectively. Bland-Altman plots showed a high level of agreement between pairs of repeated measures. CONCLUSION: The S390L Firefly WDR slitlamp has moderate intraobserver repeatability for F-NIBUT and A-NIBUT, which suggests that F-NIBUT and A-NIBUT are tests with high variability. The remaining variables show satisfactory intraobserver repeatability.

Intense Pulse Light Combined With Low-Level Light Therapy in Dry Eye Disease: A Systematic Review.

OBJECTIVES: To evaluate the improvement in symptoms and signs associated with intense pulse light (IPL) combined with low-level light therapy (LLLT) in the treatment of dry eye disease (DED). METHODS: A systematic review of full-length original studies reporting the effects of IPL combined with LLLT for DED in two databases, PubMed and Scopus, was performed according to the PRISMA statement. The quality assessment tool for case series studies from the National Heart, Lung, and Blood Institute was used to analyze the quality of the studies selected. RESULTS: The search provided a total of 393 articles, of which six were included. Significant decreases in the Ocular Surface Disease Index (OSDI) score, meibomian gland dysfunction (MGD) score, MGD grade, and meiboscore and increases in tear film stability, lipid layer thickness, and loss area of the meibomian gland have been reported. Concerning tear volume, tear meniscus height, and Schirmer test remained unchanged. In relation to tear osmolarity and corneal fluorescein staining, contradictory outcomes were found. CONCLUSIONS: Intense pulse light combined with LLLT for the treatment of dry eye improves OSDI, tear film stability, and meibomian gland function; thus, this treatment may be recommended for DED patients due to MGD.

Choroidal thickness assessment in keratoconus patients treated with cross-linking compared to healthy population.

PURPOSE: To analyze the choroidal thickness between patients with keratoconus undergoing cross-linking treatment and a healthy population, as well as to determine the factors that influence choroidal thickness. METHODS: This was an observational, analytical, case-control study that was conducted from February 2021 to June 2021. Choroidal thickness was measured at different locations, including the subfoveal, nasal (1000 µm), temporal (1000 µm), superior (1000 µm) and inferior (1000 µm) locations using a Spectral-domain optical coherence tomography with enhanced depth imaging, which allowed us to obtain horizontal and vertical B-scans centered on the fovea. RESULTS: This study included 21 patients with keratoconus (mean age, 21.86 ± 5.28 years) and 28 healthy patients (mean age, 24.21 ± 4.71 years). Choroidal thickness was significantly greater in patients with keratoconus than in healthy patients in each of the following measured locations: subfoveal (P < 0.001); nasal (1000 µm) (P < 0.001), temporal (1000 µm) (P < 0.001), superior (1000 µm) (P < 0.001) and inferior (1000 µm) (P < 0.001) locations. Variables such as age (ρ = - 0.09; P = 0.50) and refraction (ρ = 0.14; P = 0.34) were not found to be associated with choroidal thickness. In a stepwise multiple linear regression, the group was the single variable correlated with choroidal thickness (ß = 0.88; P < 0.001). CONCLUSION: Choroidal thickness is thicker in keratoconus patients treated with cross-linking than in the healthy population. This finding could be associated with inflammatory choroidal mechanisms in keratoconus patients, but more studies are needed. Age and refractive error do not seem to influence choroidal thickness.

Renal function and clinical outcome of patients with cancer-associated venous thromboembolism randomized to receive apixaban or dalteparin. Results from the Caravaggio trial

The effect of renal impairment (RI) on risk of bleeding and recurrent thrombosis in cancer patients treated with direct oral anticoagulants for venous thromboembolism (VTE) is undefined. We ran a prespecified analysis of the randomized Caravaggio study to evaluate the role of RI as a risk factor for bleeding or recurrence in patients treated with dalteparin or apixaban for cancerassociated VTE. RI was graded as moderate (creatinine clearance between 30-59 mL/minute; 275 patients) and mild (between 60- 89 mL/minute; 444 patients). In the 1142 patients included in this analysis, the incidence of major bleeding was similar in patients with moderate vs. no or mild RI (HR 1.06-95% CI: 0.53-2.11), with no difference in the relative safety of apixaban and dalteparin. Recurrent VTE was not different in moderate vs. no or mild RI (HR=0.67, 95% CI: 0.38-1.20); in moderate RI, apixaban reduced recurrent VTE compared to dalteparin (HR=0.27, 95% CI: 0.08-0.96; P for interaction 0.1085). At multivariate analysis, no association was found between variation of renal function over time and major bleeding or recurrent VTE. Advanced or metastatic cancer was the only independent predictor of major bleeding (HR=2.84, 95% CI: 1.20-6.71), with no effect of treatment with apixaban or dalteparin. In our study, in cancer patients treated with apixaban or dalteparin, moderate RI was not associated with major bleeding or recurrent VTE. In patients with moderate renal failure, the safety profile of apixaban was confirmed with the potential for improved efficacy in comparison to dalteparin. ClinicalTrials.gov identifier: NCT03045406.