Intraoperative thyroid frozen section: indications, results and consequences.

Background: Frozen section (FS) analysis is strongly influenced by the experience of surgeons and pathologists. We analyzed its performance in a secondary care hospital with surgical and pathologic experience transferred from a university hospital. Methods: Indications, results, and consequences of all thyroid FS performed between January 1, 2021 and December 31, 2022 were critically reviewed. Results: FS was performed in 90 (26.5%) of 340 procedures. Indications consisted in a suspicious fine needle biopsy in 28 (31.1%) cases, (99m) Technetium-Methoxy-Isobutyl-Isonitrile (MIBI) retaining hypofunctional nodules in 25 (27.8%), the intraoperative appearance in 18 (20%), the sonographic appearance in 18 (20%) and a positron emission tomography (PET) positive result in 1 case (1.1%). Malignancy was diagnosed in 21 (23.3%) and confirmed by final histology in all cases (100%). In the remaining 69 (76.7%) FS displaying no positive malignancy criteria, final histology delivered benign in 62 (89.8%) and malignant diagnoses in 7 cases (10.1%). 25% of thyroid carcinomas could not be diagnosed by FS. FS sensitivity was consequently 75% (95% CI: 55.1-89.3%). All missed malignancies were papillary thyroid carcinomas of follicular variant (fvPTC). FS sensitivity was lowest in MIBI positive hypofunctional nodules (33%) and Bethesda III (50%) as opposed to Bethesda V (92.9%) and to those cases with suspicious sonographic or intraoperative appearance (71.4%). Two-staged surgery was necessary in 10 (15.8%) of carcinomas. Conclusions: Sensitivity of FS in a secondary care hospital offering surgical and pathologic experience from a specialized university center is 75% and mainly reduced by the prevalence of fvPTC. Omitting FS in Bethesda III and MIBI positive hypofunctional nodules might improve FS performance.

Light chain mutations contribute to defining the fibril morphology in systemic AL amyloidosis.

Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent.

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole.

The presence and accumulation of both, plastics and antibiotics in soils may lead to the colonization, selection, and propagation of soil bacteria with certain metabolic traits, e.g., antibiotic resistance, in the plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and degrade 13C-labeled sulfamethoxazole (SMX). Using single-cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polyethylene (PE) and polystyrene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse microorganisms were colonizing both plastic types, with a slight preference for PE substrate. CARD-FISH bacterial cell counts on PE and PS surfaces formed under SMX amendment ranged from 5.36 × 103 to 2.06 × 104, and 2.06 × 103 to 3.43 × 103 hybridized cells mm-2, respectively. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom%, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 ‰). Independent Mann-Whitney U test showed a significant difference between the control plastisphere samples incubated without SMX and those in 13C-SMX incubations (P < 0.001). Our results provide direct evidence demonstrating, at single-cell level, the capacity of bacterial colonizers of plastics to assimilate 13C-SMX from contaminated soils. These findings expand our knowledge on the role of soil-seeded plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Clinical Impact of Pelvic Sentinel Lymph Node Biopsy for Vulvar Squamous Cell Carcinoma.

BACKGROUND/AIM: This study investigated the clinical impact of resection of pelvic sentinel lymph nodes (PSLNs) in squamous cell vulvar cancer (SCVC). PATIENTS AND METHODS: Sixty-two groins of 33 patients with SCVC who underwent sentinel lymph node (SLN) resection between 2010 and 2021 at the University Hospital of Cologne, Germany, were analyzed in this retrospective cohort study. The frequency of additionally resectable PSLNs, histological findings, and count rates were analyzed and compared to the findings for inguinal sentinel lymph nodes (ISLNs). RESULTS: In all patients and in 61 (98%) of the 62 radiolabeled groins, at least one SLN could be resected. Five (8%) of the 62 groins had histologically confirmed lymph node metastases (4/33 patients, 12%). Twenty (33%) of the 62 groins underwent additional PSLN resection. Resection of these PSLNs was feasible without causing an additional burden for the patients. None of the PSLNs showed signs of tumor infiltration. Information on the extent of radioactivity for ISLNs and simultaneously for PSLNs, expressed as count rate of intraoperative measurement with the gamma probe, was available for 20 (32%) groins. In three (15%) of these cases, the highest count rate in a SLN was found in a PSLN and not in an ISLN. CONCLUSION: Resection of PSLNs is feasible and can be performed without short-term complications. In patients with early SCVC, resection of PSLNs is not necessary, even in those with early infiltration of inguinal lymph nodes. The intraoperative count rate of SLN is not relevant for the decision to perform resection.

Literature review and protocol for a prospective multicentre cohort study on multimodal prediction of seizure recurrence after unprovoked first seizure.

INTRODUCTION: Epilepsy is a common neurological disorder characterised by recurrent seizures. Almost half of patients who have an unprovoked first seizure (UFS) have additional seizures and develop epilepsy. No current predictive models exist to determine who has a higher risk of recurrence to guide treatment. Emerging evidence suggests alterations in cognition, mood and brain connectivity exist in the population with UFS. Baseline evaluations of these factors following a UFS will enable the development of the first multimodal biomarker-based predictive model of seizure recurrence in adults with UFS. METHODS AND ANALYSIS: 200 patients and 75 matched healthy controls (aged 18-65) from the Kingston and Halifax First Seizure Clinics will undergo neuropsychological assessments, structural and functional MRI, and electroencephalography. Seizure recurrence will be assessed prospectively. Regular follow-ups will occur at 3, 6, 9 and 12 months to monitor recurrence. Comparisons will be made between patients with UFS and healthy control groups, as well as between patients with and without seizure recurrence at follow-up. A multimodal machine-learning model will be trained to predict seizure recurrence at 12 months. ETHICS AND DISSEMINATION: This study was approved by the Health Sciences and Affiliated Teaching Hospitals Research Ethics Board at Queen's University (DMED-2681-22) and the Nova Scotia Research Ethics Board (1028519). It is supported by the Canadian Institutes of Health Research (PJT-183906). Findings will be presented at national and international conferences, published in peer-reviewed journals and presented to the public via patient support organisation newsletters and talks. TRIAL REGISTRATION NUMBER: NCT05724719.

Cognitive dysfunction at epilepsy onset as a marker for seizure recurrence.

BACKGROUND: Cognitive dysfunction has been correlated with seizure control in chronic epilepsy and in newly diagnosed epilepsy, which potentially makes it a good marker for predicting disease course and seizure control. However, there is a lack of prospective studies examining the role of cognitive dysfunction in predicting seizure recurrence at the earliest stages of the disease, such as following the first unprovoked seizure (UFS) or new onset epilepsy (NOE). METHODS: Thirty three adult participants (FS=18, NOE=15) from the Halifax First Seizure Clinic (HFSC) completed a cognitive screening assessment at baseline (typically 3 months following diagnosis); seizure-recurrence was evaluated one year after the initial HFSC visit. RESULTS: Cognitive impairment, defined as at least one z-score in the impaired range (≤-1.5) relative to published test norms, was documented in 76% of the patients with seizure recurrence at follow-up and in 55% without seizure recurrence. Speed/executive functions and Memory were the most frequently affected domains, with impaired performance noted in 35% and 29% of the entire sample, respectively. Although the seizure recurrence vs. non-recurrence groups did not differ significantly on likelihood of impairment in any specific cognitive domains, a regression model of seizure recurrence that included years of education, baseline mood and anxiety scores, normal vs. abnormal baseline MRI, and impaired (vs. unimpaired) function in six cognitive domains was significant overall (Χ2 (10) = 24.04, p =.007*, R2N =.77). The regression model was no longer significant with the cognitive variables removed. CONCLUSIONS: Subtle cognitive dysfunction, especially in the domains of executive functions and memory are prevalent in individuals at the earliest stages of epilepsy. In addition to abnormal MRI and EEG findings at baseline, which are far less prevalent in FS and NOE, cognitive factors show promise in helping predict seizure recurrence in these populations.

Active crystallization from power functional theory.

We address the gas, liquid, and crystal phase behaviors of active Brownian particles in three dimensions. The nonequilibrium force balance at coexistence leads to equality of state functions for which we use power functional approximations. Motility-induced phase separation starts at a critical point and quickly becomes metastable against active freezing for Péclet numbers above a nonequilibrium triple point. The mean swim speed acts as a state variable, similar to the density of depletion agents in colloidal demixing. We obtain agreement with recent simulation results and correctly predict the strength of particle number fluctuations in active fluids.

Why neural functionals suit statistical mechanics.

We describe recent progress in the statistical mechanical description of many-body systems via machine learning combined with concepts from density functional theory and many-body simulations. We argue that the neural functional theory by Sammülleret al(2023Proc. Natl Acad. Sci.120e2312484120) gives a functional representation of direct correlations and of thermodynamics that allows for thorough quality control and consistency checking of the involved methods of artificial intelligence. Addressing a prototypical system we here present a pedagogical application to hard core particle in one spatial dimension, where Percus' exact solution for the free energy functional provides an unambiguous reference. A corresponding standalone numerical tutorial that demonstrates the neural functional concepts together with the underlying fundamentals of Monte Carlo simulations, classical density functional theory, machine learning, and differential programming is available online athttps://github.com/sfalmo/NeuralDFT-Tutorial.

Intracortical Myelin in Youths at Risk for Depression.

Background: Major depressive disorder (MDD) is a leading cause of disability. To understand why depression develops, it is important to distinguish between early neural markers of vulnerability that precede the onset of MDD and features that develop during depression. Recent neuroimaging findings suggest that reduced global and regional intracortical myelination (ICM), especially in the lateral prefrontal cortex, may be associated with depression, but it is unknown whether it is a precursor or a consequence of MDD. The study of offspring of affected parents offers the opportunity to distinguish between precursors and consequences by examining individuals who carry high risk at a time when they have not experienced depression. Methods: We acquired 129 T1-weighted and T2-weighted scans from 56 (25 female) unaffected offspring of parents with depression and 114 scans from 63 (34 female) unaffected offspring of parents without a history of depression (ages 9 to 16 years). To assess scan quality, we calculated test-retest reliability. We used the scan ratios to calculate myelin maps for 68 cortical regions. We analyzed data using mixed-effects modeling. Results: ICM did not differ between high and low familial risk youths in global (B = 0.06, SE = 0.03, p = .06) or regional (B = 0.05, SE = 0.03, p = .08) analyses. Our pediatric sample had high ICM reliability (intraclass correlation coefficient = 0.79; 95% CI, 0.55-0.88). Conclusions: Based on our results, reduced ICM does not appear to be a precursor of MDD. Future studies should examine ICM in familial high-risk youths across a broad developmental period.

Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the ataxia telangiectasia and RAD3-related (ATR) kinase.

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells, it reduced ATR to 40 % of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells.

Leaching of tire particles and simultaneous biodegradation of leachables.

The fate of organic compounds released from tire wear particle (TWP) in the aquatic environment is still poorly understood. This is especially true near sources where biotic and abiotic transformation and leaching from TWP are simultaneous and competing processes. To address this knowledge-gap an experiment was performed, allowing for biodegradation (a) during the leaching from a suspension of cryo-milled tire tread (CMTT) and (b) subsequent to leaching. Besides measuring the Dissolved Organic Carbon (DOC) content, 19 tire-related chemicals were quantified, and non-target screening was performed by LC-HRMS. The non-inoculated control experiment exhibited a DOC of up to 4 mg g-1, with up to 700 µg g-1 of 1,3-diphenylguanidine (DPG) as the most prominent compound, followed by three benzothiazoles (2-mercaptobenzothiazole (2-MBT), 2-hydroxybenzothiazole (2-OHBT) and benzothiazole-2-sulfonic acid (BTSA); 50 µg g-1 each) and 4-hydroxydiphenylamine (4-HDPA) (50 µg g-1). Biodegradation reduced the DOC by 88 % and the concentration of most organic compounds by more than 85 %. At the end of the experiment hexamethoxymethylmelamine (HMMM) was the most prominent single compounds (18 µg g-1). Non-target screening showed a more complex picture. Another 25 transformation products (TPs) of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) and 44 TPs and derivatives related to DPG were detected in solution, of which 11 and 28 were still present after or formed by biodegradation, respectively. Of these 39 TPs and derivatives, 31 could be detected in road runoff samples. This study provides a more comprehensive picture of the leachables of tire particles that are of environmental relevance. It also outlines that derivatives of tire additives formed during tire production and use may deserve more attention as leachables. The large extent of biodegradation of tire leachables suggests that settling ponds may be a useful treatment option for road runoff.

Cryo-EM observation of the amyloid key structure of polymorphic TDP-43 amyloid fibrils.

The transactive response DNA-binding protein-43 (TDP-43) is a multi-facet protein involved in phase separation, RNA-binding, and alternative splicing. In the context of neurodegenerative diseases, abnormal aggregation of TDP-43 has been linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration through the aggregation of its C-terminal domain. Here, we report a cryo-electron microscopy (cryo-EM)-based structural characterization of TDP-43 fibrils obtained from the full-length protein. We find that the fibrils are polymorphic and contain three different amyloid structures. The structures differ in the number and relative orientation of the protofilaments, although they share a similar fold containing an amyloid key motif. The observed fibril structures differ from previously described conformations of TDP-43 fibrils and help to better understand the structural landscape of the amyloid fibril structures derived from this protein.

Insights into the Structural Basis of Amyloid Resistance Provided by Cryo-EM Structures of AApoAII Amyloid Fibrils.

Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.

Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing.

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Cryo-EM Analysis of the Effect of Seeding with Brain-derived Aß Amyloid Fibrils.

Aß amyloid fibrils from Alzheimer's brain tissue are polymorphic and structurally different from typical in vitro formed Aß fibrils. Here, we show that brain-derived (ex vivo) fibril structures can be proliferated by seeding in vitro. The proliferation reaction is only efficient for one of the three abundant ex vivo Aß fibril morphologies, which consists of two peptide stacks, while the inefficiently proliferated fibril morphologies contain four or six peptide stacks. In addition to the seeded fibril structures, we find that de novo nucleated fibril structures can emerge in seeded samples if the seeding reaction is continued over multiple generations. These data imply a competition between de novo nucleation and seed extension and suggest further that seeding favours the outgrowth of fibril morphologies that contain fewer peptide stacks.

Neural functional theory for inhomogeneous fluids: Fundamentals and applications.

We present a hybrid scheme based on classical density functional theory and machine learning for determining the equilibrium structure and thermodynamics of inhomogeneous fluids. The exact functional map from the density profile to the one-body direct correlation function is represented locally by a deep neural network. We substantiate the general framework for the hard sphere fluid and use grand canonical Monte Carlo simulation data of systems in randomized external environments during training and as reference. Functional calculus is implemented on the basis of the neural network to access higher-order correlation functions via automatic differentiation and the free energy via functional line integration. Thermal Noether sum rules are validated explicitly. We demonstrate the use of the neural functional in the self-consistent calculation of density profiles. The results outperform those from state-of-the-art fundamental measure density functional theory. The low cost of solving an associated Euler-Lagrange equation allows to bridge the gap from the system size of the original training data to macroscopic predictions upon maintaining near-simulation microscopic precision. These results establish the machine learning of functionals as an effective tool in the multiscale description of soft matter.

Common transthyretin-derived amyloid fibril structures in patients with hereditary ATTR amyloidosis.

Systemic ATTR amyloidosis is an increasingly important protein misfolding disease that is provoked by the formation of amyloid fibrils from transthyretin protein. The pathological and clinical disease manifestations and the number of pathogenic mutational changes in transthyretin are highly diverse, raising the question whether the different mutations may lead to different fibril morphologies. Using cryo-electron microscopy, however, we show here that the fibril structure is remarkably similar in patients that are affected by different mutations. Our data suggest that the circumstances under which these fibrils are formed and deposited inside the body - and not only the fibril morphology - are crucial for defining the phenotypic variability in many patients.

Correction: High resolution microscopy to evaluate the efficiency of surface sterilization of Zea Mays seeds.

[This corrects the article DOI: 10.1371/journal.pone.0242247.].