The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini.

The non-heme iron-dependent dioxygenase 2-aminoethanethiol dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyses Nt-cys sulfinylation, which promotes O2-dependent proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signalling (RGS) 4 and 5, and the pro-inflammatory cytokine interleukin-32 (IL32), all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. Furthermore, the role of individual chemical groups, active site metal, amino acid composition and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and IL32. We demonstrate, using surface plasmon response (SPR) and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence (15N-HSQC) nuclear magnetic resonance (NMR) titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human ß-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

Ki-67 Labelling Index as a Predictor of Invasive Features in Thyroid Cancer: Retrospective Analysis and Implications.

BACKGROUND: Ki-67 immunostaining is commonly used in neuroendocrine tumors to estimate the proliferative index and for grading. This study investigates its association with the invasiveness of follicular-derived thyroid carcinomas (TCs). METHODS: A retrospective analysis of patients with TC at three McGill University teaching hospitals between January 2018 and November 2023 was conducted. The inclusion criteria included patients with malignant thyroid tumors and accessible Ki-67 LI data from final pathology specimens. The data collected included patient demographics, Ki-67 LI values, and different invasiveness attributes, such as molecular mutations, the histological subtype, lymphovascular invasion (LVI), extrathyroidal extension (ETE), and positive lymph nodes (LNs). RESULTS: In total, 212 patients met the inclusion criteria, of which 80.7% were females and 19.3% were males. The Ki-67 LI ranged from 1% to 30%, with the majority of the cases within the range of 1-15%. A significant association was observed between higher Ki-67 LI and high-risk histological subtypes of thyroid carcinoma (p < 0.001). Similarly, Ki-67 LI was significantly associated with LVI and positive LN metastasis (p < 0.001 and p = 0.036, respectively). However, no significant association was found between the Ki-67 LI and gene mutations or ETE (p = 0.133 and p = 0.190, respectively). Using percentiles to establish a cutoff, patients with a Ki-67 LI higher than 6.7 showed a higher likelihood of being associated with invasive features. CONCLUSION: Elevated Ki-67 LI can serve as an indicator of aggressiveness in follicular-derived TC, especially when associated with distinct histological subtypes, LVI and positive LNs.

A large-scale and PCR-referenced vocal audio dataset for COVID-19.

The UK COVID-19 Vocal Audio Dataset is designed for the training and evaluation of machine learning models that classify SARS-CoV-2 infection status or associated respiratory symptoms using vocal audio. The UK Health Security Agency recruited voluntary participants through the national Test and Trace programme and the REACT-1 survey in England from March 2021 to March 2022, during dominant transmission of the Alpha and Delta SARS-CoV-2 variants and some Omicron variant sublineages. Audio recordings of volitional coughs, exhalations, and speech were collected in the 'Speak up and help beat coronavirus' digital survey alongside demographic, symptom and self-reported respiratory condition data. Digital survey submissions were linked to SARS-CoV-2 test results. The UK COVID-19 Vocal Audio Dataset represents the largest collection of SARS-CoV-2 PCR-referenced audio recordings to date. PCR results were linked to 70,565 of 72,999 participants and 24,105 of 25,706 positive cases. Respiratory symptoms were reported by 45.6% of participants. This dataset has additional potential uses for bioacoustics research, with 11.3% participants self-reporting asthma, and 27.2% with linked influenza PCR test results.

Tyrosine Sulfation Modulates the Binding Affinity of Chemokine-Targeting Nanobodies.

Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.

Structural basis of chemokine recognition by the class A3 tick evasin EVA-ACA1001.

Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.

A Prospective Study of Publicly Funded Molecular Testing of Indeterminate Thyroid Nodules: Canada's Experience.

CONTEXT: Indeterminate thyroid nodules (ITNs) lead to diagnostic surgeries in many countries. Use of molecular testing (MT) is endorsed by several guidelines, but costs are limitative, especially in public healthcare systems like in Canada. OBJECTIVES: Primary objective: evaluate the clinical value of Thyroseq® v3 (TSv3) using benign call rate (BCR) in a real-world practice. Secondary objective: assess cost-effectiveness of MT. DESIGN: This is a multicentric prospective study. SETTING: This study was conducted in 5 academic centers in Quebec, Canada. PATIENTS OR OTHER PARTICIPANTS: 500 consecutive patients with Bethesda III (on 2 consecutive cytopathologies) or IV and TIRADS 3 or 4 nodules measuring 1 to 4 cm were included. INTERVENTION: MT was performed between November 2021 and November 2022. Patients with a positive TSv3 were referred to surgery. Patients with a negative TSv3 were planned for follow-up by ultrasonography for a minimum of 2 years. MAIN OUTCOME MEASURE: The BCR, corresponding to the proportion of ITNs with negative TSv3 results, was assessed. RESULTS: 500 patients underwent TSv3 testing, with a BCR of 72.6% (95% CI: 68.5-76.5; p<0.001). 99.7% of patients with a negative result avoided surgery. The positive predictive value of TSv3 was 68.2% (95% CI: 58.5-76.9). The cost-benefit analysis identified that the implementation of MT would yield cost savings of $6.1 million over the next 10 years. CONCLUSIONS: Use of MT (TSv3) in a well-selected population with ITNs led to a BCR of 72.6%. It is cost-effective and prevents unnecessary surgeries in a public healthcare setting.

Small bowel lipoma and intussusception: a case report.

Intussusception is defined as the telescoping of bowel into itself. Intussusception is the leading cause of bowel obstruction in children, but it is rare in adults [1, 2]. It has a pathological intramural or extramural lead point. In adults, it accounts for 1%-5% of cases of bowel obstruction [3, 4]. Unlike presentation in the paediatric population of cramping abdominal pain, bloody mucus, and palpable mass in right iliac fossa, presentation in adults can be more varied and non-specific [1, 4]. Hence, diagnosis of small bowel intussusception (SBI) can be challenging, requiring a higher degree of clinical suspicion [5]. While cases of paediatric intestinal intussusception are often primary, most adult cases are secondary to structural lesions [5]. This case is of a 57-year-old lady who presented with SBI secondary to a small bowel lipoma (SBL), reflecting the importance of considering SBL as a differential in the causes of SBI.

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

Development of supramolecular anticoagulants with on-demand reversibility.

Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.

Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Reporting outcome measures in veterinary physiotherapy with particular reference to the treatment of canine and equine joint cases in the UK.

BACKGROUND: Outcome measures are extensively used within human physiotherapy, but a widely accepted issue in veterinary physiotherapy is that outcome measures lack sufficient evaluation and standardisation in terms of how they are implemented. This cross-sectional study aimed to provide clarity on (1) the current selection of outcome measures in canine and equine physiotherapy and (2) investigate external influences on outcome measure selection, including comparative literature availability, professional memberships and background. METHODS: A structured scoping literature review consolidated current understanding and limitations. This informed a survey of qualified veterinary physiotherapists (n = 40). The statistical analysis comprised descriptive statistics. RESULTS: Key observations included (1) a lack of difference in outcome measure application between veterinary physiotherapists with and without a human physiotherapy background, (2) enhanced outcome measure utilisation by registry body members and (3) an overall skew towards subjective, rather than objective, outcome measure use. LIMITATIONS: The study was limited by the absence of a defined veterinary physiotherapist population and subsequent convenience sample size. CONCLUSION: The apparent skew towards subjective outcome measures highlights objective outcome measure underutilisation and the need for a more extensive evidence base. In conclusion, there is a need to develop comprehensive professional development resources promoting the use of repeatable outcome measures such as goniometers and the Liverpool osteoarthritis scoring.

A Bayesian survival treed hazards model using latent Gaussian processes.

Survival models are used to analyze time-to-event data in a variety of disciplines. Proportional hazard models provide interpretable parameter estimates, but proportional hazard assumptions are not always appropriate. Non-parametric models are more flexible but often lack a clear inferential framework. We propose a Bayesian treed hazards partition model that is both flexible and inferential. Inference is obtained through the posterior tree structure and flexibility is preserved by modeling the log-hazard function in each partition using a latent Gaussian process. An efficient reversible jump Markov chain Monte Carlo algorithm is accomplished by marginalizing the parameters in each partition element via a Laplace approximation. Consistency properties for the estimator are established. The method can be used to help determine subgroups as well as prognostic and/or predictive biomarkers in time-to-event data. The method is compared with some existing methods on simulated data and a liver cirrhosis dataset.

Molecular Profiling for Bethesda III to VI Nodules: Results of a Multicenter International Retrospective Study.

OBJECTIVE: Molecular testing is a well-established tool that assists in the management of thyroid nodules. We describe our experience using molecular testing of thyroid nodules with Bethesda III to VI cytology. METHODS: This is a retrospective multicenter, multinational study of thyroid nodules that underwent preoperative molecular profiling with ThyGenX/ThyGeNEXT or ThyroSeq V3 between 2015 and 2022. The clinical characteristics and mutational profiles of tumors were compared. Collected data included demographics, cytology results, surgical pathology, and molecular alterations. Molecular alterations were categorized into 3 main phenotypes: BRAF-like, RAS-like, and non-BRAF-non-RAS (NBNR). RESULTS: Overall, 784 patients who had surgery were included, of which 603 (76.2%) were females. The most common histologic type was papillary thyroid cancer (PTC) with 727 (91.9%) cases. In total, 205 (28.2%) cases showed an aggressive subtype of PTC (eg, tall cell and hobnail). BRAF-like alterations were most likely to be found in Bethesda V and VI nodules and show extrathyroidal extension (ETE), nodal disease, and/or aggressive subtypes of PTC (P < .001 for all). RAS-like alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive histology (P < .001 for all). NBNR alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive subtypes of PTC. However, they were rarely but significantly associated with poorly differentiated thyroid cancer (P < .005). CONCLUSION: Molecular testing of thyroid nodules can help determine the likelihood of malignancy and classify nodules into several tumor phenotypes, predicting their behaviors and potentially allowing for a more tailored treatment. NBNR alterations should be managed with caution.

Bayesian model averaging of longitudinal dose-response models.

Selecting a safe and clinically beneficial dose can be difficult in drug development. Dose justification often relies on dose-response modeling where parametric assumptions are made in advance which may not adequately fit the data. This is especially problematic in longitudinal dose-response models, where additional parametric assumptions must be made. This paper proposes a class of longitudinal dose-response models to be used in the Bayesian model averaging paradigm which improve trial operating characteristics while maintaining flexibility a priori. A new longitudinal model for non-monotonic longitudinal profiles is proposed. The benefits and trade-offs of the proposed approach are demonstrated through a case study and simulation.

The Impact of BRAF V600E Mutation Allele Frequency on the Histopathological Characteristics of Thyroid Cancer.

BACKGROUND: A BRAF V600E mutation in papillary thyroid cancer (PTC) has been shown to be associated with aggressive behavior. Nevertheless, not all BRAF V600E PTCs behave aggressively. Allele frequency (AF) is the number of mutated molecules divided by the total number of wild-type molecules at a specific location in the genome. The relationship between BRAF V600E AF and the histopathological features of thyroid malignancies is not well understood. We hypothesized that the BRAF V600E AF will correlate directly with aggressive histopathological behavior. The aim of this study was to examine this relationship. METHODS: A retrospective chart review was performed for patients treated for BRAF V600E thyroid malignancies from 2019 to 2022 at McGill University tertiary care hospitals (n = 317). Patients with BRAF V600E-positive malignancies that included information on AF were included (n = 44). The correlation between AF and tumor histopathological features was analyzed. RESULTS: Out of the 44 nodules with a BRAF V600E mutation, those with aggressive features of PTC had a mean AF of 25.8%, which was significantly higher than the non-aggressive group with a mean AF of 10.25% (p = 0.020). Additionally, there was a statistically significant difference in mean AF between patients with a positive sentinel LN (29%) and those with a negative sentinel LN (17.8%) (p = 0.021). Classical PTC was present in 29.5% (13/44) of nodules, with a mean AF of 15.6%. The tall cell subtype was found in 64% (28/44) of nodules, with a mean AF of 23%. Solid and hobnail subtypes were less common in this study, and there was no statistically significant relationship between AF and histopathological subtypes (p = 0.107). Nodules smaller than 1cm had a mean AF of 13.3%, while nodules ranging from 1 2cm had a mean AF of 20.6%, and those larger than 2cm had a mean AF of 27.7%. However, no statistical difference was observed between AF and nodule size (p = 0.160). CONCLUSION: In this study, BRAF V600E mutations in conjunction with AF help to determine whether thyroid malignancies will display aggressive behavior. This pre-operative finding can help thyroid specialists to determine the extent of thyroidectomy and whether lymph node dissection is required.

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Prostatitis: ¿infección, alteración neuromuscular o síndrome doloroso? La adecuada clasificación del paciente es clave / Prostatitis: infection, altered or neuromuscular pain syndrome? The proper classification of the patient is key

La prostatitis en un término muy amplio utilizado para describir la inflamación de la próstata con una variedad de síntoma urinarios bajos, molestias en la actividad sexual y disfunción. Es una condición que afecta entre el 5 y 10 por cien de la población masculina y es el diagnostico urológico más común en menores de 50 años. La prostatitis se clasifica en cuatro categorías, incluyendo sus formas aguda y crónica bacterianas, una crónica abacteriana y una asintomática. Las formas bacterianas son más fáciles de diagnosticar y tratar, pero no es usual que los síntomas del paciente estén claramente relacionados con una condición infecciosa, tanto así que la prostatitis crónica abacteriana (también conocida como síndrome de dolor pélvico crónico), es la forma más prevalente y menos entendida, así como también es la que implica un mayor reto diagnóstico y terapéutico. Esta forma de prostatitis puede responder a terapias no centradas en la próstata como terapia física, liberación de puntos gatillo miofasciales y terapias de relajación. Teniendo en cuenta que hay múltiples formas de prostatitis, es necesario tener presentes las múltiples modalidades terapéuticas, hacer un enfoque diagnostico apropiado y hacer un diagnóstico diferencial adecuado , con miras a tener un manejo efectivo.

Terapéutica del dolor / Therapeutic of pain

Conjunto de aportaciones hechas por especialistas en materia del dolor, comenzando por los síntomas, el diagnóstico y los posibles tratamientos existentes en la clasificación que aquí se presenta con el título Terapéutica del dolor, dividida en: 1. Dolor agudo y crónico: aspectos generales 2. Síndromes dolorosos no malignos 3. Origen del dolor maligno 4. Nuevos procedimientos farmacológicos para el tratamiento del dolor 5. Recursos no farmacológicos para el control del dolor