A native mass spectrometry approach to qualitatively elucidate interfacial epitopes of transient protein-protein interactions.

Native mass spectrometric analysis of TPR2A and GrpE with unpurified peptides derived from limited proteolysis of their respective PPI partners (HSP90 C-terminus and DnaK) facilitated efficient, qualitative identification of interfacial epitopes involved in transient PPI formation. Application of this approach can assist in elucidating interfaces of currently uncharacterised transient PPIs.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs. "Crab"-like configuration of two catalytic peptides ("pincers") flanking the recognition oligonucleotide was instrumental here in providing increased catalytic turnover, leading to ≈30-fold decrease in miRNA half-life as compared with that for "single-pincer" conjugates. Dynamic modeling of miRNA cleavage illustrated how such design enabled "Crabs" to drive catalytic turnover through simultaneous attacks at different locations of the RNA-DNA heteroduplex, presumably by producing smaller cleavage products and by providing toeholds for competitive displacement by intact miRNA strands. miRNA cleavage at the 5'-site, spreading further into double-stranded region, likely provided a synergy for RNase H1 through demolition of its loading region, thus facilitating enzyme turnover. Such synergy was critical for sustaining persistent disposal of continually-emerging oncogenic miRNAs. A single exposure to the best structural variant (Crab-p-21) prior to transplantation into mice suppressed their malignant properties and reduced primary tumor volume (by 85 %) in MCF-7 murine xenograft models.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity.

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD - human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117-1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD - hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD - hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions.

Joint pain, physical function, and balance self-confidence in acromegaly versus nonfunctioning pituitary adenoma patients.

CONTEXT: Patients with acromegaly (PWA) experience balance issues, despite achieving biochemical remission, that may significantly impair their quality of life. OBJECTIVE: We sought to assess the prevalence of falls and balance self-confidence in PWA in comparison with a control group. Furthermore, we investigated the effect of joint pain and function as predictors for their balance self-confidence. DESIGN: Cross-sectional, case-controlled. SETTING: Tertiary care centers. PARTICIPANTS: In this case-control study, we surveyed PWA (n = 94) and nonfunctioning pituitary adenoma (PNA; n = 82) with similar age, sex, and body mass index from two Canadian centers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Data were obtained on number of falls during the past 12 months, self-confidence to maintain balance, joint pain, joint surgery, pain medication usage, and upper and lower extremity musculoskeletal disability. RESULTS: While both PWA and PNA had a similarly high risk of falls, PWA had lower self-confidence to maintain balance (P < .01). Patients with acromegaly had higher joint pain scores and more functional impairment in upper extremity, hip, knee, and ankle joints (all P < .01). In both groups, age, sex, and ankle functional score were predictors of balance self-confidence. For PWA, hip functional score was also a predictor of balance self-confidence in contrast to knee and back pain scores being predictors for the PNA group. CONCLUSIONS: We confirmed an increased prevalence of falls in both groups with diminished balance confidence in PWA. This reduced balance self-confidence seems to be related to their increased hip functional impairment in comparison with PNA.

Intraosseous Cavernous Hemangioma of the Middle Turbinate: A Case Report.

BACKGROUND: We report a case of an intraosseous cavernous hemangioma originating from the middle turbinate that expanded into the anterior skull base, without traversing the cribriform plate. METHODS: The mass was found incidentally after a computed tomography head was ordered for unrelated reasons. On questioning, the patient denied any nasal symptoms. Magnetic resonance imaging showed an enhancing mass and the radiological imaging supported a broad differential. RESULTS: The lesion was removed by endoscopic image-guided surgery, and the pathology was that of a benign intraosseous cavernous hemangioma. There was no residual hemangioma on postoperative imaging and the nasal mucosa healed well. This is the first report of an intraosseous cavernous hemangioma of the middle turbinate showing superior expansion to the anterior skull base. CONCLUSION: This case demonstrates the extent to which cavernous hemangiomas may expand into surrounding tissues. While these lesions are uncommon, they can be considered as part of a broad differential diagnosis of sinonasal tumors.

A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally complex natural products with diverse bioactivities. Here we report discovery of a RiPP, kintamdin, for which the structure is determined through spectroscopy, spectrometry and genomic analysis to feature a bis-thioether macrocyclic ring and a ß-enamino acid residue. Biosynthetic investigation demonstrated that its pathway relies on four dedicated proteins: phosphotransferase KinD, Lyase KinC, kinase homolog KinH and flavoprotein KinI, which share low homologues to enzymes known in other RiPP biosynthesis. During the posttranslational modifications, KinCD is responsible for the formation of the characteristic dehydroamino acid residues including the ß-enamino acid residue, followed by oxidative decarboxylation on the C-terminal Cys and subsequent cyclization to provide the bis-thioether ring moiety mediated by coordinated action of KinH and KinI. Finally, conserved genomic investigation allows further identification of two kintamdin-like peptides among the kin-like BGCs, suggesting the occurrence of RiPPs from actinobacteria.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Bulge-Forming miRNases Cleave Oncogenic miRNAs at the Central Loop Region in a Sequence-Specific Manner.

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)2Gly]2 (BC-miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC-miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA. A thorough study of mono- and bis-BC-miRNases (containing one or two catalytic peptides, respectively) revealed that: (i) the sequence of miRNA bulge loops and neighbouring motifs are of fundamental importance for efficient miRNA cleavage (i.e., motifs containing repeating pyrimidine-A bonds are more susceptible to cleavage); (ii) the incorporation of the second catalytic peptide in the same molecular scaffold increases the potency of BC-miRNase, providing a complete degradation of miR-17 within 72 h; (iii) the synergetic co-operation of BC-miRNases with RNase H accelerates the rate of miRNA catalytic cleavage by both the conjugate and the enzyme. Such synergy allows the rapid destruction of constantly emerging miRNA to maintain sufficient knockdown and achieve a desired therapeutic effect.

Establishing the Socio-Economic Impact of Degenerative Cervical Myelopathy Is Fundamental to Improving Outcomes [AO Spine RECODE-DCM Research Priority Number 8].

STUDY DESIGN: Literature Review (Narrative). OBJECTIVE: To contextualize AO Spine RECODE-DCM research priority number 5: What is the socio-economic impact of DCM? (The financial impact of living with DCM to the individual, their supporters, and society as a whole). METHODS: In this review, we introduce the methodology of health-economic investigation, including potential techniques and approaches. We summarize the current health-economic evidence within DCM, so far focused on surgical treatment. We also cover the first national estimate, in partnership with Myelopathy.org from the United Kingdom, of the cost of DCM to society. We then demonstrate the significance of this question to advancing care and outcomes in the field. RESULTS: DCM is a common and often disabling condition, with a significant lack of recognition. While evidence demonstrates the cost-effectives of surgery, even among higher income countries, health inequalities exist. Further the prevalent residual disability in myelopathy, despite treatment affects both the individual and society as a whole. A report from the United Kingdom provides the first cost-estimate to their society; an annual cost of ∼£681.6 million per year, but this is likely a significant underestimate. CONCLUSION: A clear quantification of the impact of DCM is needed to raise the profile of a common and disabling condition. Current evidence suggests this is likely to be globally substantial.

"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

A native mass spectrometry platform identifies HOP inhibitors that modulate the HSP90-HOP protein-protein interaction.

Herein we describe a native mass spectromery protein-peptide model as a competent surrogate for the HOP-HSP90 protein-protein interaction (PPI), application of which led to the qualititive identification of two new peptides capable of in vitro PPI disruption. This proof of concept study offers a viable alternative for PPI inhibitor screening.

Staged reflexive artificial intelligence driven testing algorithms for early diagnosis of pituitary disorders.

BACKGROUND: Sellar masses (SM) frequently present with insidious hormonal dysfunction. We previously showed that, by utilizing a combined reflex/reflecting approach involving a laboratory clinician (LC) on common endocrine test results requested by non-specialists, and subsequently adding further warranted tests, previously undiagnosed pituitary disorders can be identified. However, manually employing these strategies by an LC is not feasible for wider screening of pituitary disorders. OBJECTIVE: The aim of this study was to compare the accuracy and financial impact of an Artificial Intelligence (AI) based, fully computerized reflex protocol with manual reflex/reflective intervention protocol led by an LC. METHODS: We developed a proof-of-concept AI-based framework to fully computerize multi-stage reflex testing protocols for pituitary dysfunction using automated reasoning methods. We compared the efficacy of this AI-based protocol with a reflex/reflective protocol based on manually curated retrospective data in identifying pituitary dysfunction based on 12 months of laboratory testing. RESULTS: The AI-based reflex protocol, as compared with the manual protocol, would have identified laboratory tests for add-on that either directly matched or included all manual add-on tests in 92% of cases, and recommended a similar specialist referral in 90% of the cases. The AI-based protocol would have issued 2.8 times the total number of manual add-on laboratory tests at an 85% lower operation cost than the manual protocol when considering marginal test costs, technical staff and specialist salary. CONCLUSION/DISCUSSION: Our AI-based reflex protocol can successfully identify patients with pituitary dysfunction, with lower estimated laboratory cost. Future research will focus on enhancing the protocol's accuracy and incorporating the AI-based reflex protocol into institutional laboratory and hospital information systems for the detection of undiagnosed pituitary disorders.

Loss of SNAI2 in Prostate Cancer Correlates With Clinical Response to Androgen Deprivation Therapy.

PURPOSE: Androgen receptor (AR) signaling is important in prostate cancer progression, and therapies that target this pathway have been the mainstay of treatment for advanced disease for over 70 years. Tumors eventually progress despite castration through a number of well-characterized mechanisms; however, little is known about what determines the magnitude of response to short-term pathway inhibition. METHODS: We evaluated a novel combination of AR-targeting therapies (degarelix, abiraterone, and bicalutamide) and noted that the objective patient response to therapy was highly variable. To investigate what was driving treatment resistance in poorly responding patients, as a secondary outcome we comprehensively characterized pre- and post-treatment samples using both whole-genome and RNA sequencing. RESULTS: We find that resistance following short-term treatment differs molecularly from typical progressive castration-resistant disease, associated with transcriptional reprogramming, to a transitional epithelial-to-mesenchymal transition (EMT) phenotype rather than an upregulation of AR signaling. Unexpectedly, tolerance to therapy appears to be the default state, with treatment response correlating with the prevalence of tumor cells deficient for SNAI2, a key regulator of EMT reprogramming. CONCLUSION: We show that EMT characterizes acutely resistant prostate tumors and that deletion of SNAI2, a key transcriptional regulator of EMT, correlates with clinical response.

Improved identification and quantification of peptides in mass spectrometry data via chemical and random additive noise elimination (CRANE).

MOTIVATION: The output of electrospray ionization-liquid chromatography mass spectrometry (ESI-LC-MS) is influenced by multiple sources of noise and major contributors can be broadly categorized as baseline, random and chemical noise. Noise has a negative impact on the identification and quantification of peptides, which influences the reliability and reproducibility of MS-based proteomics data. Most attempts at denoising have been made on either spectra or chromatograms independently, thus, important 2D information is lost because the mass-to-charge ratio and retention time dimensions are not considered jointly. RESULTS: This article presents a novel technique for denoising raw ESI-LC-MS data via 2D undecimated wavelet transform, which is applied to proteomics data acquired by data-independent acquisition MS (DIA-MS). We demonstrate that denoising DIA-MS data results in the improvement of peptide identification and quantification in complex biological samples. AVAILABILITY AND IMPLEMENTATION: The software is available on Github (https://github.com/CMRI-ProCan/CRANE). The datasets were obtained from ProteomeXchange (Identifiers-PXD002952 and PXD008651). Preliminary data and intermediate files are available via ProteomeXchange (Identifiers-PXD020529 and PXD025103). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Host-Guest Induced Peptide Folding with Sequence-Specific Structural Chirality.

Controlling the spatial and temporal behavior of peptide segments is essential in the fabrication of functional peptide-based materials and nanostructures. To achieve a desired structure, complex sequence design is often required, coupled with the inclusion of unnatural amino acids or synthetic modifications. Herein, we investigate the structural properties of 1:1 inclusion complexes between specific oligopeptides and cucurbit[8]uril (CB[8]), inducing the formation of turns, and by alteration of the peptide sequence, tunable structural chirality. We also explore extended peptide sequence binding with CB[8], demonstrating a simple approach to construct a peptide hairpin.

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, ß-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

Preclinical Evaluation of the Stealth Autoguide Robotic Guidance Device for Stereotactic Cranial Surgery: A Human Cadaveric Study.

INTRODUCTION: Stereotactic procedures are routinely performed for brain biopsies, deep brain stimulation, and placement of stereoelectroencephalography (SEEG) electrodes for epilepsy. The recently developed Stealth Autoguide (Medtronic, Minneapolis, MN, USA) device does not require patients to don a stereotactic frame. In this preclinical study, we sought to quantitatively compare the Stealth Autoguide robotic system to 2 devices commonly used in clinical practice: the Navigus biopsy system (Medtronic) and the Leksell stereotactic frame (Elekta Ltd., Stockholm, Sweden). METHODS: In the first experimental setup, we compared target accuracy of the Stealth Autoguide to the Navigus system by using phantom heads filled with gelatin to simulate the brain tissue. In the second experimental setup, we inserted SEEG electrodes to targets within cadaveric heads in a simulated operating room environment. RESULTS: Using a homogeneous gelatin-filled phantom 3D reconstruction of a human head, we found that using the Stealth Autoguide system, while maintaining accuracy, was faster to use than the Navigus system. In our simulated operating room environment using nonliving human cadaveric heads, we found the accuracy of the Stealth Autoguide robotic device to be comparable to that of the Leksell frame. DISCUSSION/CONCLUSION: These results compare the use of the Stealth Autoguide robotic guidance system with commonly used stereotactic devices, and this is the first study to compare its use and accuracy with the Leksell frame. These findings provide mounting evidence that Stealth Autoguide will have potential clinical uses in various stereotactic neurosurgical procedures.