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Pain is a common symptom in patients with cancer. Up to two thirds of patients with cancer experience pain that requires a strong opioid for pain relief. Cancer pain management can be challenging as the pain experience differs among patients. A patient’s response to opioids and other analgesia depends on several factors such as the character and cause of pain, and the pharmacodynamics and pharmacokinetics of the drug. Optimizing pain control is important, as pain can impact negatively on many aspects of a patient’s life such as mood, sleep, social interactions, cognition and physical function, resulting in physical, emotional and existential suffering. We will use the following case study to illustrate the process of pain assessment and the importance of understanding the aetiology of the pain in a patient with cancer. We will also discuss the pharmacological and non-pharmacological management of cancer pain.

ObjectiveThis work aims to determine the AVEM duration and number of captured seizures required to resolve different clinical questions, using a retrospective review of ictal recordings.MethodsPatients who underwent home-based AVEM had event data analyzed retrospectively. Studies were grouped by clinical indication: seizure differential diagnosis, classification, or treatment assessment. The proportion of studies where the conclusion was changed after the first seizure was determined, as was the AVEM duration needed for at least 99% of studies to reach a diagnostic conclusion.ResultsThe referring clinical question was not answered entirely by the first event in 29.56% (n=227) of studies. Diagnostic and classification indications required a minimum of 7 days for at least 99% of studies to be answered, whilst treatment-assessment required at least 6 days.ConclusionsAt least 7 days of monitoring, and potentially multiple events, are required to adequately answer these clinical questions in at least 99% of patients. The widely applied 72 hours or single event recording cut-offs may be insufficient to correctly answer these three indications in a substantial proportion of patients.SignificanceExtended duration of monitoring and capturing multiple events should be considered when attempting to capture seizures on AVEM.

Many arguments have been advanced for the integration of mathematics and science education including economic arguments, integration being logical, being engaging, increasing transfer, increasing conceptual learning, and that the real world is interdisciplinary in nature. This chapter explores some of the issues and contradictions with each of these arguments drawing on Bernstein's theory of boundaries. An example of integration in the policy sphere (STEM policy in England) is first discussed, and some of the tensions arising are explored. Crossing the boundary is more challenging than is often implied in discussions of integration with issues of epistemology, status, and language needing to be addressed. Further, integration may not yield the expected benefits and could even decrease conceptual learning in the disciplines. The author argues that the policy context should be considered when advocating integration and that careful consideration be given as to whether integration is genuinely the most appropriate solution to identified educational issues.

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion inAkkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology.IMPORTANCETaken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2 it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

ObjectivesRecording electrographic and behavioral information during epileptic and other paroxysmal events is important during video EEG monitoring. This study was undertaken to measure the event capture rate of an ambulatory service operating across Australia using a shoulder-worn EEG device and telescopic pole-mounted camera.MethodsNeurologist reports were accessed retrospectively. Studies with confirmed events were identified and assessed for event capture by recording modality, whether events were reported or discovered, and wakefulness.Results6,265 studies were identified, of which 2,788 (44.50%) had events. A total of 15,691 events were captured, of which 77.89% were reported. The EEG-ECG amplifier was active for 99.83% of events. The patient was in view of the camera for 94.90% of events. 84.89% of studies had all events on camera, and 2.65% had zero events on camera (mean=93.66%, median=100.00%). 84.42% of events from wakefulness were reported, compared to 54.27% from sleep.ConclusionEvent capture was similar to previously reported rates from ambulatory studies, with higher capture rates on video. Most patients have all events captured on camera.SignificanceAmbulatory monitoring is capable of high rates of event capture, and the use of wide-angle cameras allows for all events to be captured in the majority of studies.HighlightsA review was undertaken of an Australia-wide ambulatory video-EEG monitoring servicePatients were in view of camera for 94.90% of events, and 84.89% of studies had all events on camera84.42% of events from wakefulness were reported, compared to 54.27% from sleep

SummaryFerroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Ferroptosis is driven by excessive iron-dependent peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid. Here, we reveal that an understudied Golgi membrane scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells. Upregulation of MMD correlates with sensitization to ferroptosis upon monocyte-to-macrophage differentiation. Mechanistically, MMD interacts with ACSL4 and MBOAT7, two enzymes that catalyze consecutive reactions in the biosynthesis of phosphatidylinositol (PI) containing arachidonic acid. MMD increases cellular levels of arachidonoyl-phospholipids and heightens susceptibility to ferroptosis in an ACSL4- and MBOAT7-dependent manner. We propose that MMD potentiates the synthesis of arachidonoyl-PI by bridging ACSL4 with MBOAT7. This molecular mechanism not only clarifies the biochemical underpinnings of ferroptosis susceptibility, with potential therapeutic implications, but also contributes to our understanding of the regulation of cellular lipid metabolism.Graphical Abstract

The twenty-first century brought unprecedented rates of teenage psychological disorder (PD), with conditions such as depression, anxiety, and schizophrenia reaching all-time highs. Young adolescents, despite having been recognized as a psychologically vulnerable demographic, are constantly excluded in their own mental health evaluations, and teachers in the American education system play a pivotal role in addressing this gap. Thus, in this paper, I specifically explore how high school students and teachers perceive teenage PD by examining their opinions on literary portrayals of mentally ill adolescents. This article thoroughly investigates this inquiry by utilizing quantitative, open-ended survey research followed by a thematic and comparative analysis. A total of 43 students and 24 teachers participated in the study, and I identified five recurring themes throughout their responses: the validity of teenagers’ struggles, PD’s relation to teenagers’ school life, PD’s relation to teenagers’ social life, trust in professional treatment, and the potential of recovery. My findings demonstrated that although both groups showed a mutual concern for the mental welfare of teenagers, there were stark differences between students’ and teachers’ perceptions of teenage PD. These results present a fundamental ideological dichotomy that must be bridged in order to help adolescents benefit from their schools, and an implementation of comprehensive mental health and social and emotional well-being (SEWB) training for all teachers may be the first step to achieving this goal.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease, notably cancer. Since their discovery, several mechanisms of RTK dysregulation have been identified, resulting in multiple cancer types displaying 'oncogenic addiction' to RTKs. As a result, RTKs have represented a major class for targeted therapeutics over the past two decades, with numerous small molecule-based tyrosine kinase inhibitor (TKI) therapeutics having been developed and clinically approved for several cancers. However, many of the current RTK inhibitor treatments eventually result in the rapid development of acquired resistance and subsequent tumor relapse. Recent technological advances and tools are being generated for the identification of novel RTK small molecule therapeutics. These newer technologies will be important for the identification of diverse types of RTK inhibitors, targeting both the receptors themselves as well as key cellular factors that play important roles in the RTK signaling cascade.

Immune checkpoint inhibitors target suppressor receptors, including cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1). The activated T cells are not antigen specific; therefore, the blockade of the immune checkpoint may result in the development of autoimmune adverse events. The most common immune-related adverse events (irAEs) are rash, colitis, and endocrinopathies. However, irAEs that affect the hematologic system are rare and can affect red blood cells (e.g., autoimmune hemolytic anemia), white blood cells, and platelets (e.g., immune thrombocytopenia). Usually one cell line is affected; however, in some cases, multiple cell lines can be affected. Other changes in the hematologic system can also be affected (e.g., cryoglobulinemia, cytokine release syndrome). Due to the rarity and lack of recognition of these AEs, the timing, spectrum of events, and clinical presentation are poorly understood. Management of hematologic irAEs usually involves the use of steroids; however, other agents (e.g., IVIG, cyclosporine, rituximab) or procedures (e.g., plasma exchange, transfusions) can also be used.

Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor

The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.

Nematode parasites of humans, livestock and crops pose a significant burden on human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we present a pipeline that exploits multiple motor outputs of the model nematode C. elegans for nematicide discovery. This pipeline yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call Nementin. We find that Nementin agonizes neuronal dense core vesicle release, which in turn agonizes cholinergic signaling. Consequently, Nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase inhibitors (AChEIs), but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChEI pesticides used to control nematode infections and infestations.Significance StatementParasitic nematodes pose a considerable burden to human health and food security. Small molecules that have traditionally been used to control these parasites have either been banned because of toxicity concerns or are being rendered ineffective because of the evolution of resistance. Significant gaps in our nematicidal toolkit are therefore becoming an alarming problem. Here, we describe our discovery of Nementin, a small molecule that disrupts the nematode nervous system but is ineffective against non-targeted organisms. We find that Nementin also enhances the activity of non-selective pesticides but does so in a nematode-selective manner. Hence, Nementin is an innovative solution to combat parasitic nematodes in a safe and phylum-selective manner.One-Sentence SummaryA C. elegans-based screening pipeline identifies a selective nematicide that also potentiates acetylcholinesterase inhibitors.

The majority of germ cell tumors (GCT) arise from the testicles and are often self-diagnosed. Extra-gonadal germ cell tumors (EGGCT) are rare and vary greatly in their clinical presentations. This case report describes a 24-year-old male with an unusual presentation for an EGGCT.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes. This work was supported by European Union FP7 Grant No. 278568 “PRIMES” and Science Foundation Ireland Investigator Program Grant 14/IA/2395 to W.K. B.K. is supported by SmartNanoTox (Grant no. 686098), NanoCommons (Grant no. 731032), O.R. by MSCA-IF-2016 SAMNets (Grant no. 750688). D.M. is supported by Science Foundation Ireland Career Development award 15-CDA-3495. I.J. is supported by the Canada Research Chair Program (CRC #225404), Krembil Foundation, Ontario Research Fund (GL2-01-030 and #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), and IBM. O.S. is supported by ERC investigator Award ColonCan 311301 and CRUK. I.S. is supported by the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/ DIG-501411 & RE08-009), Consortium Québécois sur la Découverte du Médicament (CQDM Quantum Leap) & Brain Canada (Quantum Leap), and CQDM Explore and OCE (#23929). T.C. was supported by a Teagasc Walsh Fellowship

Leptomeningeal disease in patients with melanoma historically portends a grim prognosis, with median survival measured in weeks to months. The advent of effective immunotherapy and targeted agents may modify the outcome of such patients. This case report describes a 43-year-old patient diagnosed with stage IIIa BRAF-positive cutaneous melanoma in 2012 who subsequently developed leptomeningeal involvement as her sole site of melanotic metastasis. She received multiple systemic therapies and radiotherapy and survived 2.5 years after her diagnosis with central nervous system involvement. This case report highlights the importance of a multidisciplinary team and the advent of effective agents, which offers the potential for significantly improved outcomes for patients with metastatic melanoma involving the central nervous system.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The disease-initiating event in most CRC cases is the loss of function (LOF) of the adenomatous polyposis coli (APC) gene, accounting for 70-80% of CRC. Strategies to target cells that harbor the truncation mutation in APC, which leads to its LOF, may provide the opportunity to intercept tumorigenesis in CRC patients. High-risk CRC populations include patients with familial adenomatous polyposis (FAP), the inherited disorder caused by germline APC mutations. Aurora Kinase (AK), a family of serine/threonine kinases, is essential for proper cell division, and overexpression of AK has been associated with multiple heme and solid tumor malignancies, including CRC. Aurora kinases have been well-studied, both preclinically and clinically, as a target for therapeutic intervention across multiple cancers; however, the role of AK in CRC disease progression has not been fully explored. Here we report a synthetic lethal dependency between APC and AK which was identified in a human isogenic colon organoid model containing a genetically engineered APC LOF (APCLOF). Aurora A-specific, Aurora B-specific, and Pan-AK inhibitors were evaluated in isogenic APCLOF or APCWT colon organoids derived from multiple donors. All AK inhibitors demonstrated selective growth inhibition of the APCLOF organoid. The strongest synthetic lethal effect was observed with a pan-AK inhibitor showing greater than 1000-fold selectivity in the APCLOF colon organoids compared to APCWT. Interestingly, this activity diminished as oncogenic driver mutations in KRAS and/or p53 genes were introduced into the APCLOF organoids. These findings suggest that therapeutic benefit of AK inhibitors would be greater in early APCLOF driven adenomas as opposed to advanced CRC. Despite these encouraging results indicating therapeutic opportunity in early adenoma disease, on-target genotoxicity was observed in vitro through increases in micronuclei at pharmacologic doses. Therefore, although there is a high unmet need in FAP for therapeutic options, potential genotoxicity associated with AK inhibitor treatments may limit utility as an intervention strategy. Citation Format: Dana S. Gaffney, Eileen Vesely, David Pocalyko, Kurtis Bachman, Karla Wiehagen, Glenn Cowley, Victoria Wong, Prashanth Gokare, Peggy Guzzie-Peck, Sandy Weiner, Ann De Smedt, Jacqueline Kinyamu-Akunda, Tammy Bush, Zhuming Zhang, Robert Schulingkamp, Sylvia F. Boj, Rene Overmeer, Carla Verissimo, Roshni Nair. Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1416.

The author reflects on her decades of cultural and political activism in the fight for Ethnic Studies—from her role in the 1969 Third World Strike at UC Berkeley, to her community activism after her graduation, to her participation in the fiftieth anniversary of the strike, including a transcription of her speech at the event.

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.

Surface structures that trap light leading to near complete structural absorption creates an appearance of "super black.'' Well known in the natural world from bird feathers and butterfly scales, super black has evolved independently from various anatomical structures. Due to an exceptional ability to reduce specular reflection, these biological materials have garnered interest from optical industries. Here we describe the false eyes of the eyed elater click beetle, which, while not classified as super black, still attains near complete absorption of light partly due to an array of vertically-aligned microtubules. These cone-shaped microtubules are modified hairs (setae) that are localized to eyespots on the dorsum of the beetle, and absorb 96.1% of incident light (at a 24.8 degrees collection angle) in the spectrum between 300-700 nm. Filled with melanin, the setae combine structure and pigment to generate multiple reflections and refractions causing light to travel a greater distance. This light-capturing architecture leaves little light available to receivers and the false eyes appear as deep black making them appear more conspicuous to predators. National Science Foundation (DBI)National Science Foundation (NSF) [1458045]; Virginia Tech, Department of Entomology, College of Life Sciences; USDA NIFA Hatch Project [VA-160028] This work was supported by the National Science Foundation (DBI #1458045), and by Virginia Tech, Department of Entomology, College of Life Sciences, and by a USDA NIFA Hatch Project (VA-160028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2' deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Background: There are frequent calls in the literature for school science and mathematics departments to collaborate, largely in response to perceived overlaps between the two subjects in the context of Science, Technology, Engineering and Mathematics (STEM). Yet few studies explore how such collaborations might work. This paper is unusual both in its focus on mathematics/science collaborations which have not arisen from a specific short-term intervention and in its focus on the views of practising teachers rather than policy-makers or curriculum developers. Purpose: We ask how and why collaborations get started and explore how mathematics and science departments actually work together in secondary (high) schools in England. We ask what some of the affordances and challenges are in both initiating and sustaining collaborative practice. Design and methods: Six schools were identified and visited and semi-structured interviews carried out with the 15 teachers most closely involved in collaborating, to explore their perspectives and insights. Results: The findings show that collaborations are possible, though they are challenging to sustain, and they can be approached in a number of ways. Mathematics/science collaboration can be a key site of professional learning for teachers, particularly about the ‘other’ curriculum. Informal conversations across departments were highly valued but tended to be between those with a well-established pre-existing relationship. While physical structures can promote collaboration, it needs strong support from senior leadership teams to both begin and continue. Conclusion: Contrary to the commonly espoused view that there are many overlaps between mathematics and science in school, it can be a significant challenge for teachers to find them. Collaboration is neither straightforward to begin nor to sustain. Researchers and policy-makers should thus be cautious about recommending collaboration as straightforward for science and mathematics teachers to adopt. These conclusions provide a major challenge to simplistic advocacy of STEM in schools.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes., Kras is often mutated in colorectal cancer but how this oncogenic mutation alters signalling pathways globally is undetermined. Here, the authors analyse how this mutation affects protein interaction networks and signal flow showing an extensive re‐wiring of signalling in response to KRas mutation

The accurate determination of protein–protein interactions has been an important focus of molecular biology toward which much progress has been made due to the continuous development of existing and new technologies. However, current methodscan have limitations, including scale and restriction to high affinity interactions, limiting our understanding of a large subset of these interactions. Here, we describe a modified bacterial-hybrid assay that employs combined selectable and scalable reporters that enable the sensitive screening of large peptide libraries followed by the sorting of positive interactions by the level of reporter output. We have applied this tool to characterize a set of human and E.coli PDZ domains. Our results are consistent with prior characterization of these proteins, and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the Membrane Yeast Two-Hybrid (MYTH) and the Mammalian Membrane Two-Hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.