Mutant clones in normal epithelium outcompete and eliminate emerging tumours.

Human epithelial tissues accumulate cancer-driver mutations with age1-9, yet tumour formation remains rare. The positive selection of these mutations suggests that they alter the behaviour and fitness of proliferating cells10-12. Thus, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less competitive neighbours11-14. However, little is known about how such dynamic competition in normal epithelia influences early tumorigenesis. Here we show that the majority of newly formed oesophageal tumours are eliminated through competition with mutant clones in the adjacent normal epithelium. We followed the fate of nascent, microscopic, pre-malignant tumours in a mouse model of oesophageal carcinogenesis and found that most were rapidly lost with no indication of tumour cell death, decreased proliferation or an anti-tumour immune response. However, deep sequencing of ten-day-old and one-year-old tumours showed evidence of selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased early tumour removal, whereas pharmacological inhibition of clonal competition reduced tumour loss. These results support a model in which survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the surrounding normal tissue. Mutant clones in normal epithelium have an unexpected anti-tumorigenic role in purging early tumours through cell competition, thereby preserving tissue integrity.

Identifying eating disorders at the earliest opportunity: Testing the reliability of an online eating disorder screener (IOI-S) in primary care and youth mental health settings.

AIM: Eating disorders (EDs) are associated with significant disease burden and unacceptably high mortality rates. Early intervention significantly improves prognosis and can prevent chronic suffering; however, large numbers of people with the illness are not being identified or managed in primary healthcare. The current study aimed to test the reliability of the face-to-face, clinician delivery of a previously validated, co-designed, online screening tool for eating disorders. METHODS: Individuals aged 14 and over who read, English were recruited from the community in either primary care (general practice) settings or headspace youth mental health centres. They completed the InsideOut Institute Screener (IOI-S) face-to-face, delivered verbally by the study researcher clinician and then online by self-report. The primary outcome was test-retest reliability as measured by two-way mixed effects model Intraclass Correlation Coefficient (ICC) with absolute agreement. RESULTS: A total of 83 participants aged 14-81 (M 36.2) completed the study in New South Wales and the Northern Territory, Australia, between April and November 2022. The ICC between successive iterations of the test was significantly positive (0.980), demonstrating strong internal validity and test-retest reliability of the scale. CONCLUSIONS: The IOI-S is an adaptive 6-item screening tool designed to 'start a conversation' and determine risk using gentle language conceived by individuals with lived experience. Originally designed for online use, the current study broadens its versatility to clinical settings. The screener performs equally well when delivered face-to-face in clinical practice. In conjunction with increased practitioner education and improved treatment referral pathways, broad implementation of the screener in early healthcare settings can support timely identification and intervention for those with EDs.

Effects on blood pressure and cardiovascular risk of variations in patients' adherence to prescribed antihypertensive drugs: role of duration of drug action.

AIM: To determine the effects of imperfect adherence (i.e. occasionally missing prescribed doses), and the influence of rate of loss of antihypertensive effect during treatment interruption, on the predicted clinical effectiveness of antihypertensive drugs in reducing mean systolic blood pressure (SBP) and cardiovascular disease (CVD) risk. METHOD: The effects of imperfect adherence to antihypertensive treatment regimens were estimated using published patterns of missed doses, and taking into account the rate of loss of antihypertensive effect when doses are missed (loss of BP reduction in mmHg/day; the off-rate), which varies between drugs. Outcome measures were the predicted mean SBP reduction and CVD risk, determined from the Framingham Risk Equation for CVD. RESULTS: In patients taking 75% of prescribed doses (typical of clinical practice), only long-acting drugs with an off-rate of ∼1 mmHg/day were predicted to maintain almost the full mean SBP-lowering effect throughout the modelled period. In such patients, using shorter-acting drugs (e.g. an off-rate of ∼5-6 mmHg/day) was predicted to lead to a clinically relevant loss of mean SBP reduction of > 2 mmHg. This change also influenced the predicted CVD risk reduction; in patients with a baseline 10-year CVD risk of 27.0% and who were taking 75% of prescribed doses, a difference in off-rate from 1 to 5 mmHg/day led to a predicted 0.5% absolute increase in 10-year CVD risk. CONCLUSIONS: In patients who occasionally miss doses of antihypertensives, modest differences in the rate of loss of antihypertensive effect following treatment interruption may have a clinically relevant impact on SBP reduction and CVD risk. While clinicians must make every effort to counsel and encourage each of their patients to adhere to their prescribed medication, it may also be prudent to prescribe drugs with a low off-rate to mitigate the potential consequences of missing doses.

Study protocol for an open labelled randomised controlled trial of perioperative oral nutrition supplement in breast and colorectal cancer patients undergoing elective surgery.

BACKGROUND: While it is well established that perioperative use of oral nutrition supplement (ONS) improves nutrition status among severely malnourished surgical cancer patients, the evidence requires further substantiation for non-severely malnourished patients with cancer. This protocol paper presents the rationale and design of a randomised controlled trial to evaluate the effectiveness of preoperative as well as an extended 90-day postoperative use of ONS on nutritional and clinical outcomes among patients undergoing elective surgery for breast and colorectal cancer. METHODS: Patients with primary breast and colorectal cancer undergoing elective surgery are recruited from two tertiary hospitals. Eligible patients are assigned into one of the three intervention arms: (i) Group SS will receive ONS in addition to their normal diet up to 14 days preoperatively and postoperatively up to discharge; (ii) Group SS-E will receive ONS in addition to their normal diet up to 14 days preoperatively, postoperatively up to discharge and for an extended 90 days after discharge; and (iii) Group DS will receive ONS in addition to their normal diet postoperatively up to discharge from the hospital. The ONS is a standard formula fortified with lactium to aid in sleep for recovery. The primary endpoints include changes in weight, body mass index (BMI), serum albumin and prealbumin levels, while secondary endpoints are body composition (muscle and fat mass), muscle strength (handgrip strength), energy and protein intake, sleep quality, haemoglobin, inflammatory markers (transferrin, high sensitivity C-reactive protein, interleukin-6), stress marker (saliva cortisol), length of hospital stay and postoperative complication rate. DISCUSSION: This trial is expected to provide evidence on whether perioperative supplementation in breast and colorectal cancer patients presenting with high BMI and not severely malnourished but undergoing the stress of surgery would be beneficial in terms of nutritional and clinical outcomes. TRIAL REGISTRATION: ClinicalTrial.gov NCT04400552. Registered on 22 May 2020, retrospectively registered.

Localization of cyclic GMP and cyclic AMP in cardiac and skeletal muscle: immunocytochemical demonstration.

When rat cardiac and skeletal muscle are explored by immunocytochemical procedures designed to show sites of localization of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP), distinct staining patterns for the two nucleotides are seen. Antibody to cyclic AMP is found in the area of the sarcoplasmic reticulum, while antibody to cyclic GMP is found with a periodic distribution corresponding to that of the A band. This suggests a role for cyclic GMP in the regulation of myosin.

Issues in blood pressure control and the potential role of single-pill combination therapies.

Hypertension (HTN) is a major risk factor for cardiovascular mortality, yet only a small proportion of hypertensive individuals receive appropriate therapy and achieve target blood pressure (BP) values. Factors influencing the success of antihypertensive therapy include physicians' acceptance of guideline BP targets, the efficacy and tolerability of the drug regimen, and patient compliance and persistence with therapy. It is now well recognised that most hypertensive patients require at least two antihypertensive agents to achieve their target BP. However, complicated treatment regimens are a major contributory factor to poor patient compliance. The use of combination therapy for HTN offers a number of advantages over the use of monotherapy, including improved efficacy, as drug combinations with a synergistic mechanism of action can be used. This additive effect means that lower doses of the individual components can be used, which may translate into a decreased likelihood of adverse events. The use of single-pill combination therapy, in which two or more agents are combined in a single dosage form, offers all the benefits of free combination therapy (improved efficacy and tolerability over monotherapy) together with the added benefit of improved patient compliance because of the simplified treatment regimen. The use of single-pill combination therapy may also be associated with cost savings compared with the use of free combinations for reasons of cheaper drug costs, fewer physician visits and fewer hospitalisations for uncontrolled HTN and cardiovascular events. Thus, the use of single-pill combination therapy for HTN should help improve BP goal attainment through improved patient compliance, leading to reduced costs for cardiovascular-related care.

3D segmentation and quantification of a masticatory muscle from MR data using patient-specific models and matching distributions.

A method is proposed for 3D segmentation and quantification of the masseter muscle from magnetic resonance (MR) images, which is often performed in pre-surgical planning and diagnosis. Because of a lack of suitable automatic techniques, a common practice is for clinicians to manually trace out all relevant regions from the image slices which is extremely time-consuming. The proposed method allows significant time savings. In the proposed method, a patient-specific masseter model is built from a test dataset after determining the dominant slices that represent the salient features of the 3D muscle shape from training datasets. Segmentation is carried out only on these slices in the test dataset, with shape-based interpolation then applied to build the patient-specific model, which serves as a coarse segmentation of the masseter. This is first refined by matching the intensity distribution within the masseter volume against the distribution estimated from the segmentations in the dominant slices, and further refined through boundary analysis where the homogeneity of the intensities of the boundary pixels is analyzed and outliers removed. It was observed that the left and right masseter muscles' volumes in young adults (28.54 and 27.72 cm(3)) are higher than those of older (ethnic group removed) adults (23.16 and 22.13 cm(3)). Evaluation indicates good agreement between the segmentations and manual tracings, with average overlap indexes for the left and right masseters at 86.6% and 87.5% respectively.

Spectrogram denoising and automated extraction of the fundamental frequency variation of dolphin whistles.

Marine mammal vocalizations are often analyzed using time-frequency representations (TFRs) which highlight their nonstationarities. One commonly used TFR is the spectrogram. The characteristic spectrogram time-frequency (TF) contours of marine mammal vocalizations play a significant role in whistle classification and individual or group identification. A major hurdle in the robust automated extraction of TF contours from spectrograms is underwater noise. An image-based algorithm has been developed for denoising and extraction of TF contours from noisy underwater recordings. An objective procedure for measuring the accuracy of extracted spectrogram contours is also proposed. This method is shown to perform well when dealing with the challenging problem of denoising broadband transients commonly encountered in warm shallow waters inhabited by snapping shrimp. Furthermore, it would also be useful with other types of broadband transient noise.

Masseter segmentation using an improved watershed algorithm with unsupervised classification.

The watershed algorithm always produces a complete division of the image. However, it is susceptible to over-segmentation and sensitivity to false edges. In medical images this leads to unfavorable representations of the anatomy. We address these drawbacks by introducing automated thresholding and post-segmentation merging. The automated thresholding step is based on the histogram of the gradient magnitude map while post-segmentation merging is based on a criterion which measures the similarity in intensity values between two neighboring partitions. Our improved watershed algorithm is able to merge more than 90% of the initial partitions, which indicates that a large amount of over-segmentation has been reduced. To further improve the segmentation results, we make use of K-means clustering to provide an initial coarse segmentation of the highly textured image before the improved watershed algorithm is applied to it. When applied to the segmentation of the masseter from 60 magnetic resonance images of 10 subjects, the proposed algorithm achieved an overlap index (kappa) of 90.6%, and was able to merge 98% of the initial partitions on average. The segmentation results are comparable to those obtained using the gradient vector flow snake.

Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis.

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.

Control and localization of rat adrenal cyclic guanosine 3', 5'-monophosphate. Comparison with adrenal cyclic adenosine 3', 5'-monophosphate.

Cyclic AMP and cyclic GMP were measured in rat adrenal glands after either hypophysectomy alone or after hypophysectomy and treatment with ACTH. Adrenal cyclic GMP levels rise in acutely hypophysectomized rats to a maximum at 1 h of approximately 200% of control levels; there is a return to base line at 4-12 h after hypophysectomy. In contrast, adrenal cyclic AMP falls immediately to about 50% of control levels after hypophysectomy and remains at approximately 1 pmol per mg tissue. Doses of ACTH beyond the physiological range markedly suppress adrenal cyclic GMP while producing a 50-fold or greater rise in cyclic AMP in hypophysectomized rats. This pattern of adrenal cyclic GMP rise was unchanged in acutely hypophysectomized animals treated with desamethasone. N-6-2'-0 dibutyryl cyclic AMP acted similarly to the effect of ACTH in bringing about a suppression of adrenal cyclic GMP levels. Physiological i.v. pulse doses of ACTH produced a rapid dose related increase in adrenal cyclic GMP. In vitro incubation of quartered adrenal pairs with 500 mU ACTH produced elevated cyclic AMP levels and suppression of cyclic GMP. Whereas adrenal cyclic AMP fell rapidly to 50% of control levels after hypophysectomy and remained at about 1 pmol per mg tissue for 7 days, adrenal cyclic GMP showed a biphasic rhythm in long-term hypophysectomized animals. After an initial peak at 1 h after hypophysectomy, adrenal cyclic GMP declined to baseline at 4-12 h but thereafter progressively rose with time, eventually reaching levels over 1 pmol per mg tissue. Fluorescent immunocytochemical staining of rat adrenal zona fasciculata showed cyclic AMP largely confined to cytoplasmic elements with little fluorescence contained in nuclei. In constant, cyclic GMP was found discretely positioned in nuclei with prominent fluorescence in nucleoli in addition to cytoplasmic localization. It is concluded that in hypophysectomized rats ACTH, either directly or in conjunction with altertion of adrenal cyclic AMP, appears to be one factor which regulates adrenal cyclic GMP. The direction of cyclic GMP change and the different subcellular localization of the nucleotides suggest divergent roles for cyclic AMP and cyclic GMP in adrenocortical function. Furthermore, our observations suggest a role for adrenal cyclic GMP in nuclear directed events.

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling.

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.

Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade.

The receptor tyrosine kinase RET functions as the signal transducing receptor for the GDNF (for "glial cell-derived neurotrophic factors") family of ligands. Mutations in the RET gene were implicated in Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN 2), and thyroid carcinomas. In this report we demonstrate that the docking protein FRS2 is tyrosine phosphorylated by ligand-stimulated and by constitutively activated oncogenic forms of RET. Complex formation between RET and FRS2 is mediated by binding of the phosphotyrosine-binding domain of FRS2 to pY1062, a residue in RET that also functions as a binding site for Shc. However, overexpression of FRS2 but not Shc potentiates mitogen-activated protein (MAP) kinase activation by RET oncoproteins. We demonstrate that oncogenic RET-PTC proteins are associated with FRS2 constitutively, leading to tyrosine phosphorylation of FRS2, MAP kinase stimulation, and cell proliferation. However, loss-of-function HSCR-associated RET mutants exhibit impaired FRS2 binding and reduced MAP kinase activation. These experiments demonstrate that FRS2 couples both ligand-regulated and oncogenic forms of RET, with the MAP kinase signaling cascade as part of the response of RET under normal biological conditions and pathological conditions, such as MEN 2 and papillary thyroid carcinomas.

New insights into image processing of cortical blood flow monitors using laser speckle imaging.

Laser speckle imaging has increasingly become a viable technique for real-time medical imaging. However, the computational intricacies and the viewing experience involved limit its usefulness for real-time monitors such as those intended for neurosurgical applications. In this paper, we propose a new technique, tLASCA, which processes statistics primarily in the temporal direction using the laser speckle contrast analysis (LASCA) equation, proposed by Briers and Webster. This technique is thoroughly compared with the existing techniques for signal processing of laser speckle images, including, the spatial-based sLASCA and the temporal-based modified laser speckle imaging (mLSI) techniques. sLASCA is an improvement of the basic LASCA technique. In sLASCA, the derived contrasts are further averaged over a predetermined number of raw speckle images. mLSI, on the other hand, is the technique in which temporal statistics are processed using the equation described by Ohtsubo and Asakura. tLASCA preserves the original image resolution similar to mLSI. tLASCA outperforms sLASCA (window size M = 5) with faster convergence of K values (5.32 versus 20.56 s), shorter per-frame processing time (0.34 versus 2.51 s), and better subjective and objective quality evaluations of contrast images. tLASCA also outperforms mLSI with faster convergence of K values (5.32 s) compared to N values (10.44 s), shorter per-frame processing time (0.34 versus 0.91 s), smaller intensity fluctuations among frames (8%-10% versus 15%-35%), and better subjective and objective quality evaluations of contrast images. As laser speckle imaging becomes an important tool for real-time monitoring of blood flows and vascular perfusion, tLASCA is proven to be the technique of choice.

Heterogeneous meshing and biomechanical modeling of human spine.

We aim to develop a patient-specific biomechanical model of human spine for the purpose of surgical training and planning. In this paper, we describe the development of a finite-element model of the spine from the VHD Male Data. The finite-element spine model comprises volumetric elements suitable for deformation and other finite-element analysis using ABAQUS. The mesh generation solution accepts segmented radiological slices as input, and outputs three-dimensional (3D) volumetric finite element meshes that are ABAQUS compliant. The proposed mesh generation method first uses a grid plane to divide the contours of the anatomical boundaries and its inclusions into discrete meshes. A grid frame is then built to connect the grid planes between any two adjacent planes using a novel scheme. The meshes produced consist of brick elements in the interior of the contours and with tetrahedral and wedge elements at the boundaries. The nodal points are classified according to their materials and hence, elements can be assigned different properties. The resultant spine model comprises a detailed model of the 7 cervical vertebrae, 12 thoracic vertebrae, 5 lumbar vertebrae, and S1. Each of the vertebrae and intervertebral disc has between 1200 and 6000 elements, and approximately 1200 elements, respectively. The accuracy of the resultant VHD finite element spine model was good based on visual comparison of volume-rendered images of the original CT data, and has been used in a computational analysis involving needle insertion and static deformation. We also compared the mesh generated using our method against two automatically generated models; one consists of purely tetrahedral elements and the other hexahedral elements.

3D statistical models for tooth surface reconstruction.

This paper presents a method to reconstruct the 3D surface of a tooth given partial information about its shape. A statistical model comprising a mean shape and a series of deformation modes is obtained offline using a set of specimens. During reconstruction, rigid registration is performed to align the mean shape with the target. The mean shape is then deformed to approximate the target by minimizing the sum of squared distances between the two surfaces according to the deformation modes. The method is shown to be efficient for the recovery of tooth shape given crown information.