G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Developing a personalised, evidence-based and inclusive learning (PEBIL) model of blended learning: A cross-sectional survey.

Whilst the use of various blended learning models preceded the COVID-19 pandemic, the abrupt shift to remote delivery served as catalyst within the sector in enhancing digital solutions to meet immediate student needs. As we emerge from the pandemic, a return to purely didactic and impersonal in-person teaching seems anticlimactic, with the return to the lecture theatre seeing many lecturers trialling various digital tools in creating more interactive in-person, synchronous, and asynchronous sessions. In evaluating students' experiences of the various tools and approaches applied by academic staff, a survey was developed by a multidisciplinary team of educators at Cardiff University's School of Medicine exploring student perceptions of e-learning resources (ELRs), as well as student experiences of various blended learning approaches. The primary aim of this study was to evaluate student experience, satisfaction, and engagement with ELRs and blended learning. A total of 179 students (undergraduate and postgraduate) completed the survey. 97% confirmed that e-learning resources were blended within the teaching they received, with 77% rating the quality of e-learning as good-to-excellent and 66% reporting a preference for asynchronous resources that enable them to learn at their own pace. A variety of platforms, tools, and approaches were identified by students as meeting their diverse learning needs. We therefore propose a personalised, evidence-based and inclusive learning (PEBIL) model enabling the application of digital technologies both on and offline.

Pathogenic germline variants in patients with features of hereditary renal cell carcinoma: Evidence for further locus heterogeneity.

Inherited renal cell carcinoma (RCC) is associated with multiple familial cancer syndromes but most individuals with features of non-syndromic inherited RCC do not harbor variants in the most commonly tested renal cancer predisposition genes (CPGs). We investigated whether undiagnosed cases might harbor mutations in CPGs that are not routinely tested for by testing 118 individuals with features suggestive of inherited RCC (family history of RCC, two or more primary RCC aged <60 years, or early onset RCC ≤46 years) for the presence of pathogenic variants in a large panel of CPGs. All individuals had been prescreened for pathogenic variants in the major RCC genes. We detected pathogenic or likely pathogenic (P/LP) variants of potential clinical relevance in 16.1% (19/118) of individuals, including P/LP variants in BRIP1 (n = 4), CHEK2 (n = 3), MITF (n = 1), and BRCA1 (n = 1). Though the power to detect rare variants was limited by sample size the frequency of truncating variants in BRIP1, 4/118, was significantly higher than in controls (P = 5.92E-03). These findings suggest that the application of genetic testing for larger inherited cancer gene panels in patients with indicators of a potential inherited RCC can increase the diagnostic yield for P/LP variants. However, the clinical utility of such a diagnostic strategy requires validation and further evaluation and in particular, confirmation of rarer RCC genotype-phenotype associations is required.

Loss of growth hormone signaling in the mouse germline or in adulthood reduces islet mass and alters islet function with notable sex differences.

In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared with wild-type controls. Also, islets from GHRKO mice secreted ∼50% less glucose-stimulated insulin compared with size-matched islets from wild-type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at 6 mo of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12 mo of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls, whereas isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.NEW & NOTEWORTHY Growth hormone (GH) is important for more than just growth. GH helps to maintain pancreatic islet mass and insulin secretion throughout life. Sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose regulation despite losing islet mass.

Genome-wide association studies of toxicity to oxaliplatin and fluoropyrimidine chemotherapy with or without cetuximab in 1800 patients with advanced colorectal cancer.

Chemotherapies administered at normal therapeutic dosages can cause significant side-effects and may result in early treatment discontinuation. Inter-individual variation in toxicity highlights the need for biomarkers to personalise treatment. We sought to identify such biomarkers by conducting 40 genome-wide association studies, together with gene and gene set analyses, for any toxicity and 10 individual toxicities in 1800 patients with advanced colorectal cancer treated with oxaliplatin and fluoropyrimidine chemotherapy ± cetuximab from the MRC COIN and COIN-B trials (385 patients received FOLFOX, 360 FOLFOX + cetuximab, 707 XELOX and 348 XELOX + cetuximab). Single nucleotide polymorphisms (SNPs), genes and gene sets that reached genome-wide or suggestive significance were validated in independent patient groups. We found that MROH5 was significantly associated with neutropenia in MAGMA gene analyses in patients treated with XELOX (P = 6.6 × 10-7 ) and was independently validated in those receiving XELOX + cetuximab; pooled P = 3.7 × 10-7 . rs13260246 at 8q21.13 was significantly associated with vomiting in patients treated with XELOX (odds ratio = 5.0, 95% confidence interval = 3.0-8.3, P = 9.8 × 10-10 ) but was not independently replicated. SNPs at 139 loci had suggestive associations for toxicities and lead SNPs at five of these were independently validated (rs6030266 with diarrhoea, rs1546161 with hand-foot syndrome, rs9601722 with neutropenia, rs13413764 with lethargy and rs4600090 with nausea; all with pooled P's < 5.0 × 10-6 ). In conclusion, the association of MROH5 with neutropenia and five other putative biomarkers warrant further investigation for their potential clinical utility. Despite our comprehensive genome-wide analyses of large, well-characterised, clinical trials, we found a lack of common variants with modest effect sizes associated with toxicities.

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.

A Practical Guide to Rodent Islet Isolation and Assessment Revisited.

Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.

Characterization of renal cell carcinoma-associated constitutional chromosome abnormalities by genome sequencing.

Constitutional translocations, typically involving chromosome 3, have been recognized as a rare cause of inherited predisposition to renal cell carcinoma (RCC) for four decades. However, knowledge of the molecular basis of this association is limited. We have characterized the breakpoints by genome sequencing (GS) of constitutional chromosome abnormalities in five individuals who presented with RCC. In one individual with constitutional t(10;17)(q11.21;p11.2), the translocation breakpoint disrupted two genes: the known renal tumor suppressor gene (TSG) FLCN (and clinical features of Birt-Hogg-Dubé syndrome were detected) and RASGEF1A. In four cases, the rearrangement breakpoints did not disrupt known inherited RCC genes. In the second case without chromosome 3 involvement, the translocation breakpoint in an individual with a constitutional t(2;17)(q21.1;q11.2) mapped 12 Kb upstream of NLK. Interestingly, NLK has been reported to interact indirectly with FBXW7 and a previously reported RCC-associated translocation breakpoint disrupted FBXW7. In two cases of constitutional chromosome 3 translocations, no candidate TSGs were identified in the vicinity of the breakpoints. However, in an individual with a constitutional chromosome 3 inversion, the 3p breakpoint disrupted the FHIT TSG (which has been reported previously to be disrupted in two apparently unrelated families with an RCC-associated t(3;8)(p14.2;q24.1). These findings (a) expand the range of constitutional chromosome rearrangements that may be associated with predisposition to RCC, (b) confirm that chromosome rearrangements not involving chromosome 3 can predispose to RCC, (c) suggest that a variety of molecular mechanisms are involved the pathogenesis of translocation-associated RCC, and (d) demonstrate the utility of GS for investigating such cases.

Common genetic variation and survival after colorectal cancer diagnosis: a genome-wide analysis.

Genome-wide association studies have identified several germline single nucleotide polymorphisms (SNPs) significantly associated with colorectal cancer (CRC) incidence. Common germline genetic variation may also be related to CRC survival. We used a discovery-based approach to identify SNPs related to survival outcomes after CRC diagnosis. Genome-wide genotyping arrays were conducted for 3494 individuals with invasive CRC enrolled in six prospective cohort studies (median study-specific follow-up = 4.2-8.1 years). In pooled analyses, we used Cox regression to assess SNP-specific associations with CRC-specific and overall survival, with additional analyses stratified by stage at diagnosis. Top findings were followed-up in independent studies. A P value threshold of P < 5×10(-8) in analyses combining discovery and follow-up studies was required for genome-wide significance. Among individuals with distant-metastatic CRC, several SNPs at 6p12.1, nearest the ELOVL5 gene, were statistically significantly associated with poorer survival, with the strongest associations noted for rs209489 [hazard ratio (HR) = 1.8, P = 7.6×10(-10) and HR = 1.8, P = 3.7×10(-9) for CRC-specific and overall survival, respectively). No SNPs were statistically significantly associated with survival among all cases combined or in cases without distant-metastases. SNPs in 6p12.1/ELOVL5 were associated with survival outcomes in individuals with distant-metastatic CRC, and merit further follow-up for functional significance. Findings from this genome-wide association study highlight the potential importance of genetic variation in CRC prognosis and provide clues to genomic regions of potential interest.

Psychological sequelae of surgery in a prospective cohort of patients from three intraoperative awareness prevention trials.

BACKGROUND: Elective surgery can have long-term psychological sequelae, especially for patients who experience intraoperative awareness. However, risk factors, other than awareness, for symptoms of posttraumatic stress disorder (PTSD) after surgery are poorly defined, and practical screening methods have not been applied to a broad population of surgical patients. METHODS: The Psychological Sequelae of Surgery study was a prospective cohort study of patients previously enrolled in the United States and Canada in 3 trials for the prevention of intraoperative awareness. The 68 patients who experienced definite or possible awareness were matched with 418 patients who denied awareness based on age, sex, surgery type, and awareness risk. Participants completed the PTSD Checklist-Specific (PCL-S) and/or a modified Mini-International Neuropsychiatric Interview telephone assessment to identify symptoms of PTSD and symptom complexes consistent with a PTSD diagnosis. We then used structural equation modeling to produce a composite PTSD score and examined potential risk factors. RESULTS: One hundred forty patients were unreachable; of those contacted, 303 (88%) participated a median of 2 years postoperatively. Forty-four of the 219 patients (20.1%) who completed the PCL-S exceeded the civilian screening cutoff score for PTSD symptoms resulting from their surgery (15 of 35 [43%] with awareness and 29 of 184 [16%] without). Nineteen patients (8.7%; 5 of 35 [14%] with awareness and 14 of 184 [7.6%] without) both exceeded the cutoff and endorsed a breadth of symptoms consistent with the Diagnostic and Statistical Manual Fourth Edition diagnosis of PTSD attributable to their surgery. Factors independently associated with PTSD symptoms were poor social support, previous PTSD symptoms, previous mental health treatment, dissociation related to surgery, perceiving that one's life was threatened during surgery, and intraoperative awareness (all P ≤ 0.017). Perioperative dissociation was identified as a potential mediator for perioperative PTSD symptoms. CONCLUSIONS: Events in the perioperative period can precipitate psychological symptoms consistent with subsyndromal and syndromal PTSD. We not only confirmed the high rate of postoperative PTSD in awareness patients but also identified a significant rate in matched nonawareness controls. Screening surgical patients, especially those with potentially mediating risk factors such as intraoperative awareness or perioperative dissociation, for postoperative PTSD symptoms with the PCL-S is practical and could promote early referral, evaluation, and treatment.

Signal propagation in multi-layer DNAzyme cascades using structured chimeric substrates.

Signal propagation through enzyme cascades is a critical component of information processing in cellular systems. Although such systems have potential as biomolecular computing tools, rational design of synthetic protein networks remains infeasible. DNA strands with catalytic activity (DNAzymes) are an attractive alternative, enabling rational cascade design through predictable base-pair hybridization principles. Multi-layered DNAzyme signaling and logic cascades are now reported. Signaling between DNAzymes was achieved using a structured chimeric substrate (SCS) that releases a downstream activator after cleavage by an upstream DNAzyme. The SCS can be activated by various upstream DNAzymes, can be coupled to DNA strand-displacement devices, and is highly resistant to interference from background DNA. This work enables the rational design of synthetic DNAzyme regulatory networks, with potential applications in biomolecular computing, biodetection, and autonomous theranostics.

Reconstructing microvascular network skeletons from 3D images: What is the ground truth?

Structural changes to microvascular networks are increasingly highlighted as markers of pathogenesis in a wide range of disease, e.g. Alzheimer's disease, vascular dementia and tumour growth. This has motivated the development of dedicated 3D imaging techniques, alongside the creation of computational modelling frameworks capable of using 3D reconstructed networks to simulate functional behaviours such as blood flow or transport processes. Extraction of 3D networks from imaging data broadly consists of two image processing steps: segmentation followed by skeletonisation. Much research effort has been devoted to segmentation field, and there are standard and widely-applied methodologies for creating and assessing gold standards or ground truths produced by manual annotation or automated algorithms. The Skeletonisation field, however, lacks widely applied, simple to compute metrics for the validation or optimisation of the numerous algorithms that exist to extract skeletons from binary images. This is particularly problematic as 3D imaging datasets increase in size and visual inspection becomes an insufficient validation approach. In this work, we first demonstrate the extent of the problem by applying 4 widely-used skeletonisation algorithms to 3 different imaging datasets. In doing so we show significant variability between reconstructed skeletons of the same segmented imaging dataset. Moreover, we show that such a structural variability propagates to simulated metrics such as blood flow. To mitigate this variability we introduce a new, fast and easy to compute super metric that compares the volume, connectivity, medialness, bifurcation point identification and homology of the reconstructed skeletons to the original segmented data. We then show that such a metric can be used to select the best performing skeletonisation algorithm for a given dataset, as well as to optimise its parameters. Finally, we demonstrate that the super metric can also be used to quickly identify how a particular skeletonisation algorithm could be improved, becoming a powerful tool in understanding the complex implication of small structural changes in a network.

PIGA Mutations and Glycosylphosphatidylinositol Anchor Dysregulation in Polyposis-Associated Duodenal Tumorigenesis.

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA. IMPLICATIONS: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis.

Exome resequencing identifies potential tumor-suppressor genes that predispose to colorectal cancer.

Inherited factors account for around one third of all colorectal cancers (CRCs) and include rare high penetrance mutations in APC, MSH2, MSH6, and POLE. Here, we sought novel tumor-suppressor genes that predispose to CRC by exome resequencing 50 sporadic patients with advanced CRC (18 diagnosed ≤35 years of age) at a mean coverage of 30×. To help identify potentially pathogenic alleles, we initially sought rare or novel germline truncating mutations in 1,138 genes that were likely to play a role in colorectal tumorigenesis. In total, 32 such mutations were identified and confirmed, and included an insertion in APC and a deletion in POLE, thereby validating our approach for identifying disease alleles. We sought somatic mutations in the corresponding genes in the CRCs of the patients harboring the germline lesions and found biallelic inactivation of FANCM, LAMB4, PTCHD3, LAMC3, and TREX2, potentially implicating these genes as tumor suppressors. We also identified a patient who carried a germline truncating mutation in NOTCH3, part of the Notch signaling cascade that maintains intestinal homeostasis. Our whole exome analyses provided further gene lists to facilitate the identification of potential predisposition alleles.

Influence of chronic hypoxia on the hypoxic ventilatory response of juvenile and adult rats.

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition - from blunting to augmenting the HVR - occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 - 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one "typical" pattern for VAH in adult rats.

Relationship between 233 colorectal cancer risk loci and survival in 1926 patients with advanced disease.

Background: Genome, transcriptome and methylome-wide association studies have identified single-nucleotide polymorphisms (SNPs) or genes at 258 loci associated with colorectal cancer (CRC) risk. We studied the relationship between these and patient outcome. Methods: We studied 1926 unrelated patients with advanced CRC from COIN and COIN-B. Of 205 CRC-risk SNPs, 19 were directly genotyped and 162 were imputed, and of 53 risk genes, 52 were tested. An additive Cox model for overall survival was adjusted for known prognostic factors. For nominally significant SNPs or genes, we considered a recessive model with a Bonferroni corrected threshold of P = 2.1 × 10-4. We examined SNPs as expression quantitative trait loci (eQTL) and the relationship between gene expression in colorectal tumours and survival in 597 unrelated patients. Results: Eleven SNPs or genes were nominally associated with survival under an additive model. Only rs117079142 mapping to UTP23 and EIF3H (Hazard Ratio [HR] = 2.79, 95% Confidence Intervals [CI] = 1.70-4.58, P = 4.7 × 10-5) and rs9924886 mapping to CDH1 and CDH3 (HR = 1.24, 95% CI = 1.12-1.38, P = 5.2 × 10-5) passed the multiple testing threshold under a recessive model. rs117079142 was an eQTL for UTP23 and rs9924886 for CDH1, CDH3 and ZFP90. Decreased CDH1 expression in CRCs was associated with worse survival (HR = 2.18, 95% CI = 1.3-3.5, P = 1.8 × 10-3). Conclusion: rs117079142 and rs9924886 may represent potential prognostic biomarkers for CRC.

Challenges and opportunities of integrating imaging and mathematical modelling to interrogate biological processes.

Advances in biological imaging have accelerated our understanding of human physiology in both health and disease. As these advances have developed, the opportunities gained by integrating with cutting-edge mathematical models have become apparent yet remain challenging. Combined imaging-modelling approaches provide unprecedented opportunity to correlate data on tissue architecture and function, across length and time scales, to better understand the mechanisms that underpin fundamental biology and also to inform clinical decisions. Here we discuss the opportunities and challenges of such approaches, providing literature examples across a range of organ systems. Given the breadth of the field we focus on the intersection of continuum modelling and in vivo imaging applied to the vasculature and blood flow, though our rationale and conclusions extend widely. We propose three key research pillars (image acquisition, image processing, mathematical modelling) and present their respective advances as well as future opportunity via better integration. Multidisciplinary efforts that develop imaging and modelling tools concurrently, and share them open-source with the research community, provide exciting opportunity for advancing these fields.

Similarities in Calcium Oscillations Between Neonatal Mouse Islets and Mature Islets Exposed to Chronic Hyperglycemia.

Pulsatility is important to islet function. As islets mature into fully developed insulin-secreting micro-organs, their ability to produce oscillatory intracellular calcium ([Ca2+]i) patterns in response to glucose also matures. In this study, we measured [Ca2+]i using fluorescence imaging to characterize oscillations from neonatal mice on postnatal (PN) days 0, 4, and 12 in comparison to adult islets. Under substimulatory (3-mM) glucose levels, [Ca2+]i was low and quiescent for adult islets as expected, as well as for PN day 12 islets. In contrast, one-third of islets on PN day 0 and 4 displayed robust [Ca2+]i oscillations in low glucose. In stimulatory glucose (11 mM) conditions, oscillations were present on all neonatal days but differed from patterns in adults. By PN day 12, [Ca2+]i oscillations were approaching characteristics of fully developed islets. The immature response pattern of neonatal islets was due, at least in part, to differences in adenosine 5'-triphosphate (ATP)-sensitive K+-channel activity estimated by [Ca2+]i responses to KATP channel agents diazoxide and tolbutamide. Neonatal [Ca2+]i patterns were also strikingly similar to patterns observed in mature islets exposed to hyperglycemic conditions (20 mM glucose for 48 hours): elevated [Ca2+]i and oscillations in low glucose along with reduced pulse mass in high glucose. Since a hallmark of diabetic islets is dedifferentiation, we propose that diabetic islets display features of "reverse maturation," demonstrating similar [Ca2+]i dynamics as neonatal islets. Pulsatility is thus an important emergent feature of neonatal islets. Our findings may provide insight into reversing ß-cell dedifferentiation and to producing better functioning ß cells from pluripotent stem cells.