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A single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof-of-principle system capable of single-shot tomography of a ~1 mm

We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5,000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.

Of current obesity treatments, bariatric surgery induces the most weight loss. Given the marked increase in the number of bariatric surgeries performed, elucidating the mechanisms of action is a key research goal. We compared whole brain activation in response to high-energy dense (HED) vs. low-energy dense (LED) visual and auditory food cues before and approximately 4 months after Roux-en-Y Gastric Bypass (RYGB) (n = 16) and Sleeve Gastrectomy (SG) (n = 9). We included two control groups: a low-calorie diet weight loss group (WL) (n = 14) and a non-treatment group (NT) (n = 16). Relative to the control groups, the surgery groups showed increased dorsolateral prefrontal cortex (dlPFC) and decreased parahippocampal/fusiform gyrus (PHG/fusiform) activation in response to HED vs. LED, suggesting greater cognitive dietary inhibition and decreased rewarding effects and attention related to HED foods. dlPFC activation was significantly more increased in RYGB vs. SG. We also found that postprandial increases in GLP-1 concentrations (pre to postsurgery) correlated with postsurgical decreases in RYGB brain activity in the inferior temporal gyrus and the right middle occipital gyrus in addition to increases in the right medial prefrontal gyrus/paracingulate for HED > LED stimuli, suggesting involvement of these attention and inhibitory regions in satiety signaling postsurgery.

We present a single-shot volumetric imaging method, utilising optical projection tomography. We record projections simultaneously, implementing compressive sensing and machine learning to record up to 70 (camera limited) 1x1x1.9mm volumes/second.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, histone H3K9me3, H3K27me3, H4K16ac and DNA methylation1-3. In higher vertebrates, epidemiological and experimental evidence supports similar trans-generational effects4,5 although the mechanisms that underpin these are incompletely understood6-9. We generated a luciferase reporter knock-in mouse for the imprinted Dlk1 locus, to visualise and track epigenetic fidelity across generations. We showed that exposure to high-fat diet (HFD) in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 allele in offspring, coincident with increased DNA methylation at the Dlk1 sDMR. Interestingly, maternal Dlk1 mis-expression was also evident in the next generation (F2), exclusively in animals derived from F1-exposed females. Oocytes from these females showed altered microRNA and gene expression, without any major changes in underlying DNA methylation, and correctly imprinted Dlk1 expression resumed in subsequent generations (F3 onwards). Our results reveal how canonical and non-canonical imprinting mechanisms enable the foetal epigenome to adapt to in utero challenge to modulate the properties of two successive generations of offspring.

Aims/hypothesis Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. Methods Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results Glucose-stimulated mitochondrial Ca2+ accumulation (pppMcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (pMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (ppMcu-KO ( Conclusions/interpretation MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain to be established.

The present study was a 4-week randomized trial to assess the efficacy and safety of sitagliptin, a dipeptidyl-peptidase-4 inhibitor, in persistent or recurring type 2 diabetes after Roux-en-Y gastric bypass surgery (RYGB). Participants (n = 32) completed a mixed meal test (MMT) and self-monitoring of plasma glucose (SMPG) before and 4 weeks after randomization to either sitagliptin 100 mg daily or placebo daily. Questionnaires were administered to assess gastrointestinal discomfort. Outcome variables were glucose, active glucagon-like peptide-1 and β-cell function during the MMT, and glucose levels during SMPG. Age (56.3 ± 8.2 years), body mass index (34.4 ± 6.7 kg/m2 ), glycated haemoglobin (7.21 ± 0.77%), diabetes duration (12.9 ± 10.0 years), years since RYGB (5.6 ± 3.3 years) and β-cell function did not differ between the placebo and sitagliptin groups at pre-intervention. Sitagliptin was well tolerated, decreased postprandial glucose levels during the MMT (from 8.31 ± 1.92 mmol/L to 7.67 ± 1.59 mmol/L, P = 0.03) and mean SMPG levels, but had no effect on β-cell function. In patients with diabetes and mild hyperglycemia after RYGB, a short course of sitagliptin provided a small but significant glucose-lowering effect, with no identified improvement in β-cell function.

Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+uptake into pancreatic β-cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial importer (MCU) complex is thought to represent the main route for Ca2+transport across the inner mitochondrial membrane, its role in β-cells has not previously been examinedin vivo. Here, we inactivated the pore-forming subunit MCUa (MCU) selectively in the β-cell in mice usingIns1Cre-mediated recombination. Glucose-stimulated mitochondrial Ca2+accumulation, ATP production and insulin secretion were strongly (pMCUnull animals (βMCU-KO)in vitro. Interestingly, cytosolic Ca2+concentrations increased (pin vivoinsulin secretion at 5 (p2+uptake in pancreatic β-cells and is required for normal GSIS. The apparent compensatory mechanisms which maintain glucose tolerance in βMCU-KO mice remain to be established.

Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm‐to‐cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts—reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN‐based reconstructions of OPT data of other species including live zebrafish., Optical projection tomography is typically used for 3D imaging of fixed cleared tissue or whole organisms. in vivo applications require faster imaging, which is realized by acquiring less image data and reconstructing the tomographic images using iterative compressive sensing algorithms. However, these algorithms can be prohibitively slow for systematic studies. Instead, a neural network can reconstruct images much faster, with improved quality, and can be trained on ex vivo data.

Optical Projection Tomography (OPT), the optical equivalent of x-ray computed tomography, reconstructs the 3D structure of a sample from a series of wide-field 2D projections acquired at different angles [1]. OPT is used to map the optical attenuation and/or fluorescence distributions of intact transparent samples without the need for mechanical sectioning. While it is typically applied to chemically cleared samples, it can also be used to image inherently transparent or weakly scattering live organisms including adult zebrafish up to ~1cm in diameter [2]. When applying OPT to live samples it is important to minimise the data acquisition time while maximising the image quality in the presence of scattering. The former issue can be addressed using compressive sensing to reduce the number of projections required [3]. Scattered light can be rejected using structured illumination [4], but this removes emission from regions the excitation modulation does not reach and reduces the available dynamic range. To address this, we have explored the rejection of scattered light by acquiring projections with parallel semi-confocal line illumination and detection in an approach we describe as slice-OPT (sl-OPT). The impact of optical scattering can also be reduced by imaging at longer wavelengths [5]. We are exploring OPT in the NIR 1&2 spectral windows. However, exotic array detectors, e.g. for short wave infrared light, are costly and so we are also developing a single pixel camera [6] approach. We will present our progress applying these techniques to 3D imaging of vasculature and tumour burden in live adult zebrafish. [1] Sharpe et al, Science, vol. 296, Issue 5567, pp. 541-545, 2002. [2] Kumar et al, Oncotarget, vol. 7, no.28, pp. 43939-43948, 2016. [3] Correia et al, PloS one, vol. 10, no. 8, p. e0136213, 2015. [4] Kristensson et al, Optics express, vol. 20, no. 13, pp. 14437-14450, 2012. [5] Shi et al., Journal of Biophotonics, vol. 9, no. 1-2, pp. 38-43, 2016. [6] Duarte et al., IEEE Signal Processing Magazine, vol. 25, no. 2, pp. 83-91, 2008.

β-cell glucose sensitivity (BCGS) improves after gastric bypass (RYGB) in individuals in diabetes (DM2) remission. We sought to assess BCGS (pmol/kg/min/mM) after an oral glucose test (OGT) and an IV graded glucose infusion (GGI) in subjects with and without DM2 remission after RYGB. Twenty five subjects with DM2 were studied with GGI and 75g-OGT before and 3 months after RYGB. Glucose, insulin, C-peptide, insulin secretion rate, GLP-1 and BCGS were assessed during the OGT and the GGI. Data are presented as mean±SD. Within and between group comparisons with paired and unpaired t-test; GLM with repeated measure to assess change during OGT or GGI were (SPSS 24). Of the 25 subjects studied pre-surgery, 10 experienced DM2 remission (REM), 9 remained with DM2 (N-REM) at 3 months; 6 remained glucose intolerant and were not included in the analysis. Pre-surgery BMI (41.5±4.7 kg/m2), weight (108.3±15.9 kg), age (43.8±8.8 years), gender (95% female), HOMA-IR (12.1±6.7) and Matsuda index did not differ between groups; N-REM had longer DM2 duration (14 vs. 2.5 y p Weight loss did not differ for REM and N-REM (17±5%). HOMA-IR, GLP-1 and GGI-BCGS improved similarly in both groups. OGT-BCGS increased in both groups, but more in REM vs. N-REM (+1.31±1.0 vs. +0.30±0.28, p=0.01). The difference between OGT-BCGS and GGI-BCGS observed pre-RYGB (0.58±0.54 vs. 0.38±.035, p=ns) was exaggerated at 3 months by 4x in REM (2.13±1.vs. 0.58±0.15, p=0.001) and ∼2.5x in N-REM (0.60±.0.43 vs. 0.24±0.17, p=0.07). In conclusion, β-cell function assessed during an IV GGI improves by the same magnitude regardless of DM2 remission status after RYGB. However, β-cell function improved more in REM than N-REM after oral glucose, suggesting a greater incretin effect, or other gut-related mechanisms, in REM after RYGB. Disclosure A. Shah: Employee; Spouse/Partner; Daiichi Sankyo Company, Limited. K. Levesque: None. M. Ahlers: None. M.M. Holter: None. F. Alam: None. E. Pierini: None. B.L. Rojas: None. V. Mark: None. K. Patel: None. R. Dutia: None. E.J. Harvey: None. K. Park: None. N. Koshy: None. S.J. Belsley: None. J.J. Mcginty: None. B. Laferrere: None.

OBJECTIVE Hepatic insulin clearance is a significant regulator of glucose homestasis. We hypothesized that the improvement in insulin clearance rates (ICRs) under fasting conditions and in response to oral and intravenous (IV) glucose would improve similarly after Roux-en-Y gastric bypass (RYGB) and adjustable gastric banding (AGB) as a function of weight loss; the difference in ICR after oral and IV glucose stimulation will be enhanced after RYGB compared with AGB, an effect mediated by glucagon-like peptide 1 (GLP-1). RESEARCH DESIGN AND METHODS In study 1, the ICR was calculated under fasting condition (F-ICR), after oral glucose (O-ICR), and after an isoglycemic IV glucose clamp (IV-ICR) in individuals from an established cohort with type 2 diabetes mellitus (T2DM) before, after 10% matched weight loss, and 1 year after either RYGB (n = 22) or AGB (n = 12). In study 2, O-ICR was studied in a separate cohort of individuals with T2DM (n = 22), before and 3 months after RYGB, with and without exendin(9-39) infusion. RESULTS In study 1, age, BMI, T2DM duration and control, and ICR did not differ between RYGB and AGB preintervention. Weight loss at 1 year was two times greater after RYGB than after AGB (31.6 ± 5.9% vs. 16.6 ± 9.8%; P < 0.05). RYGB and AGB both significantly increased F-ICR, O-ICR, and IV-ICR at 1 year. ICR was inversely associated with insulinemia. The difference between IV-ICR and O-ICR was significantly greater after RYGB versus AGB. GLP-1 antagonism with exendin(9-39) led to an increase in O-ICR in subjects post-RYGB. CONCLUSIONS Weight loss increased ICR, an effect more pronounced after RYGB compared with AGB. Our data support a potential role for endogenous GLP-1 in the control of postprandial ICR after RYGB.

Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering.

Fluorescence lifetime measurements can provide quantitative assays of the local fluorophore environment and can be applied to read out biomolecular interactions via Forster resonance energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can be automated for high content analysis (HCA) to map protein–protein interactions with applications in drug discovery, systems biology and basic research. The automated acquisition of FLIM data over 100's of fields of view provides statistical power to overcome noise in instrumentation and biological systems and thus exploit relatively small changes in mean lifetime to provide useful readouts that would not be practically achievable in manual microscopy experiments. We present here an automated HCA system with the ability to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential through exemplar applications of different FRET readouts.

Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing – with the latter typically requiring weeks for cm scale tissue samples. To address this challenge, we present a new, convenient, aqueous optical clearing agent, termed UbasM: Urea-Based Amino-Sugar Mixture, that rapidly renders fixed tissue samples highly transparent and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues. UbasM is simple, inexpensive, reproducible and compatible with all labeling methods that we have encountered. It can enable convenient, volumetric imaging of tissue up to the scale of whole adult mouse organs and should be useful for a wide range of light microscopy and tomography techniques applied to biomedical research, especially the study on organism-level systems biology at multiple levels.

We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 μm). All three methods use the microscope's existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations' viability.

Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Forster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB- based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

The mechanisms responsible for the remarkable remission of type 2 diabetes after Roux-en-Y gastric bypass (RYGBP) are still puzzling. To elucidate the role of the gut, we compared β-cell function assessed during an oral glucose tolerance test (OGTT) and an isoglycemic intravenous glucose clamp (iso-IVGC) in: 1) 16 severely obese patients with type 2 diabetes, up to 3 years post-RYGBP; 2) 11 severely obese normal glucose-tolerant control subjects; and 3) 7 lean control subjects. Diabetes remission was observed after RYGBP. β-Cell function during the OGTT, significantly blunted prior to RYGBP, normalized to levels of both control groups after RYGBP. In contrast, during the iso-IVGC, β-cell function improved minimally and remained significantly impaired compared with lean control subjects up to 3 years post-RYGBP. Presurgery, β-cell function, weight loss, and glucagon-like peptide 1 response were all predictors of postsurgery β-cell function, although weight loss appeared to be the strongest predictor. These data show that β-cell dysfunction persists after RYGBP, even in patients in clinical diabetes remission. This impairment can be rescued by oral glucose stimulation, suggesting that RYGBP leads to an important gastrointestinal effect, critical for improved β-cell function after surgery.

More than 65loci, encoding up to 500 different genes, have been implicated by genome-wide association studies (GWAS) as conferring an increased risk of developing type 2 diabetes (T2D). Whilst mouse models have in the past been central to understanding the mechanisms through which more penetrant risk genes for T2D, for example, those responsible for neonatal or maturity-onset diabetes of the young, only a few of those identified by GWAS, notablyTCF7L2andZnT8/SLC30A8, have to date been examined in mouse models. We discuss here the animal models available for the latter genes and provide perspectives for future, higher throughput approaches towards efficiently mining the information provided by human genetics.

Summary Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact., Graphical Abstract, Highlights • Allele-specific expression of imprinted Cdkn1c imaged in vivo using bioluminescence • Chromatin-modifying drugs applied in utero transiently de-repress Cdkn1c imprinting • In utero exposure to low-protein diet permanently disrupts the Cdkn1c imprint • Folate supplements during gestation protect against loss of Cdkn1c imprinting, Van de Pette et al. use sensitive allele-specific reporters to longitudinally image imprinted Cdkn1c expression in mice and show that expression is modulated by environmental factors encountered in utero. These results establish imprinting deregulation as a mechanism linking early-life adversity to later-life outcomes and provide tools to detect imprinting changes in vivo.

OBJECTIVE The superior effect of Roux-en-Y gastric bypass (RYGB) on glucose control compared with laparoscopic adjustable gastric banding (LAGB) is confounded by the greater weight loss after RYGB. We therefore examined the effect of these two surgeries on metabolic parameters matched on small and large amounts of weight loss. RESEARCH DESIGN AND METHODS Severely obese individuals with type 2 diabetes were tested for glucose metabolism, β-cell function, and insulin sensitivity after oral and intravenous glucose stimuli, before and 1 year after RYGB and LAGB, and at 10% and 20% weight loss after each surgery. RESULTS RYGB resulted in greater glucagon-like peptide 1 release and incretin effect, compared with LAGB, at any level of weight loss. RYGB decreased glucose levels (120 min and area under the curve for glucose) more than LAGB at 10% weight loss. However, the improvement in glucose metabolism, the rate of diabetes remission and use of diabetes medications, insulin sensitivity, and β-cell function were similar after the two types of surgery after 20% equivalent weight loss. CONCLUSIONS Although RYGB retained its unique effect on incretins, the superiority of the effect of RYGB over that of LAGB on glucose metabolism, which is apparent after 10% weight loss, was attenuated after larger weight loss.

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.

Background Laparoscopic sleeve gastrectomy is commonly performed using multiple ports. The quest to minimize surgical trauma has led to the development of single port laparoscopy, which has been shown to be a safe, less-invasive method of performing a variety of abdominal surgeries. We describe the feasibility and safety of single port sleeve gastrectomy (SPSG) for morbid obesity at an academic affiliate of a university hospital. Methods A total of 25 patients undergoing elective SPSG were compared with a demographically similar contemporaneous cohort of 9 patients who underwent standard multiple port laparoscopic sleeve gastrectomy. The data collected included the operative time, narcotic consumption, duration of patient controlled analgesia use, subjective pain scores, and length of stay. Results The patients undergoing SPSG experienced significantly less pain at 1 hour postoperatively ( P = .039). No statistically significant difference was found in pain between the 2 groups at 12 and 24 hours ( P = .519 and P = .403, respectively). The quantity of narcotic use ( P = .538), duration of patient controlled analgesia use ( P = .820), and length of stay ( P = .571) were not significantly different between the 2 groups. The operative time for SPSG was 118 minutes versus 101 minutes for multiple port surgery ( P = .160). Conclusions SPSG is safe and feasible for selected patients. The patients undergoing SPSG reported significantly less pain at the first postoperative hour. No significant differences between the 2 groups were seen in any of the other postoperative parameters.

Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p

Following gastric bypass surgery (GBP), there is a post-prandial rise of incretin and satiety gut peptides. The mechanisms of enhanced incretin release in response to nutrients after GBP is not elucidated and may be in relation to altered nutrient transit time and/or malabsorption.Seven morbidly obese subjects (BMI = 44.5 ± 2.8 kg/m(2)) were studied before and 1 year after GBP with a D: -xylose test. After ingestion of 25 g of D: -xylose in 200 mL of non-carbonated water, blood samples were collected at frequent time intervals to determine gastric emptying (time to appearance of D: -xylose) and carbohydrate absorption using standard criteria.One year after GBP, subjects lost 45.0 ± 9.7 kg and had a BMI of 27.1 ± 4.7 kg/m(2). Gastric emptying was more rapid after GBP. The mean time to appearance of D: -xylose in serum decreased from 18.6 ± 6.9 min prior to GBP to 7.9 ± 2.7 min after GBP (p = 0.006). There was no significant difference in absorption before (serum D: -xylose concentrations = 35.6 ± 12.6 mg/dL at 60 min and 33.9 ± 9.1 mg/dL at 180 min) or 1 year after GBP (serum D: -xylose = 31.5 ± 18.1 mg/dL at 60 min and 27.2 ± 11.9 mg/dL at 180 min).These data confirm the acceleration of gastric emptying for liquid and the absence of carbohydrate malabsorption 1 year after GBP. Rapid gastric emptying may play a role in incretin response after GBP and the resulting improved glucose homeostasis.

In order to study physical relationships within tissue volumes or even organism-level systems, the spatial distribution of multiple fluorescent markers needs to be resolved efficiently in three dimensions. Here, rather than acquiring discrete spectral images sequentially using multiple emission filters, a hyperspectral scanning laser optical tomography system is developed to obtain hyperspectral volumetric data sets with 2-nm spectral resolution of optically transparent mesoscopic (millimeter-centimeter) specimens. This is achieved by acquiring a series of point-scanning hyperspectral extended depth of field images at different angles and subsequently tomographically reconstructing the 3D intensity distribution for each wavelength. This technique is demonstrated to provide robust measurements via the comparison of spectral and intensity profiles of fluorescent bead phantoms. Due to its enhanced spectral resolving ability, this technique is also demonstrated to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual-labeled mouse thymus gland sample and the ability to distinguish tumorous and normal tissues of an unlabeled mouse intestine sample.

Optical imaging of tissue autofluorescence has the potential to provide rapid label-free screening and detection of surface tumors for clinical applications, including when combined with endoscopy. Quantitative imaging of intensity-based contrast is notoriously difficult and spectrally resolved imaging does not always provide sufficient contrast. We demonstrate that fluorescence lifetime imaging (FLIM) applied to intrinsic tissue autofluorescence can directly contrast a range of surface tissue tumors, including in gastrointestinal tissues, using compact, clinically deployable instrumentation achieving wide-field fluorescence lifetime images of unprecedented clarity. Statistically significant contrast is observed between cancerous and healthy colon tissue for FLIM with excitation at 355 nm. To illustrate the clinical potential, wide-field fluorescence lifetime images of unstained ex vivo tissue have been acquired at near video rate, which is an important step towards real-time FLIM for diagnostic and interoperative imaging, including for screening and image-guided biopsy applications.

The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic β-cell and hypothalamic neurons using the rat insulin promoter (RIP2). Cre transgene (βAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the β-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the β-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to βAMPKdKO mice, βLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1- CreER mice, total β-cell mass and the size of individual islets and β-cells were increased and islet architecture was markedly altered in βLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1- CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca2+ were sharply reduced in βLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on β-cell function.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain.The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques.Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.

We describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono-exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We report angular multiplexed OPT and FLIM OPT applied to in vivo imaging of cancer and FRET biosensors in adult zebrafish. Multiple-spectral 3-D datasets of entire adult zebrafish can be acquired in 3 minutes.

We present sub-volume optical projection tomography utilising an electrically tunable lens and tracking technology. Applied to 3D fluorescent bead phantoms and zebrafish embryos, we demonstrate an improvement in resolution over conventional OPT.

We present the application of FLIM-OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed cleavable FRET biosensor for Caspase-3 as an indicator of gamma radiation induced apoptosis.

Background This study examined the feasibility of using Polyflex stents in the treatment of enteric leaks after various bariatric operations. Chronic and acute leaks were treated. Methods We performed a retrospective case series review. Four patients received 6 Polyflex stents to treat complications of bariatric surgery. Two presented with early sepsis before stenting. One presented with abdominal pain. One presented with a chronic persistent fistula with an associated abscess. Stenting was performed under endoscopy with fluoroscopic guidance. The stents were left in place for 6 weeks. Results All patients tolerated a clear liquid diet within 24 hours of stenting and were able to be advanced to a pureed diet. All patients improved clinically after stenting. Three patients with acute leaks sealed their leaks after stent placement. One patient with a chronic leak persisted and required operative closure after a second stent was placed and failed. All patients experienced short-term nausea, as well as early satiety that lasted the duration of the stenting. One patient experienced hypersialisis while the stent was in place. Two stents migrated, although this had no effect on leak closure. One patient had an anastomotic stenosis successfully treated with a second stent. Conclusions Polyflex stents are useful in bypassing acute upper intestinal leaks after various bariatric operations. They provide a temporary bridge for wound healing with continued oral intake. Stenting provides a minimally invasive option in the management of acute leaks and, in our experience, had no serious associated morbidity.

We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

Rachel Fetner, M.D.*, James McGinty, M.D., Colleen Russell, Ph.D., F. Xavier Pi-Sunyer, M.D., M.P.H., Blandine Laferrere, M.D. Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, St. Luke’s-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York Department of Surgery, Division of Bariatric Surgery, St. Luke’s-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York Obesity Research Center, St. Luke’s-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York Manuscript received April 28, 2005; revised August 5, 2005; accepted September 2, 2005