Open-top multisample dual-view light-sheet microscope for live imaging of large multicellular systems.

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids.

Self-organization and symmetry breaking in intestinal organoid development.

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.

Evaluation of a Voltametric E-Tongue Combined with Data Preprocessing for Fast and Effective Machine Learning-Based Classification of Tomato Purées by Cultivar.

The potential of a voltametric E-tongue coupled with a custom data pre-processing stage to improve the performance of machine learning techniques for rapid discrimination of tomato purées between cultivars of different economic value has been investigated. To this aim, a sensor array with screen-printed carbon electrodes modified with gold nanoparticles (GNP), copper nanoparticles (CNP) and bulk gold subsequently modified with poly(3,4-ethylenedioxythiophene) (PEDOT), was developed to acquire data to be transformed by a custom pre-processing pipeline and then processed by a set of commonly used classifiers. The GNP and CNP-modified electrodes, selected based on their sensitivity to soluble monosaccharides, demonstrated good ability in discriminating samples of different cultivars. Among the different data analysis methods tested, Linear Discriminant Analysis (LDA) proved to be particularly suitable, obtaining an average F1 score of 99.26%. The pre-processing stage was beneficial in reducing the number of input features, decreasing the computational cost, i.e., the number of computing operations to be performed, of the entire method and aiding future cost-efficient hardware implementation. These findings proved that coupling the multi-sensing platform featuring properly modified sensors with the custom pre-processing method developed and LDA provided an optimal tradeoff between analytical problem solving and reliable chemical information, as well as accuracy and computational complexity. These results can be preliminary to the design of hardware solutions that could be embedded into low-cost portable devices.

Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity.

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors' sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological data. The overall performance of the sensing devices, combined with the portability of the IoT-based device, enables their suitability as a high-throughput diagnostic tool. Both of the immunosensors were validated using clinical human serum specimens from SARS-CoV-2 infected patients, provided by IRCCS Ospedale San Raffaele.

A Self-Calibrating IoT Portable Electrochemical Immunosensor for Serum Human Epididymis Protein 4 as a Tumor Biomarker for Ovarian Cancer.

Nowadays, analytical techniques are moving towards the development of smart biosensing strategies for the point-of-care accurate screening of disease biomarkers, such as human epididymis protein 4 (HE4), a recently discovered serum marker for early ovarian cancer diagnosis. In this context, the present work represents the first implementation of a competitive enzyme-labelled magneto-immunoassay exploiting a homemade IoT Wi-Fi cloud-based portable potentiostat for differential pulse voltammetry readout. The electrochemical device was specifically designed to be capable of autonomous calibration and data processing, switching between calibration, and measurement modes: in particular, firstly, a baseline estimation algorithm is applied for correct peak computation, then calibration function is built by interpolating data with a four-parameter logistic function. The calibration function parameters are stored on the cloud for inverse prediction to determine the concentration of unknown samples. Interpolation function calibration and concentration evaluation are performed directly on-board, thus reducing the power consumption. The analytical device was validated in human serum, demonstrating good sensing performance for analysis of HE4 with detection and quantitation limits in human serum of 3.5 and 29.2 pM, respectively, reaching the sensitivity that is required for diagnostic purposes, with high potential for applications as portable and smart diagnostic tool for point-of-care testing.

Robotic treatment of oligometastatic kidney tumor with synchronous pancreatic metastasis: case report and review of the literature.

BACKGROUND: The management of metastatic Renal Cell Carcinoma (RCC) has changed dramatically in the last 20 years, and the role of surgery in the immunotherapy's era is under debate. Metastatic lesions interesting pancreas are infrequent, but those harbouring from RCC have an high incidence. If metachronous resections are not rare, synchronous resection of primary RCC and its pancreatic metastasis is uncommonly reported, and accounts for a bad prognosis. CASE PRESENTATION: We report the case of a 68 years old woman, who presented hematuria at hospital incoming, with radiological appearance of a 13 cm left renal mass, with a 2.5 cm single pancreatic tail metastasis. Work-up of staging ruled out other distant metastases, urothelial cancer and there was no evidence of inferior vena cava thrombosis. We choose a 5-port trans-peritoneal robotic approach using lazy right lateral decubitus. Synchronous robotic radical nephrectomy and spleen-sparing pancreatic resection was performed. The pancreatic mass was completely enucleated from pancreatic parenchyma using a latero-medial dissection. Peri-operative hemoglobine loss was 2.4 g/dL. Total operative time was 213 min. No post-operative complications were recorded and patient was discharged in 7th post-operative day. Histopathological examination showed a pT2b N0 M1 RCC, Fuhrman grade II, with pancreatic tail metastasis; both, primary and metastatic lesions had the same histological characteristics with negative surgical margins. After 9 months patient had no evidence of disease recurrence at radiological studies. CONCLUSIONS: The rationale for surgical removal of disseminated tumor, followed by immunotherapy, includes improving prognosis and enhancing the potential of an immune-mediated response to systemic treatment. A spleen-sparing procedure can adequately preserve post-operative immunologic capabilities. In our experience, the correct assessment of pre-operative imaging data and surgeon skills in robotic surgery seem to play a key role in the success of these procedures. Robotic surgery seems to enhance the possibility to control multiple vessels encountered during dissection. Such a conservative approach may be helpful in future research aimed at uncovering biological features, and also leading to better targeted preventive interventions and more individualized and effective treatments.

Robot assisted laparoscopic excision of a paraganglioma: new therapeutic approach.

The Paraganglioma is the most common extra-adrenal pheochromocytoma arising from neural crest (1) (It will better to write: The paraganglioma is an extra-adrenal pheocromocytoma arising from the neural crest. 10% of pheocromocytomas are extra-adrenal and can arise form chromaffin tissue derived from primitive neuroectoderm). Minimally invasive techniques allow surgeons to perform the procedure without wide exposure and mobilization of intra abdominal organs. To our knowledge we present the third case of robotic excision of a retroperitoneal paraganglioma.

Acute uropathogen-related cowperitis with sepsis: case report and literature review.

Acute cowperitis, which was previously known as a common complication of sexually transmitted infections (STIs), is now commonly associated with bacterial urinary tract infections, particularly Escherichia coli. Patients often have a history of STIs, and the symptoms resemble other male accessory gland infections (MAGIs). Recent cases associated with sepsis have been managed with percutaneous drainage and/or surgery. We present a case of acute cowperitis with sepsis and an abscess in the right small gland. The diagnosis was made using transperineal ultrasound, and the patient was successfully treated only with a long-term antibiotic therapy.

Urologic latency time during uroflow stop test with electromyography: an incontinence detector in rehabilitation after robotic radical prostatectomy.

BACKGROUND: Stress urinary incontinence (UI) is the most common presentation following robot-assisted radical prostatectomy (RARP), but a postoperative non-invasive and objective test is still lacking. To assess pelvic floor integrity after RARP, we recently proposed Uroflow Stop Test (UST) with surface electromyography (EMG). AIM: Here we provide two new clinical parameters: the neurologic latency time (NLT) and the urologic latency time (ULT) derived from UST-EMG Test. Principal outcome was to evaluate their variation during one year follow-up and ULT ability to predict post-RARP UI. DESIGN: Observational and longitudinal study. SETTING: Interdivisional Urology Clinic (Perugia-Terni, Italy). POPULATION: Patients with prostate cancer treated with a full nerve-sparing RARP who underwent postoperative pelvic floor muscles training (PFMT): a diurnal functional home program and a weekly hospital program with the use of biofeedback, between 1 and 3 months postoperatively. METHODS: All patients consecutively performed a UST-EMG test at one, three, six, and twelve months after surgery. At each follow-up visit we collected NLT values, ULT values, 5-item 26-Expanded Prostate Cancer Index (EPIC), Incontinence Developed on Incontinence Questionnaire (ICIQ-UI) Short Form and International Prostate Symptom Score (IPSS). We analysed statistically significant differences in NLT and ULT between continent and incontinent patients and we evaluate the diagnostic ability of 1-month post-surgery ULT value to diagnose the presence of postoperative UI. RESULTS: Sixty patients were enrolled. The mean time to PFMT was 31.08 (range: 30-35) days. Overall IPSS, NLT and ULT had similar trends: progressive decrease until the six months after surgery (1-month vs. 3 months vs. 6 months, P<0.05) to plateau thereafter. When considering the two group of patients, IPSS and NLT were significantly higher in the incontinent group only one month after surgery, while ULT became similar between the two groups at 6 months after surgery. The best cut-off of 1-month ULT values that maximized the Youden function at 12-months resulted 3.13 second. CONCLUSIONS: NLT and ULT may respectively account for the nerve and the urethral closure system integrity post-RARP. In the first month after RARP, both NLT and ULT differs between incontinent vs. continent patients. NLT become similar between two group after one month, confirming the recovery from neuropraxia, but ULT remains statistically significant different until 3 months postoperatively. The value of 1-month ULT resulted a valid tool to predict incontinence status at 12 months. CLINICAL REHABILITATION IMPACT: ULT and NLT may be also useful tools to monitor the continence progressive recovery after RARP and they may help rehabilitation specialists to evaluate the ongoing results during postoperative follow-up.

Rapid Quantification of SARS-Cov-2 Spike Protein Enhanced with a Machine Learning Technique Integrated in a Smart and Portable Immunosensor.

An IoT-WiFi smart and portable electrochemical immunosensor for the quantification of SARS-CoV-2 spike protein was developed with integrated machine learning features. The immunoenzymatic sensor is based on the immobilization of monoclonal antibodies directed at the SARS-CoV-2 S1 subunit on Screen-Printed Electrodes functionalized with gold nanoparticles. The analytical protocol involves a single-step sample incubation. Immunosensor performance was validated in a viral transfer medium which is commonly used for the desorption of nasopharyngeal swabs. Remarkable specificity of the response was demonstrated by testing H1N1 Hemagglutinin from swine-origin influenza A virus and Spike Protein S1 from Middle East respiratory syndrome coronavirus. Machine learning was successfully used for data processing and analysis. Different support vector machine classifiers were evaluated, proving that algorithms affect the classifier accuracy. The test accuracy of the best classification model in terms of true positive/true negative sample classification was 97.3%. In addition, the ML algorithm can be easily integrated into cloud-based portable Wi-Fi devices. Finally, the immunosensor was successfully tested using a third generation replicating incompetent lentiviral vector pseudotyped with SARS-CoV-2 spike glycoprotein, thus proving the applicability of the immunosensor to whole virus detection.

Correlation between FA and ADC, number and length of the periprostatic neurovascular fibers.

PURPOSE: Our aim was to explore the relation between FA and ADC, number and length of the periprostatic neurovascular fibers (PNF) by means of 1.5 T Diffusion Tensor Imaging (DTI) imaging through a multivariate linear regression analysis model. METHODS: For this retrospective study, 56 patients (mean age 63.5 years), who underwent 1.5-T prostate MRI, including DTI, were enrolled between October 2014 and December 2018. Multivariate regression analysis was performed to evaluate the statistically significant correlation between FA values (dependent variable) and ADC, the number and the length of PNF (independent variables), if p-value <0.05. A value of 0.5 indicated poor agreement; 0.5-0.75, moderate agreement; 0.75-0.9, good agreement; 0.61-0.80, good agreement; and 0.9-1.00, excellent agreement. RESULTS: The overall fit of the multivariate regression model was excellent, with R2 value of 0.9445 (R2 adjusted 0.9412; p < 0.0001). Multivariate linear regression analysis showed a statistically significant correlation (p < 0.05) for all the three independent variables. The r partial value was -0.9612 for ADC values (p < 0.0001), suggesting a strong negative correlation, 0.4317 for the number of fiber tracts (p < 0.001), suggesting a moderate positive correlation, and -0.306 for the length of the fiber tracts (p < 0.05), suggesting a weak negative correlation. CONCLUSIONS: Our multivariate linear regression model has demonstrated a statistically significant correlation between FA values of PNF with other DTI parameters, in particular with ADC.

Multiscale light-sheet organoid imaging framework.

Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based "Digital Organoid Viewer" tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity.

Capturing Cardiogenesis in Gastruloids.

Organoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here, we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo fundamental steps of early heart organogenesis with an in-vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids (gastruloids) mimic embryonic development and support the generation of cardiovascular progenitors, including first and second heart fields. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling.

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.

Adoptive transfer of allogeneic tumor-specific T cells mediates effective regression of large tumors across major histocompatibility barriers.

Graft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8(+) pmel-1 or CD4(+) TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma-bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persisted at detectable levels for several weeks and mediated significant regression of large, vascularized tumors. We found that the risk of GVHD was low when tumor-specific T cells were transferred and significant toxicity was observed only when substantial numbers of open repertoire allogeneic naive T cells were mixed with the tumor-specific lymphocytes. Taken together, these data indicate that the use of tumor-specific allogeneic CD8(+) T cells or CD4(+) can result in significant antitumor effects in the absence of measurable GVHD.

Tumor-specific Th17-polarized cells eradicate large established melanoma.

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

Endoscopic Combined Intrarenal Surgery for Stone Formation After Previous Laparoscopic and Open Renal Surgery.

Background: Nonabsorbable sutures used during renal surgery represent a known substratum for stone growth. We hereby describe two cases of nephrolithiasis secondary to permanent suture material, originally placed during conservative renal surgical procedures and afterward migrated into the caliceal system, managed with endoscopic combined intrarenal surgery (ECIRS) with subsequent complete renal clearance. Case Presentation: Case 1. A 54-year-old male, with history of laparoscopic excision of a left parapelvic cyst, presenting with left inferior caliceal stone. Case 2. A 79-year-old female, who underwent open enucleation of a left renal pelvis tumor 15 years before. She presented with bilateral lithiasis, including a staghorn stone in the left renal pelvis. Both patients underwent left ECIRS by two surgeons, revealing the presence of suture devices, which were completely removed. Abdominal CT at 1 month after surgery did not show residual lithiasic fragments in both cases. Conclusion: To prevent the risk of stone formation, it is mandatory to use nonabsorbable suture agents sparingly during conservative renal surgery and furthermore to remove all foreign material from the collecting system because they have the potential for calculi growth. In this sense, ECIRS technique may also avoid further open or minimally invasive surgery and the use of suture instruments.