A modular framework for multi-scale tissue imaging and neuronal segmentation.

The development of robust tools for segmenting cellular and sub-cellular neuronal structures lags behind the massive production of high-resolution 3D images of neurons in brain tissue. The challenges are principally related to high neuronal density and low signal-to-noise characteristics in thick samples, as well as the heterogeneity of data acquired with different imaging methods. To address this issue, we design a framework which includes sample preparation for high resolution imaging and image analysis. Specifically, we set up a method for labeling thick samples and develop SENPAI, a scalable algorithm for segmenting neurons at cellular and sub-cellular scales in conventional and super-resolution STimulated Emission Depletion (STED) microscopy images of brain tissues. Further, we propose a validation paradigm for testing segmentation performance when a manual ground-truth may not exhaustively describe neuronal arborization. We show that SENPAI provides accurate multi-scale segmentation, from entire neurons down to spines, outperforming state-of-the-art tools. The framework will empower image processing of complex neuronal circuitries.

Bilateral Free Deep Inferior Epigastric Artery Perforator Flaps for Reconstruction following Mastectomy in Poland Syndrome Patients.

Background: Poland syndrome is classically described as symbrachydactyly, with hypoplasia of the pectoralis major and other upper thoracic musculoskeletal structures. It is thought to be caused by intrauterine interruption in subclavian arterial flow and often includes breast hypoplasia. Affected vasculature can pose a challenge for reconstruction with free flaps because inflow may not be reliable in this patient population. Methods: We present the rare case of a 28-year-old woman with left-sided Poland syndrome, significant family history of breast cancer, and BRCA1+ mutation who underwent bilateral prophylactic nipple-sparing mastectomies with successful immediate bilateral deep inferior epigastric artery perforator free flap reconstruction. The surgical literature in this clinical scenario is also reviewed. Results: Preoperative computed tomography angiography of the chest successfully demonstrated the patency and quantified the caliber of the internal mammary vessels to support free flap breast reconstruction. Conclusions: Free tissue transfer is a viable option for breast reconstruction in patients with Poland syndrome undergoing mastectomy guided by preoperative computed tomography angiography to characterize the internal mammary vasculature.

Sustainable Valorisation and Efficient Downstream Processing of Giant Reed by High-Pressure Carbon Dioxide Pretreatment.

This work investigated the catalytic high-pressure CO2 pretreatment of giant reed. CO2 is a renewable resource; its use does not generate chemical wastes and it can be easily removed and recycled. The effect of the addition of low concentrations of FeCl3 (0.16 wt %) and PEG 400 (1.0 wt %) on the hemicellulose hydrolysis to xylose and xylo-oligosaccharides (XOS) is reported for the first time. Under the optimised pretreatment conditions, the xylan conversion of 82 mol % and xylose and XOS yields of 43 and 20 mol % were achieved, respectively. The solid residues obtained from different pretreatments were used as the substrate for the enzymatic hydrolysis to give glucose. The total glucose yield achieved under the optimised two-step process was 67.8 mol % with respect to the glucan units in the biomass. The results demonstrated that PEG-assisted FeCl3 -catalysed scCO2 pretreatment can produce xylose- or XOS-rich hydrolysates and improve the enzymatic hydrolysis of biomass.

Long-term survival of patients with stage II and III gastric cancer who underwent gastrectomy with inadequate nodal assessment.

BACKGROUND: Gastric cancer is an aggressive disease with frequent lymph node (LN) involvement. The NCCN recommends a D2 lymphadenectomy and the harvesting of at least 16 LNs. This threshold has been the subject of great debate, not only for the extent of surgery but also for more appropriate staging. The reclassification of stage IIB through IIIC based on N3b nodal staging in the eighth edition of the American Joint Committee on Cancer (AJCC) staging system highlights the efforts to more accurately discriminate survival expectancy based on nodal number. Furthermore, studies have suggested that pathologic assessment of 30 or more LNs improve prognostic accuracy and is required for proper staging of gastric cancer. AIM: To evaluate the long-term survival of advanced gastric cancer patients who deviated from expected survival curves because of inadequate nodal evaluation. METHODS: Eligible patients were identified from the Surveillance, Epidemiology, and End Results database. Those with stage II-III gastric cancer were considered for inclusion. Three groups were compared based on the number of analyzed LNs. They were inadequate LN assessment (ILA, < 16 LNs), adequate LN assessment (ALA, 16-29 LNs), and optimal LN assessment (OLA, ≥ 30 LNs). The main outcomes were overall survival (OS) and cancer-specific survival. Data were analyzed by the Kaplan-Meier product-limit method, log-rank test, hazard risk, and Cox proportional univariate and multivariate models. Propensity score matching (PSM) was used to compare the ALA and OLA groups. RESULTS: The analysis included 11607 patients. Most had advanced T stages (T3 = 48%; T4 = 42%). The pathological AJCC stage distribution was IIA = 22%, IIB = 18%, IIIA = 26%, IIIB = 22%, and IIIC = 12%. The overall sample divided by the study objective included ILA (50%), ALA (35%), and OLA (15%). Median OS was 24 mo for the ILA group, 29 mo for the ALA group, and 34 mo for the OLA group (P < 0.001). Univariate analysis showed that the ALA and OLA groups had better OS than the ILA group [ALA hazard ratio (HR) = 0.84, 95% confidence interval (CI): 0.79-0.88, P < 0.001 and OLA HR = 0.73, 95%CI: 0.68-0.79, P < 0.001]. The OS outcome was confirmed by multivariate analysis (ALA HR = 0.68, 95%CI: 0.64-0.71, P < 0.001 and OLA: HR = 0.48, 95%CI: 0.44-0.52, P < 0.001). A 1:1 PSM analysis in 3428 patients found that the OLA group had better survival than the ALA group (OS: OLA median = 34 mo vs ALA median = 26 mo, P < 0.001, which was confirmed by univariate analysis (HR = 0.81, 95%CI: 0.75-0.89, P < 0.001) and multivariate analysis: (HR = 0.71, 95%CI: 0.65-0.78, P < 0.001). CONCLUSION: Proper nodal staging is a critical issue in gastric cancer. Assessment of an inadequate number of LNs places patients at high risk of adverse long-term survival outcomes.

Gastrectomy for stage IV gastric cancer: a comparison of different treatment strategies from the SEER database.

In the West, more than one third of newly diagnosed subjects show metastatic disease in gastric cancer (mGC) with few care options available. Gastrectomy has recently become a subject of debate, with some evidence showing advantages in survival beyond the sole purpose of treatment tumor-related complications. We investigated the survival benefit of different strategies in mGC patients, focusing on the role and timing of gastrectomy. Data were extracted from the SEER database. Groups were determined according to whether patients received gastrectomy, chemotherapy, supportive care. Patients receiving a multimodality treatment were further divided according to timing of surgery, whether performed before (primary gastrectomy, PG) or after chemotherapy (secondary gastrectomy, SG). 16,596 patients were included. Median OS was significantly higher (p < 0.001) in the SG (15 months) than in the PG (13 months), gastrectomy alone (6 months), and chemotherapy (7 months) groups. In the multivariate analysis, SG showed better OS (HR = 0.22, 95%CI = 0.18-0.26, p < 0.001) than PG (HR = 0.25, 95%CI = 0.23-0.28, p < 0.001), gastrectomy (HR = 0.40, 95%CI = 0.36-0.44, p < 0.001), and chemotherapy (HR = 0.42, 95%CI = 0.4-0.44, p < 0.001). The survival benefits persisted even after the PSM analysis. This study shows survival advantages of gastrectomy as multimodality strategy after chemotherapy. In selected patients, SG can be proposed to improve the management of stage IV disease.

The development and external validation of a nomogram predicting overall survival of gastric cancer patients with inadequate lymph nodes based on an international database.

BACKGROUND: Inadequate sampling of lymph nodes could lead to stage migration and indicate a poor prognosis for gastric cancer after curative surgery. Some emerging novel predictors and the application of a nomogram could increase the accuracy of survival prediction. METHODS: An international database regarding gastric cancer was employed as the primary cohort. The patients with inadequate (< 30) lymph nodes (LN) were analyzed by Cox proportional hazards regression. Based on the selected model, a nomogram was plotted and calibrated against an external validation database. RESULTS: A total of 1109 patients were included in the primary cohort, and there were 6584 patients in the validation cohort. There were significant differences regarding the clinical characteristics between the two cohorts. The model containing age, T stages, N stages, metastatic lymph nodes (mLN), and the number of total LN retrieved (TLN) showed superiority over the conventional TNM stages. Harrell's concordance index of the nomogram and TNM stages was 0.744 and 0.717, respectively. The external validation demonstrated a good concordance with the nomogram-predicted survival. CONCLUSIONS: The nomogram including age, T stages, N stages, mLN, and TLN had a better accuracy than the conventional TNM staging system in predicting overall survival for gastric cancer patients with inadequate (< 30) LN.

Implications of Postpancreatectomy Hypophosphatemia.

BACKGROUND: Electrolyte abnormalities are commonly found after major abdominal surgery for malignancy. We hypothesized that the severity of hypophosphatemia developed in pancreatectomy patients would be associated with the incidence of complications postoperatively. METHODS: A retrospective analysis of an institutional database was conducted for all pancreatic resections (2009-2017). Patient charts were reviewed for demographics, clinicopathologic factors, and perioperative outcomes. RESULTS: In a cohort of 283 pancreatectomy patients, 107 (37.8%) and 134 (47.3%) developed mild (2.0-2.5 mg/dL) and moderate/severe hypophosphatemia (<2.0 mg/dL), respectively. Nadir serum phosphate levels were shown to occur on postoperative day (POD) 2 for patients without complications and POD3 for patients who had at least 1 complication. Patients who developed severe hypophosphatemia were significantly more likely to suffer fistula-related complications (P = .0401). CONCLUSIONS: Assessing the severity and timing of postpancreatectomy hypophosphatemia presents an opportunity for early detection of impending fistula-related complications.

Vestibular rehabilitation has positive effects on balance, fatigue and activities of daily living in highly disabled multiple sclerosis people: A preliminary randomized controlled trial.

BACKGROUND: Balance disorders are present in patients with Multiple Sclerosis and part of these disorders could be of vestibular origin. Vestibular Rehabilitation was found to be effective in improving balance in patients with central vestibular dysfunction. OBJECTIVE: To investigate the clinical effects of vestibular rehabilitation on balance skills and secondly on fatigue and activity of daily living in highly disabled multiple sclerosis people. METHODS: Thirty hospitalized participants with severe multiple sclerosis (EDSS 6-7) were randomly assigned to the experimental group (15 patients -9F-; mean age 50.64±11.73) and the control group (15 patients -8F-; mean age 45.77±10.91). All patients were evaluated before and after treatment with the Expanded Disability Status Scale, Barthel Index Tinetti Balance and Gait scale, Berg Balance Scale, Fatigue Severity Scale, Two Minute Walking Test and Timed 25-foot walk test. Two follow-ups (i.e., at 30 and 60 days after treatment) were carried out with Barthel Index. RESULTS: Significant improvement was found in the experimental group with respect to the control group (p <  0,05) in balance, fatigue perception, activities of daily living and short distance gait. No significant improvements were found for gait endurance as measured by Two Minute Walking Test. CONCLUSIONS: Four weeks of Vestibular Rehabilitation training results in less fatigue, improved balance and performance of the activities of daily living in patients with severe Multiple Sclerosis.

Robotic Total Pelvic Exenteration: Video-Illustrated Technique.

BACKGROUND: Robotic-assisted total pelvic exenteration (TPE) can offer a minimally invasive approach to a major multi-organ operation. METHODS: In this video, we summarize a stepwise approach to robotic TPE in a 70 year-old female Jehovah's witness with a history of cervical cancer post-chemoradiation and radical hysterectomy who experienced local recurrence at the vaginal cuff involving the rectum and bladder. RESULTS: The patient was placed in the lithotomy position. A total of six robotic ports were used and the da Vinci Si robotic system was docked between the legs. We proceeded as follows: (1) the abdomen and pelvis were thoroughly explored for evidence of metastatic disease; (2) the pelvic sidewalls were mobilized and bilateral ureters identified; (3) the mesorectal plane was dissected to the level of the levators; (4) the lateral and anterior pelvic structures were completely mobilized, and parametrial tissues were mobilized to the pelvic wall; (5) the bladder was separated from the pubis symphysis, the space of Retzius entered, and the bladder and proximal urethra freed; (6) a perineal incision was made around the vagina, perineal body, and anus, which were excised; (7) an Alloderm mesh secured the pelvic floor, and an omental J flap was mobilized; and (8) a 6 cm incision was utilized for creation of an ileal conduit and a permanent-end colostomy. Final pathology was consistent with recurrent cervical squamous cell carcinoma invading into the vaginal, bladder, and rectal walls. Surgical margins and seven lymph nodes were negative for carcinoma. CONCLUSION: Robotic-assisted TPE is technically feasible in a Jehovah's witness under a multidisciplinary surgical team, even in the setting of prior radical hysterectomy and irradiated tissue.

The 30-year experience-A meta-analysis of randomised and high-quality non-randomised studies of hyperthermic intraperitoneal chemotherapy in the treatment of gastric cancer.

IMPORTANCE: Hyperthermic intraperitoneal chemotherapy (HIPEC) has been used within various multimodality strategies for the prevention and treatment of gastric cancer peritoneal carcinomatosis. OBJECTIVE: To systematically evaluate the role of HIPEC in gastric cancer and clarify its effectiveness at different stages of peritoneal disease progression. DATA SOURCES: Medline and Embase databases between January 1, 1985 and June 1, 2016. STUDY SELECTION: Randomised control trials and high-quality non-randomised control trials selected on a validated tool (methodological index for non-randomised studies) comparing HIPEC and standard oncological management for the treatment of advanced stage gastric cancer with and without peritoneal carcinomatosis were considered. DATA EXTRACTION AND SYNTHESIS: A random-effects network meta-analysis. MAIN OUTCOMES AND MEASURES: The primary outcomes were overall survival and disease recurrence. Secondary outcomes were overall complications, type of complications, and sites of recurrence. RESULTS: A total of 11 RCTs and 21 non-randomised control trials (2520 patients) were included. For patients without the presence of peritoneal carcinomatosis (PC), the overall survival rates between the HIPEC and control groups at 3 or 5 years resulted in favour of the HIPEC group (risk ratio [RR] = 0.82, P = 0.01). No difference in the 3-year overall survival (RR = 0.99, P = 0.85) in but a prolonged median survival of 4 months in favour of the HIPEC group (WMD = 4.04, P < 0.001) was seen in patients with PC. HIPEC was associated with significantly higher risk of complications for both patients with PC (RR = 2.15, P < 0.01) and without (RR = 2.17, P < 0.01). This increased risk in the HIPEC group was related to systemic drugs toxicity. Anastomotic leakage rates were found to be similar between groups. CONCLUSIONS: Our study demonstrates a survival advantage of the use of HIPEC as a prophylactic strategy and suggests that patients whose disease burden is limited to positive cytology and limited nodal involvement may benefit the most from HIPEC. For patients with extensive carcinomatosis, the completeness of cytoreductive surgery is a critical prognostic factor for survival. Future RCTs should better define patient selection criteria.

ATP citrate lyase mediates resistance of colorectal cancer cells to SN38.

Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance.

Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1.

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Overexpression of snail induces epithelial-mesenchymal transition and a cancer stem cell-like phenotype in human colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.

Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells.

Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.

Identification of cancer stem cells in human gastrointestinal carcinoid and neuroendocrine tumors.

BACKGROUND & AIMS: Metastatic gastrointestinal neuroendocrine tumors (NETs) frequently are refractory to chemotherapy. Chemoresistance in various malignancies has been attributed to cancer stem cells (CSCs). We sought to identify gastrointestinal neuroendocrine CSCs (N-CSCs) in surgical specimens and a NET cell line and to characterize novel N-CSC therapeutic targets. METHODS: Human gastrointestinal NETs were evaluated for CSCs using the Aldefluor (Stemcell Technologies, Vancouver, Canada) assay. An in vitro, sphere-forming assay was performed on primary NET cells. CNDT2.5, a human midgut carcinoid cell line, was used for in vitro (sphere-formation) and in vivo (tumorigenicity assays) CSC studies. N-CSC protein expression was characterized using Western blotting. In vivo, systemic short interfering RNA administration targeted Src. RESULTS: By using the Aldefluor assay, aldehyde dehydrogenase-positive (ALDH+) cells comprised 5.8% ± 1.4% (mean ± standard error of the mean) of cells from 19 patient samples. Although many primary cell lines failed to grow, CNDT96 ALDH+ cells formed spheres in anchorage-independent conditions, whereas ALDH- cells did not. CNDT2.5 ALDH+ cells formed spheres, whereas ALDH- cells did not. In vivo, ALDH+ CNDT2.5 cells generated more tumors, with shorter latency than ALDH- or sham-sorted cells. Compared with non-CSCs, ALDH+ cells demonstrated increased expression of activated Src, Erk, Akt, and mammalian target of rapamycin (mTOR). In vivo, anti-Src short interfering RNA treatment of ALDH+ tumors reduced tumor mass by 91%. CONCLUSIONS: CSCs are present in NETs, as shown by in vitro sphere formation and in vivo tumorigenicity assays. Src was activated in N-CSCs and represents a potential therapeutic target in gastrointestinal NETs.