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Abstract Post-resection radiologic monitoring to identify areas of new or progressive enhancement concerning for cancer recurrence is critical during patients with glioblastoma follow-up. However, treatment-related pseudoprogression presents with similar imaging features but requires different clinical management. While pathologic diagnosis is the gold standard to differentiate true progression and pseudoprogression, the lack of objective clinical standards and admixed histologic presentation creates the needs to (1) validate the accuracy of current approaches and (2) characterize differences between these entities to objectively differentiate true disease. We demonstrated using an online RNAseq repository of recurrent glioblastoma samples that cancer-immune cell activity levels correlate with heterogenous clinical outcomes in patients. Furthermore, nCounter RNA expression analysis of 48 clinical samples taken from second neurosurgical resection supports that pseudoprogression gene expression pathways are dominated with immune activation, whereas progression is predominated with cell cycle activity. Automated image processing and spatial expression analysis however highlight a failure to apply these broad expressional differences in a subset of cases with clinically challenging admixed histology. Encouragingly, applying unsupervised clustering approaches over our segmented histologic images provides novel understanding of morphologically derived differences between progression and pseudoprogression. Spatially derived data further highlighted polarization of myeloid populations that may underscore the tumorgenicity of novel lesions. These findings not only help provide further clarity of potential targets for pathologists to better assist stratification of progression and pseudoprogression, but also highlight the evolution of tumor-immune microenvironment changes which promote tumor recurrence.

Background: Bus driving is a mentally demanding activity that requires prolonged attention to ensure safety. The aim of the study was to assess mental fatigue caused by driving a public bus and to find a profile of workers at higher risk. Methods: We evaluated changes of critical flicker fusion (CFF) (index of central arousal) and heart rate variability (HRV) (index of autonomic balance) in a 6-hour driving shift on a real route, in 31 professional bus drivers, and we tested the influence of personal factors such as sleep quality, BMI, and age. Paired t-test was used to test differences of CFF and HRV between both initial and final phase of driving, while multiple linear regression tested the influence of personal variables on the indexes of mental fatigue. Results: Results showed that CFF significantly decreased after 6 hours of bus driving (41.91 Hz, sd 3.31 vs. 41.15 Hz, sd 3.15; p = 0.041), and heart rate significantly decreased in the final phase of driving, with respect to the initial phase (85 vs. 78 bpm, p = 0.027). Increasing age (beta = -0.729, p = 0.022), risk of obstructive sleep apnea syndrome (beta = -0.530, p = 0.04), and diurnal sleepiness (beta = -0.406, p = 0.017) showed a significant effect on influencing mental fatigue. Conclusion: Elderly drivers at higher risk of sleep disorders are more prone to mental fatigue, when exposed to driving activity. Monitoring indexes of central arousal and autonomic balance, coupled with the use of structured questionnaires can represent a useful strategy to detect profile of workers at higher risk of mental fatigue in such duty.

Astroblastoma (AB) is an uncommon brain tumour and its origin remains unknown. Here, the authors perform integrative molecular analysis of 35 AB-like tumours and provide evidence that these arise in the context of epigenetic and genetic changes in neural progenitors occurring during brain development.

Melanomas from gynecologic sites (MOGS) are rare and have poor survival. MicroRNAs (miRs) regulate gene expression and are dysregulated in cancer. We hypothesized that MOGS would display unique miR and mRNA expression profiles. The miR and mRNA expression profile in RNA from formalin fixed, paraffin embedded vaginal melanomas (relative to vaginal mucosa) and vulvar melanomas (relative to cutaneous melanoma) were measured with the Nanostring Human miRNA assay and Tumor Signaling mRNA assay. Differential patterns of expression were identified for 21 miRs in vaginal and 47 miRs in vulvar melanoma (fold change >2, p

Summary: Background: Mitochondrial DNA copy number (mtCN) maintains cellular function and homeostasis, and is linked to nuclear DNA methylation and gene expression. Increased mtCN in the blood is associated with smoking and respiratory disease, but has received little attention for target organ effects for smoking or electronic cigarette (EC) use. Methods: Bronchoscopy biospecimens from healthy EC users, smokers (SM), and never-smokers (NS) were assessed for associations of mtCN with mtDNA point mutations, immune responses, nuclear DNA methylation and gene expression using linear regression. Ingenuity pathway analysis was used for enriched pathways. GEO and TCGA respiratory disease datasets were used to explore the involvement of mtCN-associated signatures. Findings: mtCN was higher in SM than NS, but EC was not statistically different from either. Overall there was a negative association of mtCN with a point mutation in the D-loop but no difference within groups. Positive associations of mtCN with IL-2 and IL-4 were found in EC only. mtCN was significantly associated with 71,487 CpGs and 321 transcripts. 263 CpGs were correlated with nearby transcripts for genes enriched in the immune system. EC-specific mtCN-associated-CpGs and genes were differentially expressed in respiratory diseases compared to controls, including genes involved in cellular movement, inflammation, metabolism, and airway hyperresponsiveness. Interpretation: Smoking may elicit a lung toxic effect through mtCN. While the impact of EC is less clear, EC-specific associations of mtCN with nuclear biomarkers suggest exposure may not be harmless. Further research is needed to understand the role of smoking and EC-related mtCN on lung disease risks. Funding: The National Cancer Institute, the National Heart, Lung, and Blood Institute, the Food and Drug Administration Center for Tobacco Products, the National Center For Advancing Translational Sciences, and Pelotonia Intramural Research Funds.

Abstract Background To investigate the global expression profile of miRNAs, their impact on cellular signaling pathways, and their association with poor prognostic parameters in African-American (AA) patients with triple negative breast cancer (TNBC). Methods Twenty-five samples of AA TNBC patients were profiled for global miRNA expression and stratified considering three clinical-pathological parameters: tumor size, lymph node (LN), and recurrence (REC) status. Differential miRNA expression analysis was performed for each parameter, and their discriminatory power was determined by Receiver Operating Characteristic (ROC) curve analysis. KMplotter was assessed to determine the association of the miRNAs with survival, and functional enrichment analysis to determine the main affected pathways and miRNA/mRNA target interactions. Results A panel of eight, 23 and 27 miRNAs were associated with tumor size, LN, and REC status, respectively. Combined ROC analysis of two (miR-2117, and miR-378c), seven (let-7f-5p, miR-1255b-5p, miR-1268b, miR-200c-3p, miR-520d, miR-527, and miR-518a-5p), and three (miR-1200, miR-1249-3p, and miR-1271-3p) miRNAs showed a robust discriminatory power based on tumor size (AUC = 0.917), LN (AUC = 0.945) and REC (AUC = 0.981) status, respectively. Enrichment pathway analysis revealed their involvement in proteoglycans and glycan and cancer-associated pathways. Eight miRNAs with deregulated expressions in patients with large tumor size, positive LN metastasis, and recurrence were significantly associated with lower survival rates. Finally, the construction of miRNA/mRNA networks based in experimentally validated mRNA targets, revealed nodes of critical cancer genes, such as AKT1, BCL2, CDKN1A, EZR and PTEN. Conclusions Altogether, our data indicate that miRNA deregulated expression is a relevant biological factor that can be associated with the poor prognosis in TNBC of AA patients, by conferring to their TNBC cells aggressive phenotypes that are reflected in the clinical characteristics evaluated in this study.

Functional recovery after spinal cord injury (SCI) often proves difficult as physical and mental barriers bar survivors from enacting their designated rehabilitation programs. We recently demonstrated that adult mice administered gabapentinoids, clinically approved drugs prescribed to mitigate chronic neuropathic pain, recovered upper extremity function following cervical SCI. Given that rehabilitative training enhances neuronal plasticity and promotes motor recovery, we hypothesized that the combination of an aerobic-based rehabilitation regimen like treadmill training with gabapentin (GBP) administration will maximize recovery in SCI mice by strengthening synaptic connections along the sensorimotor axis. Whereas mice administered GBP recovered forelimb functions over the course of weeks and months following SCI, no additive forelimb recovery as the result of voluntary treadmill training was noted in these mice. To our surprise, we also failed to find an additive effect in mice administered vehicle. As motivation is crucial in rehabilitation interventions, we scored active engagement toward the rehabilitation protocol and found that mice administered GBP were consistently participating in the rehabilitation program. In contrast, mice administered vehicle exhibited a steep decline in participation, especially at chronic time points. Whereas neuroinflammatory gene expression profiles were comparable between experimental conditions, we discovered that mice administered GBP had increased hippocampal neurogenesis and exhibited less anxiety-like behavior after SCI. We also found that an external, social motivator effectively rescues participation in mice administered vehicle and promotes forelimb recovery after chronic SCI. Thus, not only does a clinically relevant treatment strategy preclude the deterioration of mental health after chronic SCI, but group intervention strategies may prove to be physically and emotionally beneficial for SCI individuals.

Abstract Background Pancreatic and peri-pancreatic neoplasms encompass a variety of histotypes characterized by a heterogeneous prognostic impact. miRNAs are considered efficient candidate biomarkers due to their high stability in tissues and body fluids. We applied Nanostring profiling of circulating exosomal miRNAs to distinct pancreatic lesions in order to establish a source for biomarker development. Methods A series of 140 plasma samples obtained from patients affected by pancreatic ductal adenocarcinoma (PDAC, n = 58), pancreatic neuroendocrine tumors (PanNET, n = 42), intraductal papillary mucinous neoplasms (IPMN, n = 20), and ampulla of Vater carcinomas (AVC, n = 20) were analyzed. Comprehensive miRNA profiling was performed on plasma-derived exosomes. Relevant miRNAs were validated by qRT-PCR and in situ hybridization (ISH). Results Lesion specific miRNAs were identified through multiple disease comparisons. Selected miRNAs were validated in the plasma by qRT-PCR and at tissue level by ISH. We leveraged the presence of clinical subtypes with each disease cohort to identify miRNAs that are differentially enriched in aggressive phenotypes. Conclusions This study shows that pancreatic lesions are characterized by specific exosomal-miRNA signatures. We also provide the basis for further explorations in order to better understand the relevance of these signatures in pancreatic neoplasms.

Summary: Lipopolysaccharides (LPSs) cause lethal endotoxemia if not rapidly cleared from blood circulation. Liver sinusoidal endothelial cells (LSEC) systemically clear LPS by unknown mechanisms. We discovered that LPS clearance through LSEC involves endocytosis and lysosomal inactivation via Stabilin-1 and 2 (Stab1 and Stab2) but does not involve TLR4. Cytokine production was inversely related to clearance/endocytosis of LPS by LSEC. When exposed to LPS, Stabilin double knockout mice (Stab DK) and Stab1 KO, but not Stab2 KO, showed significantly enhanced systemic inflammatory cytokine production and early death compared with WT mice. Stab1 KO is not significantly different from Stab DK in circulatory LPS clearance, LPS uptake and endocytosis by LSEC, and cytokine production. These data indicate that (1) Stab1 receptor primarily facilitates the proactive clearance of LPS and limits TLR4-mediated inflammation and (2) TLR4 and Stab1 are functionally opposing LPS receptors. These findings suggest that endotoxemia can be controlled by optimizing LPS clearance by Stab1.

Abstract Background Osseous‐associated cervical spondylomyelopathy (OA‐CSM) is a common condition of the cervical vertebral column that affects giant dog breeds. MicroRNAs (miRNAs) are small RNAs that regulate gene expression, and recent data suggest that circulating miRNAs present in biological fluids may serve as potential biomarkers for disease. The miRNA profiles of cerebrospinal fluid (CSF) from healthy dogs and dogs clinically affected by OA‐CSM have not been described. Objective To characterize the expression levels of miRNAs present in the CSF of normal Great Danes and identify differentially expressed miRNAs in the CSF of Great Danes clinically affected with OA‐CSM. Animals Client‐owned dogs: 12 control, 12 OA‐CSM affected. Methods Cerebrospinal fluid samples were collected prospectively. MicroRNA expression was evaluated using the NanoString nCounter platform and quantitative real‐time PCR. Results We identified 8 miRNAs with significant differential expression. MiR‐299‐5p and miR‐765 had increased expression levels in the CSF of OA‐CSM‐affected dogs, whereas miR‐494, miR‐612, miR‐302‐d, miR‐4531, miR‐4455, and miR‐6721‐5p had decreased expression levels in OA‐CSM affected dogs compared to clinically normal dogs. Quantitative real‐time PCR was performed to validate the expression levels of 2 miRNAs (miR‐494 and miR‐612), and we found a 1.5‐fold increase in miR‐494 expression and a 1.2‐fold decrease in miR‐612 in the CSF of the OA‐CSM affected group (P = .41 and .89, respectively). Conclusions and Clinical Importance Data generated from our study represent an initial characterization of the miRNA profile of normal canine CSF and suggest that a distinct CSF miRNA expression profile is associated with OA‐CSM.

Treatment of EGFR mutant non-small cell lung cancer (NSCLC) often develops resistance to EGFR TKIs. In this study, the authors discover a non-canonical activation of β-catenin signaling through Notch3 as a mechanism of adaptation to and resistance to EGFR TKI treatment in NSCLC.

The NanoString nCounter System has been widely used in basic science and translational science research for the past decade. The System consists of two instruments: the PrepStation and the Digital Analyzer, and both have been continuously improved with evolving technologies. A great amount of research data have been generated at multiple research laboratories with the employment of different generations of the System. With the need of integrating multiple datasets, researchers are interested to know whether signals are comparable between different generations of the System. Toward this end, we designed a profiling study to compare performance of two generations of the NanoString nCounter System using a common set of biological samples. Using graphical tools and statistical models, we found that two different generations of NanoString nCounter System produced equivalent signals and signal deviations are in the range of random background noises for the medium-high expression levels.

Due to the motivations of climate change, the health impacts of poor air quality, and the importance of cities for economic growth, transport policy at all levels of governance places emphasis on reducing and managing urban traffic and congestion. Whilst the majority of urban traffic is created by personal travel, freight vehicles make a relatively large contribution per vehicle to congestion, pollution and severe accidents. The European Commission (EC 2011) estimates that 6% of all EU transport carbon emissions are from urban freight. For these reasons, a well-structured portfolio of measures and policies oriented towards more sustainable and efficient management of supply chain activities carried out in urban areas is needed, in order to reduce negative externalities related to urban mobility and improve economic performance. In recent years, there has been enthusiasm amongst commentators that shared-resource economic models can both create new commercial opportunities and address policy problems, including in the transport sector. Within the city logistics subsector, this new model is exemplified by the emergence of Urban freight Consolidation Centres (UCCs). UCCs replace multiple ‘last-mile’ delivery movements, many of which involving small consignments, by a common receiving point (the consolidation centre), normally on the periphery of a city, with the final part of the delivery being shared by the consignments in a small freight vehicle. Such arrangements can represent a good compromise between the needs of city centre businesses and their customers on the one hand (i.e. high availability of a range of goods) and local and global sustainability objectives on the other. At the same time, by sharing logistics facilities and delivery vehicles, UCCs offer added-value services to both urban economic actors, such as retailers, and network logistics providers. However, UCCs add to the complexity of logistics chains, requiring additional contracts, communications and movement stages. These arrangements also introduce additional actors within the supply of delivery services, notably local authorities present as promoters and funders, rather than simply as regulators, companies specialised in the UCC operation, and companies, which provide specialist technologies, such as electric delivery vehicles. UCCs therefore also represent an example of multi-stakeholder collaboration. Drawing on the results of a 2013 survey in Bristol (United Kingdom) and a further survey carried out in 2015 in Cagliari (Italy), the present paper will provide an in-depth comparison of the differences in the perceptions of urban freight users and stakeholders towards UCCs. Retailers involved in the survey carried out in Bristol showed high satisfaction with the delivery service provided by the UCC. Different topic areas (e.g. timeliness, reliability, safety) are examined through analyses of both qualitative and quantitative data. The survey carried out in Cagliari investigated the inclination of potential users to join a UCC scheme. The comparison between the two cities considers factors such as the nature of business holding (e.g. SME versus multiple retailers), operational practices (e.g. pattern of deliveries) and operating subsector (e.g. food versus no food). An analysis on the barriers to the implementation of UCCs in Bristol and in Cagliari is provided at the end of the paper.

Freight transport in urban areas entails benefits (i.e. free access to goods when needed), but also negative externalities (environmental, social and transportation impacts). In response to these problems, the concept of city logistics emerged, for the purpose of planning, organizing, coordinating and controlling physical and information flows in order to find a compromise between efficient freight distribution in urban areas and protection of the environment. A typical city logistics initiative is the Urban Freight Consolidation Centre (UFCC), the benefits of which are significant. Its financial issues though represent a huge problem for public administrations. However, a large customer network, comprising retailers participating in the initiative, could make the UFCC a self-financing scheme. The key to expanding the scheme is closely linked with marketing campaigns and customer care. Therefore, customer care analysis represents an important tool in developing UFCC schemes. In this paper, a new Customer Satisfaction Index (CSI) is proposed for evaluating UFCC service quality. The new index, named CSImod, is a modified version of the traditional CSI, but places greater emphasis on customer dissatisfaction, so as to analyse the most critical areas of the service with a view to improving them. The index has been tested using experimental data collected within the CIVITAS RENAISSANCE Project, in which the Bristol and Bath Freight Consolidation Centre (BBFCC) scheme was evaluated. The evaluation was done from a user perspective, i.e. the participating retailers. The CSImod places more importance on the most dissatisfied customers making it possible to understand why they are dissatisfied and with what. Thus, it is possible to intervene with the aim of improving those areas of the service that are perceived as the worst. In spite of the high level of satisfaction with the overall service provided by the BBFCC, thanks to the CSImod the analysis pointed out that some retailers are dissatisfied with the delivery time arrangements and also with deliveries that were getting wet, issues about which the BBFCC manager was totally unaware. The CSImod could be used by UFCC operators to extend the network of the retailers involved and could therefore provide an implicit solution for making the scheme self-financing. First published online: 29 Feb 2016

Melanoma remains the leading cause of skin cancer–related deaths. Surgical resection and adjuvant therapies can result in disease-free intervals for stage III and stage IV disease; however, recurrence is common. Understanding microRNA (miR) dynamics following surgical resection of melanomas is critical to accurately interpret miR changes suggestive of melanoma recurrence. Plasma of 6 patients with stage III (n = 2) and stage IV (n = 4) melanoma was evaluated using the NanoString platform to determine pre- and postsurgical miR expression profiles, enabling analysis of more than 800 miRs simultaneously in 12 samples. Principal component analysis detected underlying patterns of miR expression between pre- vs postsurgical patients. Group A contained 3 of 4 patients with stage IV disease (pre- and postsurgical samples) and 2 patients with stage III disease (postsurgical samples only). The corresponding preoperative samples to both individuals with stage III disease were contained in group B along with 1 individual with stage IV disease (pre- and postsurgical samples). Group A was distinguished from group B by statistically significant analysis of variance changes in miR expression ( P < .0001). This analysis revealed that group A vs group B had downregulation of let-7b-5p, miR-520f, miR-720, miR-4454, miR-21-5p, miR-22-3p, miR-151a-3p, miR-378e, and miR-1283 and upregulation of miR-126-3p, miR-223-3p, miR-451a, let-7a-5p, let-7g-5p, miR-15b-5p, miR-16-5p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-26a-5p, miR-106a-5p, miR-17-5p, miR-130a-3p, miR-142-3p, miR-150-5p, miR-191-5p, miR-199a-3p, miR-199b-3p, and miR-1976. Changes in miR expression were not readily evident in individuals with distant metastatic disease (stage IV) as these individuals may have prolonged inflammatory responses. Thus, inflammatory-driven miRs coinciding with tumor-derived miRs can blunt anticipated changes in expression profiles following surgical resection.

The neuroendocrine effects of leptin on metabolism hold promise to be translated into a complementary therapy to traditional insulin therapy for diabetes and obesity. However, injections of leptin can provoke inflammation. We tested the effects of leptin, produced in the physiological adipocyte location, on metabolism in mouse models of genetic and dietary obesity. We generated 3T3-L1 adipocytes constitutively secreting leptin and encapsulated them in a poly-L-lysine membrane, which protects the cells from immune rejection. Ob/ob mice (OB) were injected with capsules containing no cells (empty, OB[Emp]), adipocytes (OB[3T3]), or adipocytes overexpressing leptin (OB[Lep]) into both visceral fat depots. Leptin was found in the plasma of OB[Lep], but not OB[Emp] and OB[3T3] mice at the end of treatment (72 days). The OB[Lep] and OB[3T3] mice have transiently suppressed appetite and weight loss compared to OB[Emp]. Only OB[Lep] mice have greater brown fat mass, metabolic rate, and reduced resistin plasma levels compared to OB[Emp]. Glucose tolerance was markedly better in OB[Lep] vs. OB[Emp] and OB[3T3] mice as well as in wild type mice with high-fat diet-induced obesity and insulin resistance treated with encapsulated leptin-producing adipocytes. Our proof-of-principle study provides evidence of long-term improvement of glucose tolerance with encapsulated adipocytes producing leptin.

The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out--miR29ab1b2c--has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.

One of the most important issues in Transhipment Container Terminal (TCT) management is to have fairly reliable and affordable predictions about vessel arrival. Terminal operators need to estimate the actual time of arrival in port in order to determine the daily demand for each work shift with greater accuracy. In this way, the resources required (human resources, equipment as well as spatial resources) can be allocated more efficiently. Despite contractual obligations to notify the Estimated Time of Arrival (ETA) 24 hours before arrival, ship operators often have to revise it due to unexpected events like weather conditions, delay in a previous port and so on. For planners the decision-making processes related to this topic can sometimes be so complex without the support of suitable methodological tools. Specific models should be adopted, in a daily planning scenario, to provide a useful support tool in TCTs. In this study, we discuss an exploratory analysis of the data affecting delays registered at a Mediterranean TCT. We present some preliminary results obtained using data mining techniques and propose a Classification and Regression Trees (CART) model to reduce the range of uncertainty of ship arrivals in port. This approach is compulsory to manage vast amounts of unstructured data involved in estimating of vessel arrivals.