Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene

Background: Maspin, a recently identified protein related to the family of serpins, is believed to play a role in human breast cancer. In an effort to improve the present methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for maspin transcript to identify small numbers of mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. Patients and methods: Five non-neoplastic mammary tissue samples, 13 breast cancer specimens as well as 17 peripheral blood and 4 bone marrow samples from normal subjects were screened for the presence of maspin mRNA by RT-PCR. The same assay was applied to peripheral blood or bone marrow samples obtained from 29 patients with stages I to IV breast cancer. Results: By RT-PCR it was possible to amplify maspin mRNA in all of the primary and metastatic breast cancer specimens, but in none of the normal hemopoietic samples from healthy donors. Thus, detection of maspin transcript in the peripheral blood or marrow of a patient known to have breast cancer is indicative of the presence of mammary carcinoma cells. In reconstitution experiments, maspin RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear cells (PBMCs). None of the 9 patients with stages I, II, or III breast cancer had maspin transcript in peripheral blood. Of note, 3 of 9 patients with stage TV breast cancer receiving systemic therapy at the time of sample collection, but only I of 11 patients with stage IV not receiving therapy, had detectable maspin transcript in peripheral blood. Moreover, 3 marrow specimens from stage TV patients tested positive by this assay. Conclusions: This pilot study suggests that maspin RT-PCR assay is a sensitive, specific and sufficiently rapid method for detection of small numbers of circulating cells and marrow micrometastases in breast cancer patients. The possibility of applying this assay in the detection of tumor cell contamination of both marrow and stem-cell apheresis harvests of breast cancer patients merits further investigation

An integrated approach for the analysis and modeling of road tunnel ventilation. Part I: Continuous measurement of the longitudinal airflow profile

The knowledge of the flow field inside road tunnels under normal operation, let alone fire conditions, is only approximate and partial. The reason is that while the full three-dimensional, unsteady problem is out of reach of numerical methods, on the other hand accurate measurement of the airflow in road and railway tunnels constitutes an extremely demanding task. The present work, structured as a twofold study, takes up the challenge and proposes an original integrated experimental and numerical approach for the analysis and modeling of flow inside a road tunnel and its ventilation systems, aiming at defining a methodology for the creation of “digital twins” of the system itself, on which advanced ventilation and smoke control strategies can be tested and fine-tuned. In this first part, an innovative experimental facility for the continuous acquisition of the longitudinal velocity profile along the whole length of a road tunnel has been designed and built. The facility consists of a survey rake with five bidirectional vane anemometers, which is mounted on a small electric vehicle that can travel through the tunnel at constant speed. This paper reports the design procedure of the measurement facility, with particular focus on the conception and realization of the vehicle carrying the survey rake. Results of the first experimental campaign carried out under the 11611 meters long Mont Blanc road tunnel are presented to corroborate the validity of the approach adopted and the accuracy of the measurement chain

A novel microwave and induction heating applicator for metal making: Design and testing

The use of microwave heating in primary metallurgy is gaining an increasing interest due to the possibility to selectively process ores and to volumetrically heat large amounts of low-thermal conductivity minerals. In this paper the study, development and testing of a new applicator combining the use of microwave and induction heating for rapid reduction of metal containing oxides is described. Numerical simulation was used in order to achieve the proper control over heat generation, considering the use of microwave solid state generators. A prototype, with a capacity up to 5 liters of standard input feed but with the predisposition for continuous processing has been designed, built and tested on reference loads like iron oxide powders and pellets. Results on the microwave heating part of the applicator indicate that it allows to efficiently and rapidly process these kinds of loads, which change from dielectric to conductors as reduction proceeds. The use of variable frequency solid state microwave generators allows to maximize energy efficiency and to controllably change the heating pattern inside the load

Study of the Interaction Between a High-Pressure Jet and Horizontal Tanks using CFD

Accidental high-pressure flammable gas releases are among the most relevant hazards in the process safety, and consequences could be severe. In the recent decades, there have been numerous efforts to study high-pressure jets in open field (i.e., free jets). Easy-to-use mathematical models have been developed, to rapidly assess the main physical variables involved in safety evaluations. However, in a realistic scenario, the accidental leak may involve either the ground or a piece of equipment. As demonstrated by recent works, when a jet interacts with an obstacle, its behavior can significantly change. Therefore, the mathematical models extrapolated for the free jet scenario could be a source of incorrect predictions. Focusing on the scenario of an accidental high-pressure unignited flammable jet, this work shows how the presence of one or two obstacles, placed at a different distance from the source of the leak, can influence the lower flammability limit cloud extent of methane. Varying the height of the source term, the effect of the interaction among the jet, both the obstacles, and the ground was systematically studied through a Computational Fluid Dynamics analysis

Actin and microtubules differently contribute to vacuolar targeting specificity during the export from the er

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins’ functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton

Thromopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures.

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis- regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO

Significance of glycolytic metabolism-related protein expression in colorectal cancer, lymph node and hepatic metastasis

Background: Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis. Methods: Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry. Results: All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting. Conclusion: These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘Programa Operacional Temático Factores de Competitividade’ (COMPETE) of ‘Quadro Comunitário de Apoio III’ and co-financed by the Fundo Europeu De Desenvolvimento Regional (FEDER). Ricardo Amorim was recipient of the fellowship SFRH/BD/98002/2013, from Fundação para a Ciência e a Tecnologia (FCT Portugal).info:eu-repo/semantics/publishedVersio

Myeloproliferative neoplasms and splanchnic vein thrombosis: Clinical and molecular features. A single-center cohort study

Venous thromboses account for approximately 30-40% of vascular complications in MPN, also involving the splanchnic circulation (SVT) with a prevalence of 1-23%. Here, we reported a consecutive single-center series of 54 MPN patients (pts), who developed an SVT at diagnosis or during follow-up between 1979 and 2020. We identified 13% of PV, 16.7% of ET, 44.4% of MF, and 25.9% of MPN-U. Most of the cases (83.3%) bear a JAK2V617F mutation, whereas seven (13%) pts were characterized by other molecular markers, i.e., MPL in four and CALR mutations in three cases. The remaining two (3.7%) pts were defined as triple-negative. NGS was performed in 17 (31.5%) cases: the most frequent mutations were found in TET2 (35.3%) and DNMT3A (23.5%) genes, whereas seven (41.2%) pts had no additional mutation. At the time of SVT onset, active antiplatelet therapy was documented in 18.5% of the cases. Among the 16 (29.6%) pts who suffered from SVT during follow-up, cytoreduction was already on-going in 56.3% of the cases, whereas it was then started in all but 16 pts, mainly due to a normal blood cells count. Anticoagulants were started in 43 (79.6%) pts, including ten (18.5%) cases treated with DOACs. After a median follow-up from MPN diagnosis of 8.3 years, nine (16.7%) deaths were recorded: it was due to leukemic transformation in five pts, hemorrhages in three and infections in the remaining patient. 38.9% of the pts suffered from recurrent vascular events, either involving the arterial (13%) or the venous district (25.9%), with 10 (18.5%) pts experiencing a recurrent SVT. In the present study MPN-U seems to represent a distinct clinical entity when compared to other MPN subtypes, as SVT was the initial manifestation in all these cases. Interestingly, during follow-up none of these pts developed clinical features which enabled physicians to re-classify them among one of the classical MPN. Being aware of its limitations, our study confirm that SVT associated with MPN-U represents a more indolent disease as compared with full-diagnosed MPN. Notably, all leukemic evolutions were reported among MF pts after a median follow-up of 15.6 years. Furthermore, our preliminary data support the use of NGS analysis in MPN-related SVT management as it can provide useful diagnostic and prognostic information. However, more than one third of our pts developed recurrent vascular events, confirming the limited efficacy of conventional therapeutic approaches. Updated results will be presented

Detection of antibodies to human herpesvirus 8 in Italian children: evidence for horizontal transmission

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma associated herpesvirus (KSHV), has been shown to be the causative agent for Kaposi's sarcoma (KS) and to be more prevalent in populations or risk groups at increased risk for KS. HHV-8 infection is rare in children from the US and the UK, but has been reported in African children. In this study we examine HHV-8 infection in children from Italy, a country with an elevated prevalence of HHV-8 in adults and high socio-economic conditions. © 2000 Cancer Research Campaig

Multiparametric flow cytometry for MRD monitoring in hematologic malignancies: Clinical applications and new challenges

In hematologic cancers, Minimal Residual Disease (MRD) monitoring, using either molecular (PCR) or immunophenotypic (MFC) diagnostics, allows the identification of rare cancer cells, readily detectable either in the bone marrow or in the peripheral blood at very low levels, far below the limit of classic microscopy. In this paper, we outlined the state-of-the-art of MFC-based MRD detection in different hematologic settings, highlighting main recommendations and new challenges for using such a method in patients with acute leukemias or chronic hematologic neoplasms. The combination of new molecular technologies with advanced flow cytometry is progressively allowing clinicians to design a personalized therapeutic path, proportionate to the biological aggressiveness of the disease, in particular by using novel immunotherapies, in view of a modern decision-making process, based on precision medicine. Along with the evolution of immunophenotypic and molecular diagnostics, the assessment of Minimal Residual Disease (MRD) has progressively become a keystone in the clinical management of hematologic malignancies, enabling valuable post-therapy risk stratifications and guiding risk-adapted therapeutic approaches. However, specific prognostic values of MRD in different hematological settings, as well as its appropriate clinical uses (basically, when to measure it and how to deal with different MRD levels), still need further investigations, aiming to improve standardization and harmonization of MRD monitoring protocols and MRD-driven therapeutic strategies. Currently, MRD measurement in hematological neoplasms with bone marrow involvement is based on advanced highly sensitive methods, able to detect either specific genetic abnormalities (by PCRbased techniques and next-generation sequencing) or tumor-associated immunophenotypic profiles (by multiparametric flow cytometry, MFC). In this review, we focus on the growing clinical role for MFC-MRD diagnostics in hematological malignancies-from acute myeloid and lymphoblastic leukemias (AML, B-ALL and T-ALL), to chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)-providing a comparative overview on technical aspects, clinical implications, advantages and pitfalls of MFC-MRD monitoring in different clinical settings