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In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Cervico-vaginal (CV) localization of extra-mammary Paget’s disease (EMPD) of the vulva is extremely rare. In order to investigate the incidence risk and the pathognomonic clinical and pathological features of this condition, a retrospective analysis was conducted including 94 women treated for vulvar EMPD at the European Institute of Oncology, Milan, Italy, from October 1997 to May 2020. Overall nine patients developed CV involvement from EMPD, with a cumulative incidence of 2.5% (95% CI: 0.5–8.0%) at 5 years, 6.5% (95% CI: 1.9–15.1%) at 10 years and 14.0% (95% CI: 4.8–27.8%) at 15 years, respectively. All cases except one were firstly detected by abnormal glandular cytology. None reported vaginal bleeding or other suspicious symptoms. The colposcopic findings were heterogeneous and could sometimes be misdiagnosed. Cervical and/or vaginal biopsies were always performed for histopathological diagnosis by identification of Paget cells in the epithelium or stroma. Most patients developed invasive EMPD (5/9) of the cervix and/or vagina and underwent hysterectomy with partial or total colpectomy. CV involvement from EMPD should not be underestimated in women with a long-standing history of vulvar Paget’s disease. Liquid-based cytology with immunocytochemistry represents a valuable tool for early diagnosis and should be routinely performed during the required lifelong follow-up.

Objectives: Tumor-associated antigens (TAAs) are frequently overexpressed in several cancer types. The aim of this study was to investigate the expression of TAAs in breast cancer. Material and methods: A total of 250 selected invasive breast cancers including 50 estrogen receptor (ER)-positive (Luminal B like), 50 triple-negative (TN), 50 ER-positive lobular type, 50 ER- and progesterone receptor (PgR)-positive (Luminal A like) and 50 cerbB2-positive breast cancers, were assessed for New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Wilms tumor antigen (WT-1) and PReferentially expressed Antigen of MElanoma (PRAME) antigen expression by immunohistochemistry (IHC). Results: A significantly higher expression of cancer testis (CT)-antigens NY-ESO-1 and WT-1 antigen was detected in TN breast cancers compared with ER-positive tumors. NY-ESO-1 overexpression (score 2 + and 3+) assessed by monoclonal and polyclonal antibodies was detected in 9 (18%) TN cancers as compared to 2 (4%) ER-positive tumors (p = 0.002). WT1 over-expression (score 2 + and 3+) was confirmed in 27 (54%) TN tumor samples as compared to 6 (12%) ER-positive (p < 0.0001). PRAME over-expression (score 2 + and 3+) was detected in 8 (16%) HER2 positive tumor samples as compared to no TN and ER-positive cancers (p = 0.0021). Conclusions: NY-ESO-1 and WT1 antigens are overexpressed in TN breast cancers. Because of the limited therapeutic options for this patient subgroup, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

Objectives To describe sonographic features of the microcystic elongated and fragmented (MELF) pattern of myometrial invasion (MI) using the International Endometrial Tumor Analysis (IETA) criteria; to assess the effect of the MELF pattern on preoperative ultrasound evaluation of MI; and to determine the relationship of the MELF pattern to more advanced stage (≥ IB) and lymph node metastases in women with endometrioid endometrial cancer. Methods/materials We included 850 women with endometrioid endometrial cancer from the prospective IETA study. Ultrasound experts performed all ultrasound examinations, according to the IETA protocol. Reference pathologists assessed the presence or absence of the MELF pattern. Sonographic features and accuracy of ultrasound assessment of MI were compared in cases with the presence and the absence of the MELF pattern. The MELF pattern was correlated to more advanced stage (≥IB) and lymph node metastases. Results The MELF pattern was present in 197 (23.2%) women. On preoperative ultrasound imaging the endometrium was thicker (p = 0.031), more richly vascularized (p = 0.003) with the multiple multifocal vessel pattern (p < 0.001) and the assessment of adenomyosis was more often uncertain (p < 0.001). The presence or the absence of the MELF pattern did not affect the accuracy of the assessment of MI. The MELF pattern was associated with MI ≥ 50% (p < 0.001), cervical stromal invasion (p = 0.037), more advanced stage (≥ IB) (p < 0.001) and lymph node metastases (p = 0.011). Conclusions Tumors with the MELF pattern were slightly larger, more richly vascularized with multiple multifocal vessels and assessment of adenomyosis was more uncertain on ultrasound imaging. The MELF pattern did not increase the risk of underestimating MI in preoperative ultrasound staging. Tumors with the MELF pattern were more than twice as likely to have more advanced stage (≥ IB) and lymph node metastases.

The objective of this research is to make a comprehensive description of the understanding of pragmatic meanings by speakers of Spanish as a second and foreign language. Our hypothesis is that the performance in the understanding of pragmatic contents improves if these are conveyed through a lexical or contextual item, and not grammatical one, or through a non-directive type of speech. The study is carried out through an online survey to 49 non-native Spanish speakers, which consist of 19 scenes that require a mandatory pragmatic interpretation. The statistically analyzed results confirm that performance in non-directive speech acts is better than in directive ones. However, there are no performance differences according to the level (grammatical, contextual or semantic-lexical) at which the pragmatic contents are conveyed. Furthermore, it is observed that Brazilian Portuguese speakers better understand the pragmatics of utterances as a whole and show less difficulties with directive speech acts. Other variables, such as instruction or residence time, do not contribute significantly to improving performance in understanding these statements., Esta investigación se propone realizar un diagnóstico de la comprensión de los significados pragmáticos por parte de hablantes de ELSE de Argentina. Nuestra hipótesis es que el rendimiento en comprensión de los contenidos pragmáticos mejora si estos se vehiculizan a través de un ítem léxico o contextual, y no gramatical, o a través de un tipo de habla no directivo. El estudio se lleva a cabo a través de la aplicación en línea de un cuestionario de 19 escenas que disparan una interpretación pragmática a 49 hablantes de español como lengua segunda y extranjera. Los resultados, analizados estadísticamente, arrojan resultados significativos en el rendimiento de actos de habla para el grupo total y confirman que el rendimiento en actos de habla no directivos es mejor que en los directivos. No obstante, no existen diferencias de desempeño según el nivel (gramatical, contextual o semántico léxico) en el que se vehiculizan los contenidos pragmáticos. Además, se observa que los lusoparlantes brasileños comprenden mejor la pragmática de los enunciados en general y muestran menos dificultades con los actos de habla directivos. Otras variables, como el tiempo de instrucción y el tiempo de residencia, no contribuyen significativamente a la mejora del rendimiento en la comprensión de estos enunciados.

DarkSide is a dark matter direct search experiment at Laboratori Nazionali del Gran Sasso (LNGS). DarkSide is based on the detection of rare nuclear recoils possibly induced by hypothetical dark matter particles, which are supposed to be neutral, massive (m > 10 GeV) and weakly interactive (WIMP). The dark matter detector is a two-phase time projection chamber (TPC) filled with ultra-pure liquid argon. The TPC is placed inside a muon and a neutron active vetoes to suppress the background. Using argon as active target has many advantages, the key features are the strong discriminant power between nuclear and electron recoils, the spatial reconstruction and easy scalability to multi-tons size. At the moment DarkSide-50 is filled with ultra-pure argon, extracted from underground sources, and from April 2015 it is taking data in its final configuration. When combined with the preceding search with an atmospheric argon target, it is possible to set a 90% CL upper limit on the WIMP-nucleon spin-independent cross section of 2.0×10 -44 cm2 for a WIMP mass of 100 GeV/c2. The next phase of the experiment, DarkSide-20k, will be the construction of a new detector with an active mass of ∼ 20 tons.

OBJECTIVES: To describe sonographic features of the microcystic elongated and fragmented (MELF) pattern of myometrial invasion (MI) using the International Endometrial Tumor Analysis (IETA) criteria; to assess the effect of the MELF pattern on preoperative ultrasound evaluation of MI; and to determine the relationship of the MELF pattern to more advanced stage (≥ IB) and lymph node metastases (LNM) in women with endometrioid endometrial cancer (EEC). METHODS/MATERIALS: We included 850 women with EEC from the prospective IETA 4 study. Ultrasound experts performed all ultrasound examinations, accordingto the IETA protocol. Reference pathologists assessed the presence orabsence of the MELF pattern. Sonographic features and accuracy of ultrasound assessment of MI were compared in cases with the presence and the absence of the MELF pattern. The MELF pattern was correlated to more advanced stage (≥IB) and LNM. RESULTS: The MELF pattern was present in 197 (23.2%) women. On preoperative ultrasound imaging the endometrium was thicker (p = 0.031), more richly vascularized (p = 0.003) with the multiple multifocal vessel pattern (p < 0.001) and the assessment of adenomyosis was more often uncertain (p < 0.001). The presence or the absence of the MELF pattern did not affect the accuracy of the assessment of MI. The MELF pattern was associated with MI ≥ 50% (p < 0.001), cervical stromal invasion (CSI) (p = 0.037), more advanced stage (≥ IB) (p < 0.001) and LNM (p = 0.011). CONCLUSIONS: Tumors with the MELF pattern were slightly larger, more richly vascularized with multiple multifocal vessels and assessment of adenomyosis was more uncertain on ultrasound imaging. The MELF pattern did not increase the risk of underestimating MI in preoperative ultrasound staging. Tumors with the MELF pattern were more than twice as likely to have more advanced stage (≥ IB) and LNM.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10-3) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10-3) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10 −3 ) and is undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10 −3 ) and is undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Prendendo le mosse dall’evoluzione semantica del termine «letteratura», questo articolo sostiene che l’idea di letteratura modernamente intesa, ben distinta dalle humanae litterae e non documentata prima del XVIII secolo – cioè la letteratura concepita come insieme di testi nati con specifiche finalità estetiche – nasce all’interno di una dimensione squisitamente riflessiva. Se la letteratura di secondo grado è sempre esistita, e se è verosimile che non esistano forme di intertestualità (o transtestualità) ignote alla cultura classica, è però vero che nel Settecento l’attività dello scrittore è investita come mai prima della necessità di «situarsi». Ora la creazione letteraria deve definire la propria posizione rispetto alla tradizione, alla cultura popolare, alle altre forme espressive. In questa luce, la nascita della «letteratura» è inscindibile dallo sviluppo del discorso meta-letterario: i frammenti di Friedrich Schlegel sulla poesia moderna lo testimoniano con un acume che non ha perso nulla della sua esemplarità.

Background: Cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. Patients and methods: A total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. Results: A significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. Conclusions: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements.

Background: Cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. Patients and methods: A total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. Results: A significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P ' 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P ' 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. Conclusions: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer. © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.

We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.

We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.

Item does not contain fulltext, p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and DeltaN configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the beta isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of DeltaN isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the DeltaN version, with respect to their p63alpha counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.

Item does not contain fulltext, p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and DeltaN configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the beta isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of DeltaN isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the DeltaN version, with respect to their p63alpha counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.

Item does not contain fulltext, p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and DeltaN configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the beta isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of DeltaN isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the DeltaN version, with respect to their p63alpha counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.

Il pirofosfato di vanadile (VPP, (VO)2P2O7) è il componente principale, assieme ad altri ossidi di vanadio e fosforo, del sistema catalitico spesso chiamato con il termine “V/P/O” tipicamente utilizzato nell’ossidazione di n-butano ad anidride maleica (AM). In questo lavoro di tesi, si è posta l’attenzione sulla fase di preparazione di questo sistema catalitico e, in particolare, sul processo di trattamento termico del precursore che permette di ottenere la fase attiva. Si è disposto di un unico precursore industriale costituito di (VO)HPO4·0,5H2O, il quale è stato trattato termicamente in condizioni diverse per indagare l’effetto della composizione dell’ambiente di calcinazione sulla formazione della fase attiva VPP, e quindi sulle conseguenti prestazioni catalitiche. Si è deciso innanzitutto di valutare l’effetto dato dalla presenza di vapore acqueo all’interno dell’ambiente di calcinazione, studiando atmosfere di trattamento in cui viene variato il rapporto aria/acqua: ciò ha permesso di notare come aumentando la percentuale di acqua l’ottenimento di VPP sia il fenomeno dominante, formando un catalizzatore sia attivo che selettivo in AM. Si è perciò proseguito lo studio conducendo altre prove in ambiente dalla ridotta capacità ossidante, limitando la quantità di ossigeno introducendo un flusso di azoto in alimentazione ed infine provando atmosfere costituite da miscele di aria, azoto e acqua. Questo ha permesso di determinare i ruoli relativi che rivestono sia la percentuale di ossigeno (in quanto agente ossidante del nostro precursore) sia quella dell’acqua nel trattamento termico utilizzato industrialmente per ottenere catalizzatori a base di VPP. I catalizzatori ottenuti dal trattamento termico sono perciò stati caratterizzati, sottoposti a test catalitici e caratterizzati nuovamente, al fine di correlare le caratteristiche strutturali di ciascun catalizzatore, dipendenti dall’ambiente di calcinazione dal quale derivano, al comportamento catalitico osservato.

Il pirofosfato di vanadile (VPP, (VO)2P2O7) è il componente principale, assieme ad altri ossidi di vanadio e fosforo, del sistema catalitico spesso chiamato con il termine “V/P/O” tipicamente utilizzato nell’ossidazione di n-butano ad anidride maleica (AM). In questo lavoro di tesi, si è posta l’attenzione sulla fase di preparazione di questo sistema catalitico e, in particolare, sul processo di trattamento termico del precursore che permette di ottenere la fase attiva. Si è disposto di un unico precursore industriale costituito di (VO)HPO4·0,5H2O, il quale è stato trattato termicamente in condizioni diverse per indagare l’effetto della composizione dell’ambiente di calcinazione sulla formazione della fase attiva VPP, e quindi sulle conseguenti prestazioni catalitiche. Si è deciso innanzitutto di valutare l’effetto dato dalla presenza di vapore acqueo all’interno dell’ambiente di calcinazione, studiando atmosfere di trattamento in cui viene variato il rapporto aria/acqua: ciò ha permesso di notare come aumentando la percentuale di acqua l’ottenimento di VPP sia il fenomeno dominante, formando un catalizzatore sia attivo che selettivo in AM. Si è perciò proseguito lo studio conducendo altre prove in ambiente dalla ridotta capacità ossidante, limitando la quantità di ossigeno introducendo un flusso di azoto in alimentazione ed infine provando atmosfere costituite da miscele di aria, azoto e acqua. Questo ha permesso di determinare i ruoli relativi che rivestono sia la percentuale di ossigeno (in quanto agente ossidante del nostro precursore) sia quella dell’acqua nel trattamento termico utilizzato industrialmente per ottenere catalizzatori a base di VPP. I catalizzatori ottenuti dal trattamento termico sono perciò stati caratterizzati, sottoposti a test catalitici e caratterizzati nuovamente, al fine di correlare le caratteristiche strutturali di ciascun catalizzatore, dipendenti dall’ambiente di calcinazione dal quale derivano, al comportamento catalitico osservato.

Background: Cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. Patients and methods: A total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. Results: A significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. Conclusions: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer