Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies

Soluble CD40 ligand and prolactin levels in migraine patients during interictal period

The relationship of migraine with cardiovascular diseases has been clarified by many studies, and currently, migraine is suggested to be a systematic vasculopathy. Inflammation, thrombosis and impaired vascular reactivity are the underlying pathophysiological mechanisms of the vasculopathy. In the present study, we aimed to investigate the relationship between prolactin levels and subclinical atherosclerosis risk factors such as soluble CD40 ligand (sCD40L) and high-sensitivity CRP (hsCRP) in migraine patients during interictal period. Fifty female migraine patients and age-matched 25 female control cases were enrolled in the study. Migraine diagnosis was settled according to the ICHD-II diagnostic criteria. A questionnaire was completed about the existence of vascular risk factors. Serum samples were used to measure sCD40L, hsCRP and prolactin levels. No difference was found between the prolactin levels of the migraine patients and the controls. The sCD40L levels were significantly higher in migraine patients (p < 0.001). High-sensitivity CRP levels showed no difference between the groups. There was no correlation between prolactin, sCD40L, and hs-CRP levels in migraine patients. We consider that the migraine patients are prone to subclinical atherosclerosis, but this tendency is independent of prolactin levels

Tyrosine kinase inhibitors reprogramming immunity in renal cell carcinoma: rethinking cancer immunotherapy

Review article[Abstract] The immune system regulates angiogenesis in cancer by way of both pro- and antiangiogenic activities. A bidirectional link between angiogenesis and the immune system has been clearly demonstrated. Most antiangiogenic molecules do not inhibit only VEGF signaling pathways but also other pathways which may affect immune system. Understanding of the role of these pathways in the regulation of immunosuppressive mechanisms by way of specific inhibitors is growing. Renal cell carcinoma (RCC) is an immunogenic tumor in which angiogenesis and immunosuppression work hand in hand, and its growth is associated with impaired antitumor immunity. Given the antitumor activity of selected TKIs in metastatic RCC (mRCC), it seems relevant to assess their effect on the immune system. The confirmation that TKIs improve cell cytokine response in mRCC provides a basis for the rational combination and sequential treatment of TKIs and immunotherapy

Nano-Opto-Electro-Mechanical Systems

A new class of hybrid systems that couple optical, electrical and mechanical degrees of freedom in nanoscale devices is under development in laboratories worldwide. These nano-opto-electro-mechanical systems (NOEMS) offer unprecedented opportunities to dynamically control the flow of light in nanophotonic structures, at high speed and low power consumption. Drawing on conceptual and technological advances from cavity optomechanics, they also bear the potential for highly efficient, low-noise transducers between microwave and optical signals, both in the classical and quantum domains. This Progress Article discusses the fundamental physical limits of NOEMS, reviews the recent progress in their implementation, and suggests potential avenues for further developments in this field.Comment: 27 pages, 3 figures, 2 boxe

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field