A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector.

BACKGROUND: Bile acid (BA) homeostasis is mainly regulated by bile salt excretory pump (BSEP), a hepatocyte transporter that transfers BAs to the bile. BSEP expression is regulated by BA levels through activation of farnesoid X receptor transcription factor, which binds to the inverted repeat (IR-1) element in the BSEP promoter. Gene therapy of cholestatic diseases could benefit from using vectors carrying endogenous promoters physiologically regulated by BAs, however their large size limits this approach, especially when using adeno-associated viral vector (AAV) vectors. RESULTS: We evaluated the functionality and BA-mediated regulation of minimal versions of human and mouse BSEP promoters containing IR-1 using AAV vectors expressing luciferase. Unexpectedly, a minimal mouse BSEP promoter (imPr) showed higher BA-mediated expression and inducibility than a minimal human promoter (ihPr) or than full-length BSEP promoters in human hepatic cells. In addition, in mice receiving an AAV8 vector carrying imPr promoter-driven luciferase expression was efficiently regulated by administration of a BA-enriched diet. Interestingly, this vector also expressed significantly higher luciferase levels in Abcb4-/- mice, which have high levels of BAs, compared to wild type mice, or to mice receiving a vector containing the luciferase gene downstream of the constitutive alpha-1 antitrypsin promoter. In contrast, the AAV vector containing ihPr showed very low luciferase expression with no inducibility. Finally, we optimized imPr by adding three IR-1 repeats at its 5' end. This new promoter provided higher levels of luciferase than imPr both in vitro and in vivo. CONCLUSIONS: The imPr could represent a useful tool for gene therapy approaches in which physiological BA regulation is desired.

Gene Therapy for Acquired and Genetic Cholestasis.

Cholestatic diseases can be caused by the dysfunction of transporters involved in hepatobiliary circulation. Although pharmacological treatments constitute the current standard of care for these diseases, none are curative, with liver transplantation being the only long-term solution for severe cholestasis, albeit with many disadvantages. Liver-directed gene therapy has shown promising results in clinical trials for genetic diseases, and it could constitute a potential new therapeutic approach for cholestatic diseases. Many preclinical gene therapy studies have shown positive results in animal models of both acquired and genetic cholestasis. The delivery of genes that reduce apoptosis or fibrosis or improve bile flow has shown therapeutic effects in rodents in which cholestasis was induced by drugs or bile duct ligation. Most studies targeting inherited cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), have focused on supplementing a correct version of a mutated gene to the liver using viral or non-viral vectors in order to achieve expression of the therapeutic protein. These strategies have generated promising results in treating PFIC3 in mouse models of the disease. However, important challenges remain in translating this therapy to the clinic, as well as in developing gene therapy strategies for other types of acquired and genetic cholestasis.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Microtubule-associated tumor suppressors as prognostic biomarkers in breast cancer.

PURPOSE: Breast cancer is the most common malignancy in women worldwide. Although important therapeutic progress was achieved over the past decade, this disease remains a public health problem. In light of precision medicine, the identification of new prognostic biomarkers in breast cancer is urgently needed to stratify populations of patients with poor clinical outcome who may benefit from new personalized therapies. The microtubule cytoskeleton plays a pivotal role in essential cellular functions and is an interesting target for cancer therapy. Microtubule assembly and dynamics are regulated by a wide range of microtubule-associated proteins (MAPs), some of which have oncogenic or tumor suppressor effects in breast cancer. RESULTS: This review covers current knowledge on microtubule-associated tumor suppressors (MATS) in breast cancer and their potential value as prognostic biomarkers. We present recent studies showing that combinatorial expression of ATIP3 and EB1, two microtubule-associated biomarkers with tumor suppressor and oncogenic effects, respectively, improves breast cancer prognosis compared to each biomarker alone. CONCLUSIONS: These findings are discussed regarding the increasing complexity of protein networks composed of MAPs that coordinate microtubule dynamics and functions. Further studies are warranted to evaluate the prognostic value of combined expression of different MATS and their interacting partners in breast cancer.

Timing the spinal cord development with neural progenitor cells losing their proliferative capacity: a theoretical analysis.

In the developing neural tube in chicken and mammals, neural stem cells proliferate and differentiate according to a stereotyped spatiotemporal pattern. Several actors have been identified in the control of this process, from tissue-scale morphogens patterning to intrinsic determinants in neural progenitor cells. In a previous study (Bonnet et al. eLife 7, 2018), we have shown that the CDC25B phosphatase promotes the transition from proliferation to differentiation by stimulating neurogenic divisions, suggesting that it acts as a maturating factor for neural progenitors. In this previous study, we set up a mathematical model linking fixed progenitor modes of division to the dynamics of progenitors and differentiated populations. Here, we extend this model over time to propose a complete dynamical picture of this process. We start from the standard paradigm that progenitors are homogeneous and can perform any type of divisions (proliferative division yielding two progenitors, asymmetric neurogenic divisions yielding one progenitor and one neuron, and terminal symmetric divisions yielding two neurons). We calibrate this model using data published by Saade et al. (Cell Reports 4, 2013) about mode of divisions and population dynamics of progenitors/neurons at different developmental stages. Next, we explore the scenarios in which the progenitor population is actually split into two different pools, one of which is composed of cells that have lost the capacity to perform proliferative divisions. The scenario in which asymmetric neurogenic division would induce such a loss of proliferative capacity appears very relevant.

Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase.

A fundamental issue in developmental biology and in organ homeostasis is understanding the molecular mechanisms governing the balance between stem cell maintenance and differentiation into a specific lineage. Accumulating data suggest that cell cycle dynamics play a major role in the regulation of this balance. Here we show that the G2/M cell cycle regulator CDC25B phosphatase is required in mammals to finely tune neuronal production in the neural tube. We show that in chick neural progenitors, CDC25B activity favors fast nuclei departure from the apical surface in early G1, stimulates neurogenic divisions and promotes neuronal differentiation. We design a mathematical model showing that within a limited period of time, cell cycle length modifications cannot account for changes in the ratio of the mode of division. Using a CDC25B point mutation that cannot interact with CDK, we show that part of CDC25B activity is independent of its action on the cell cycle.

Regulation of end-binding protein EB1 in the control of microtubule dynamics.

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of "endogenous EB1 antagonists" and emphasize the importance of finely regulating EB1 function at growing microtubule ends.

Playing with the cell cycle to build the spinal cord.

A fundamental issue in nervous system development and homeostasis is to understand the mechanisms governing the balance between the maintenance of proliferating progenitors versus their differentiation into post-mitotic neurons. Accumulating data suggest that the cell cycle and core regulators of the cell cycle machinery play a major role in regulating this fine balance. Here, we focus on the interplay between the cell cycle and cellular and molecular events governing spinal cord development. We describe the existing links between the cell cycle and interkinetic nuclear migration (INM). We show how the different morphogens patterning the neural tube also regulate the cell cycle machinery to coordinate proliferation and patterning. We give examples of how cell cycle core regulators regulate transcriptionally, or post-transcriptionally, genes involved in controlling the maintenance versus the differentiation of neural progenitors. Finally, we describe the changes in cell cycle kinetics occurring during neural tube patterning and at the time of neuronal differentiation, and we discuss future research directions to better understand the role of the cell cycle in cell fate decisions.

Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics.

Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study, we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression.

Synthesis, antimalarial activity and cytotoxic potential of new monocarbonyl analogues of curcumin.

A series of novel monocarbonyl analogues of curcumin have been designed, synthesized and tested for their activity against Molt4, HeLa, PC3, DU145 and KB cancer cell lines. Six of the analogues showed potent cytotoxicity towards these cell lines with IC(50) values below 1 µM, which is better than doxorubicin, a US FDA approved drug. Several analogues were also found to be active against both CQ-resistant (W2 clone) and CQ-sensitive (D6) strains of Plasmodium falciparum in an in-vitro antimalarial screening. This level of activity warrants further investigation of the compounds for development as anticancer and antimalarial agents.

Angiotensin II facilitates breast cancer cell migration and metastasis.

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

First Colombian multicentric newborn screening for congenital toxoplasmosis.

AIMS: To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern). MATERIALS AND METHODS: We collected 15,333 samples from umbilical cord blood between the period of March 2009 to May 2010 in 19 different hospitals and maternal-child health services from seven different cities. We applied an IgM ELISA assay (Vircell, Spain) to determine the frequency of IgM anti Toxoplasma. The results in blood cord samples were confirmed either by western blot and repeated ELISA IgM assay. In a sub-sample of 1,613 children that were negative by the anti-Toxoplasma IgM assay, the frequency of specific anti-Toxoplasma IgA by the ISAGA assay was determined. All children with positive samples by IgM, IgA, clinical diagnosis or treatment during pregnancy were recalled for confirmatory tests after day 10 of life. RESULTS: 61 positive samples for specific IgM (0.39%) and 9 positives for IgA (0.5%) were found. 143 questionnaires were positive for a clinical diagnosis or treatment for toxoplasmosis during pregnancy. 109 out of the 218 children that had some of the criteria for postnatal confirmatory tests were followed. Congenital toxoplasmosis infection was confirmed in 15 children: 7 were symptomatic, and three of them died before the first month of life (20% of lethality). A significant correlation was found between a high incidence of markers for congenital toxoplasmosis and higher mean annual rainfall for the city. CONCLUSIONS: Incidence for congenital toxoplasmosis is significantly different between hospitals or maternal child health services from different cities in Colombia. Mean annual rainfall was correlated with incidence of congenital toxoplasmosis.

Boronic acid-modified DNA that changes fluorescent properties upon carbohydrate binding.

A long wavelength boronic acid-modified TTP (NB-TTP) has been synthesized and enzymatically incorporated into DNA. Such DNA shows intrinsic fluorescent changes upon carbohydrate addition.