Institute for Research in Biomedicine

Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.

Brain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood-brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins.

An updated review focused on the latest progress in the field of peptides as modulators of protein–protein interactions.

This review provides an extensive range of methodologies which outline the path to achieve the synthesis of “difficult peptides”.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Proteins are fascinating supramolecular structures, which are able to recognize ligands transforming binding information into chemical signals. They can transfer information across the cell, can catalyse complex chemical reactions, and are able to transform energy into work with much more efficiency than any human engine. The unique abilities of proteins are tightly coupled with their dynamic properties, which are coded in a complex way in the sequence and carefully refined by evolution. Despite its importance, our experimental knowledge of protein dynamics is still rather limited, and mostly derived from theoretical calculations. I will review here, in a systematic way, the current state-of-the-art theoretical approaches to the study of protein dynamics, emphasizing the most recent advances, examples of use and the expected lines of development in the near future.

Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apical-basal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.

In Th1 and Th2 memory lymphocytes, the genes for the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) are imprinted for expression upon restimulation. This cytokine memory is based on expression of the transcription factors T-bet for IFN-gamma, and GATA-3 for IL-4, and epigenetic modification of the cytokine genes. In Th2 cells, expression of the cytokine IL-10 is also induced by GATA-3. Here, we show that this induction is initially not accompanied by epigenetic modification of the IL-10 gene. Only after repeated restimulation of a memory Th2 cell in the presence of IL-4, extensive histone acetylation of the IL-10 gene is detectable. This epigenetic imprinting correlates with the development of a memory for IL-10 in repeatedly restimulated Th2 cells. In Th1 cells, IL-10 expression is induced by IL-12, but the IL-10 gene lacks detectable histone acetylation. Accordingly, IL-10 expression in restimulated memory Th1 cells remains conditional on the presence of IL-12. This finding defines a potential anti-inflammatory role for IL-12 in Th1 recall responses. While in primary Th1 responses IL-12 is required to induce expression of the pro-inflammatory cytokine IFN-gamma, in secondary Th1 responses IFN-gamma re-expression is independent of IL-12, which still is able to induce expression of the anti-inflammatory cytokine IL-10.

SIGNIFICANCE: Mitochondrial dynamics is a complex process that impacts on mitochondrial biology. RECENT ADVANCES: Recent evidence indicates that proteins participating in mitochondrial dynamics have additional cellular roles. Mitofusin 2 (Mfn2) is a potent modulator of mitochondrial metabolism with an impact on energy metabolism in muscle, liver, and hypothalamic neurons. In addition, Mfn2 is subjected to tight regulation. Hence, factors such as proinflammatory cytokines, lipid availability, or glucocorticoids block its expression, whereas exercise and increased energy expenditure promote its upregulation. CRITICAL ISSUES: Importantly, Mfn2 controls cell metabolism and insulin signaling by limiting reactive oxygen species production and by modulation of endoplasmic reticulum stress. In this connection, it is critical to understand precisely the molecular mechanisms involved in the global actions of Mfn2. FUTURE DIRECTIONS: Future directions should concentrate into the analysis of those mechanisms, and to fully demonstrate that Mfn2 represents a cellular hub that senses the metabolic and hormonal milieu and drives the control of metabolic homeostasis.

OBJECTIVE: To extend the study of the chemokine receptor repertoire on human chondrocytes to receptors with reported housekeeping functions (CXCR3, CXCR4, CXCR5, and CCR6) and to evaluate whether ligands of these receptors play a role in chondrocyte phenotype modulation and proliferation. METHODS: Chemokine receptor expression was determined by flow cytometry. Subcultures of chondrocytes were collected and fixed at confluence or during the exponential phase of growth and analyzed for chemokine receptor modulation. The effects of chemokines on isolated cells as well as chondrocytes cultured within an intact extracellular matrix were investigated. Isolated human chondrocytes were stimulated with 100 nM chemokines (monokine induced by interferon-gamma, stromal cell-derived factor 1alpha [SDF-1alpha], B cell-attracting chemokine 1 [BCA-1], or macrophage inflammatory protein 3alpha), and conditioned media were assessed for matrix-degrading enzyme contents (matrix metalloproteinases [MMPs] 1, 3, and 13, and N-acetyl-beta-D-glucosaminidase [NAG]). Cell proliferation and phenotype modulation were evaluated by bromodeoxyuridine incorporation and cathepsin B production. Induction of cell proliferation was assessed in cartilage explants by immunodetection of the proliferation-associated antigen S100A4. RESULTS: CXCR3, CXCR4, CXCR5, and CCR6 were detected on human chondrocytes. CXCR3 and CXCR4 expression was increased in exponentially growing chondrocyte subcultures. Ligands of all receptors enhanced the release of MMPs 1, 3, and 13. Release of NAG and cathepsin B was significantly higher in chemokine-stimulated cultures than in unstimulated cultures. SDF-1alpha and BCA-1 also induced DNA synthesis and chondrocyte proliferation, as was shown by the up-regulation of S100A4 in cartilage explants as well. CONCLUSION: Our findings extend the repertoire of functional responses elicited by the activity of chemokines on chondrocytes and open new avenues in our understanding of the control of chondrocyte differentiation status by chemokines and their receptors.

Abstract The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Here we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility ( P. aureginosa only). We further performed targeted assays on selected genes in Drosophila melanogaster , which we suspected of being involved in long-term memory. We conclude that, while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than expectations set by baseline methods in C. albicans and D. melanogaster , it leaves considerable room and need for improvement. We finally report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bioontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

. In this work, we have evaluated the capacity of linear and branched THRre to increase the permeability of proteins in cellular models of the BBB. With this purpose, we have covalently attached peptides with one or two copies of the BBB-shuttle to proteins in order to develop chemically well-defined peptide-protein conjugates. While THRre does not enhance the uptake and transport of a model protein in BBB cellular models, branched THRre peptides displaying two copies of the BBB-shuttle result in a 2.6-fold increase.

Drugs able to treat both nociceptive and neuropathic pain effectively without major side effects are lacking. We developed a bifunctional peptide-based hybrid (KGNOP1) that structurally combines a mu-opioid receptor agonist (KGOP1) with antinociceptive activity and a weak nociceptin receptor antagonist (KGNOP3) with anti-neuropathic pain activity. We investigated KGNOP1-related behavioral effects after intravenous administration in rats by assessing thermal nociception, cold hyperalgesia in a model of neuropathic pain induced by chronic constriction injury of the sciatic nerve, and plethysmography parameters including inspiratory time (TI) and minute ventilation (VM) in comparison to the well-known opioid analgesics, tramadol and morphine. Time-course and dose-dependent effects were investigated for all behavioral parameters to determine the effective doses 50% (ED50). Pain-related effects on cold hyperalgesia were markedly increased by KGNOP1 as compared to KGNOP3 and tramadol (ED50: 0.0004, 0.32, and 12.1 μmol/kg, respectively), whereas effects on thermal nociception were significantly higher with KGNOP1 as compared to morphine (ED50: 0.41 and 14.7 μmol/kg, respectively). KGNOP1 and KGOP1 produced a larger increase in TI and deleterious decrease in VM in comparison to morphine and tramadol (ED50(TI): 0.63, 0.52, 12.2, and 50.9 μmol/kg; ED50(VM): 0.57, 0.66, 10.6, and 50.0 μmol/kg, respectively). Interestingly, the calculated ratios of anti-neuropathic pain/antinociceptive to respiratory effects revealed that KGNOP1 was safer than tramadol (ED50 ratio: 5.44 × 10 vs 0.24) and morphine (ED50 ratio: 0.72 vs 1.39). We conclude that KGNOP1 is able to treat both experimental neuropathic and nociceptive pain, more efficiently and safely than tramadol and morphine, respectively, and thus should be a candidate for future clinical developments.

The stereospecific hydrolysis of bulky aminophosphine boranes is reported for the first time. The resulting phosphinous acid boranes, upon activation, undergo stereospecific nucleophilic substitution reaction at the phosphorous center with amine nucleophiles. The combination of these two processes provides a novel access to bulky P*-ligands.

OBJECTIVE: To evaluate the apoptotic effect of the chemokine growth-related oncogene alpha (GROalpha), which we recently reported to be up-regulated in osteoarthritis (OA) chondrocytes. Chondrocyte apoptosis is considered to be a major determinant of cartilage damage in OA, a disease resulting from the aberrant production of inflammatory mediators (cytokines and chemokines) and effectors (matrix metalloproteinases and reactive oxygen and nitrogen species) by chondrocytes. METHODS: We investigated the apoptotic effect of GROalpha on isolated human cells and on in vitro-cultured cartilage explants by conventional methods (morphology, detection of DNA fragmentation in situ and in solution, exposure of phosphatidylserine) and by analysis of "early" biochemical events (plasma membrane depolarization, activation of caspase 3, and phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase). RESULTS: We clearly demonstrated that GROalpha was able to initiate a series of morphologic, biochemical, and molecular changes that led to chondrocyte apoptosis. Moreover, we found that additional signals delivered from the extracellular matrix (ECM) were essential in the control of chondrocyte susceptibility to GROalpha-induced apoptosis, since cell death was detected only when cells were stimulated after reestablishment of their proper interactions with the ECM, or in cartilage explant samples with reduced ECM, as indicated by decreased Safranin O staining. CONCLUSION: GROalpha can induce apoptosis in articular chondrocytes, and the induction is dependent upon additional signals from the ECM. These findings are relevant to understanding the pathogenesis of OA, in view of the availability of the GROalpha chemokine in the joint space in the course of this rheumatic disease.

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser(944), a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation.

The incorporation of trifluoromethylated proline analogues in the tripeptide PLG enhances its hydrophobicity and promotes passive diffusion transport.

A new class of guest-induced, bi-radical self-assembled organic capsules is reported. They are formed by the inclusion of a tetramethylammonium (TMA) cation between two monomers of the stable trityl radical OX63. OX63 is extensively used in dissolution dynamic nuclear polarization (DNP) where it leads to NMR sensitivity enhancements of several orders of magnitude. The supramolecular properties of OX63 have a strong impact on its DNP properties. An especially relevant case is the polarization of choline-containing metabolites, where complex formation between choline and OX63 results in faster relaxation.

This paper represents the extended version of the conference paper “Developing highly-integrated subcutaneous biochips for remote monitoring of human metabolism” presented at the IEEE Sensors Conference 2012, and presents data on assembly, packaging and short term in vitro and in vivo biocompatibility evaluation of a fully implantable biosensor array. The device was realized integrating three building blocks: 1) a multielectrode platform; 2) an inductive coil; and 3) an integrated circuit. The entire system measures 2.2 mm × 2.2 mm × 15 mm. Corrosion of electronic components and leaking of potentially hazardous substances in the body is prevented with a conformal coating of Parylene C, while an outer package of medical grade silicone was employed to create a soft shell suitable for implantation. Biocompatibility experiments did not show in vitro cytotoxicity in the considered period of 7 days, while comparison between 7 and 30 days in vivo implantations showed significant reduction of the inflammatory response in time, suggesting normal host recovery.

The present study aims to develop chlorotoxin (CTX), from Giant Yellow Israeli scorpion venom, as a new BBB-shuttle. Minimised versions of CTX were prepared to reduce its complexity while enhancing its BBB-shuttle capacity and preserving its protease-resistance. MiniCTX3, a monocyclic lactam-bridge peptidomimetic, was capable of transporting nanoparticles across endothelial cell monolayers. Our results reveal animal venoms as an outstanding source of new families of BBB-shuttles.