Lessons learned in a decade: Medical-toxicological view of tattooing.

Tattooing has been part of the human culture for thousands of years, yet only in the past decades has it entered the mainstream of the society. With the rise in popularity, tattoos also gained attention among researchers, with the aim to better understand the health risks posed by their application. 'A medical-toxicological view of tattooing'-a work published in The Lancet almost a decade ago, resulted from the international collaboration of various experts in the field. Since then, much understanding has been achieved regarding adverse effects, treatment of complications, as well as their regulation for improving public health. Yet major knowledge gaps remain. This review article results from the Second International Conference on Tattoo Safety hosted by the German Federal Institute for Risk Assessment (BfR) and provides a glimpse from the medical-toxicological perspective, regulatory strategies and advances in the analysis of tattoo inks.

Thrombotic Antiphospholipid Syndrome and Direct Oral Anticoagulants: Unmet Needs and Review of the Literature.

Patients with thrombotic antiphospholipid syndrome (APS) require long-term anticoagulation due to the high-thrombotic recurrence risk. Vitamin K antagonists (VKA) have been traditionally considered the standard of care in thrombotic APS. Nevertheless, the risk of recurrence persists with VKA. There are publications considering different intensities of anticoagulation with VKA; however, the standard-intensity anticoagulation (international normalized ratio between 2.0 and 3.0) is the most recommended. Furthermore, there is no consensus on the role of antiplatelet treatment in thrombotic APS. Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as an alternative to VKA for many indications. There are, however, discrepancies regarding the management with NOACs in thrombotic APS. In this review, we update the different clinical trials with NOACs in venous, arterial, and microvascular thrombosis and suggest how these patients should be managed in agreement with the expert panels. Although scarce data are published regarding the current role of NOACs in thrombotic APS, the clinical trials failed to demonstrate noninferiority of NOACs compared with VKA, especially in patients with triple antiphospholipid antibodies positivity and/or arterial thrombosis. Single or double antiphospholipid positivity should be analyzed on a case-by-case basis. In addition, we focus on different areas of uncertainty that still remain in thrombotic APS and NOACs. To summarize, emerging clinical trials are needed to provide robust data on the management of thrombotic APS.

"Vermellogens" and the Development of CB[8]-Based Supramolecular Switches Using pH-Responsive and Non-Toxic Viologen Analogues.

We present herein the "vermellogens", a new class of pH-responsive viologen analogues, which replace the direct linking between para-substituted pyridinium moieties within those by a hydrazone functional group. A series of such compounds have been efficiently synthesized in aqueous media by hydrazone exchange reactions, displaying a marked pH-responsivity. Furthermore, the parent N,N'-dimethylated "vermellogen": the "red thread", an analogue of the herbicide paraquat and used herein as a representative model of the series, showed anion-recognition abilities, non-reversible electrochemical behavior, and non-toxicity of the modified bis-pyridinium core. The host-guest chemistry for the "red thread" with the CB[7,8] macrocyclic receptors has been extensively studied experimentally and by dispersion corrected density functional theory methods, showing a parallel behavior to that previously described for the herbicide but, crucially, swapping the well-known redox reactive capabilities of the viologen-based inclusion complexes by acid-base supramolecular responsiveness.

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability.

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

Popescu Technique Revisited: Review of the Literature and Case Series.

Management of vascular malformations depends on the size, type, age of the patient, location, dissemination, and depth of penetration. Treatment options include propranolol, which reduces endothelial vessel proliferation, minimally invasive sclerotherapy to induce fibrosis, or surgery. In 1985, Valerian Popescu described a new approach to treatment consisting of intratumoral ligation by compartmentalization. This technique allows for high doses of the sclerosant agent to be delivered as systemic dissemination is restricted by a series of strangulating suture loops that divide the mass into segments. We describe the management and outcome of 2 patients who presented with vascular malformations in the orofacial region and were managed using a Popescu suturing technique. Vascular obliteration was achieved by a series of strangulating suture loops placed percutaneously throughout each lesion using a curved needle with a resorbable material (Vicryl; Ethicon, Somerville, NJ). The aim was to segment the vascular malformation into manageable sections for subsequent injection of a sclerosant. The compartmentalization also ensured that the sclerosant stayed within these compartments and was not washed out into the general circulation. Good esthetic outcomes were achieved in very visible areas such as the commissure and the vermillion border. In these areas, a surgical resection would have certainly caused a disruption of the esthetics of the lips and, in the second case, probably an alteration of function. Intratumoral ligation can be used safely to achieve control of vascular malformations with good esthetic outcomes.

Sustained spatiotemporal release of TGF-ß1 confers enhanced very early chondrogenic differentiation during osteochondral repair in specific topographic patterns.

The continuous presence of TGF-ß is critically important to induce effective chondrogenesis. To investigate chondrogenesis in a cartilage defect, we tested the hypothesis that the implantation of TGF-ß1-releasing scaffolds improves very early cartilage repair in vivo. Spatiotemporal controlled release of TGF-ß1 was achieved from multiblock scaffolds that were implanted in osteochondral defects in the medial femoral condyles of adult minipigs. We observed a sustained presence of TGF-ß1 at 4 wk in vivo, which significantly promoted structural aspects of early overall cartilage repair, especially cellularity, cellular morphology, and safranin O staining intensity. Furthermore, early aggrecan and type II collagen production were both increased in specific topographic patterns in cartilaginous repair tissue. Sustained release of TGF-ß1 also increased cell numbers and proliferation, staining intensities for the stem cell surface marker, CD105, and number of stromal cell-derived factor-1 (SDF-1) -positive cells within cartilaginous repair tissue. These data identify a mechanism by which TGF-ß1 modulates early chondrogenesis by primarily increasing the number of progenitor cells arising from the subchondral bone marrow compartment via the SDF-1/chemokine (CXC motif) receptor 4 pathway, their proliferation, differentiation, and extracellular matrix deposition in specific topographic patterns, highlighting the pivotal role played by TGF-ß1 during this crucial phase.-Asen, A.-K., Goebel, L., Rey-Rico, A., Sohier, J., Zurakowski, D., Cucchiarini, M., Madry, H. Sustained spatiotemporal release of TGF-ß1 confers enhanced very early chondrogenic differentiation during osteochondral repair in specific topographic patterns.

Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-ß and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells.

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-ß) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter (lacZ) gene condition. The rAAV-hTGF-ß and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-ß/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-ß/lacZ, IGF-I/lacZ, and lacZ treatments at equivalent vector doses. The cultures were then processed to monitor transgene (co)-overexpression, the levels of biological activities in the cells (cell proliferation, matrix synthesis), and the development of a chondrogenic versus osteogenic/hypertrophic phenotype. Effective, durable co-overexpression of TGF-ß with IGF-I via rAAV enhanced the proliferative, anabolic, and chondrogenic activities in hMSCs versus lacZ treatment and reached levels that were higher than those achieved upon single candidate gene transfer, while osteogenic/hypertrophic differentiation was delayed over the period of time evaluated. These findings demonstrate the potential of manipulating multiple therapeutic rAAV vectors as a tool to directly target bone-marrow-derived MSCs in sites of focal cartilage defects and to locally enhance the endogenous processes of cartilage repair.

Setting the stage to quit smoking in Bipolar Disorder patients: brief advice in clinical practice. / Preparando el escenario para dejar de fumar en el paciente con Trastorno Bipolar: intervención breve en la práctica clínica.

Tobacco consumption is the main preventable factor of mortality in smokers with bipolar disorder (BD), and any possible solutions are often blocked by prejudices over desire, and the possibilities and risks for these patients in giving up tobacco consumption. Adults with BD were recruited at 8 Mental Health Centres. Smokers were evaluated before and after a brief intervention based on the 3 A's and classified into a 'Stage of Change' (SOC) and their 'Readiness to Change' (RTC). A multiple linear regression was used to analyze the progression in their RTC and the independent effect of different variables (pharmacological treatment, history of psychotic symptoms, current anxiety symptoms, willingness, self-perceived capacity to quit smoking and subjective perception of cognitive functioning). Of 212 stable patients diagnosed with BD, current smokers (n=101; 47.6%) were included in the intervention phase, and 80.2% completed it. At baseline, 75.2% were considering the idea of giving up smoking and, after the brief intervention, 30.9% of the patients progressed in their SOC. A significant increase in the level of RTC was observed (53.3 vs 59.3, P=0.019). Perception of cognitive performance (ß=-0.35;P=0.002), the degree of willing to quit (ß=0.32;P=0.008), self-perceived capacity to quit tobacco smoking (ß=-0.30;P=0.012), the patient's age (ß=-0.72;P=0.004), the age of onset of smoking (ß=0.48;P=0.022) and years as a smoker (ß=0.48;P=0.025) were all factors that significantly influenced the chances of improving after the short intervention. Smokers with BD consider the idea of quitting and a brief intervention developed in the every day mental health care setting improves the level of readiness. The neurocognitive dysfunction associated with BD may limit patients' readiness to quit smoking.

El consumo de tabaco es el principal factor prevenible de mortalidad en pacientes con trastorno bipolar (TB), y las posibles soluciones se encuentran bloqueadas por prejuicios acerca del deseo, posibilidades y riesgos al dejar el consumo de tabaco en estos pacientes. En 8 Centros de Salud Mental se reclutaron consecutivamente pacientes con TB. Los fumadores fueron evaluados antes y después de una intervención breve basada en las 3 As y clasificados según los "estadios de cambio" (EC) y su "disposición para el cambio" (DC). Mediante una regresión lineal múltiple se analizó la evolución del DC y su efecto sobre otras variables independientes (tratamiento farmacológico, historias de síntomas psicóticos, presencia de síntomas de ansiedad, deseo de abandono, capacidad auto-percibida y la percepción subjetiva de funcionamiento cognitivo). Se incluyeron 212 pacientes con TB estabilizados, los fumadores activos (n=101; 47.6%) pasaron a la fase de intervención, y un 80.2% la completaron. Basalmente, 75.2% consideraban la idea de dejar de fumar, después de la intervención breve, el 30.9% de los pacientes progresó en su EC. Se observó un incremento significativo del nivel de DC (53.3 vs 59.3, P=0.019). La autopercepción del rendimiento cognitivo (ß=-0.35;P=0.002), el deseo de abandono (ß=0.32;P=0.008), la autopercepción de la capacidad para dejar de fumar (ß=-0.30;P=0.012), la edad del paciente (ß=-0.72;P=0.004), la edad de inicio del tabaquismo (ß=0.48;P=0.022) y los años fumando (ß=0.48;P=0.025) fueron los factores que influyeron significativamente en la posibilidad de cambio tras la intervención breve. Los fumadores con TB consideran la idea de dejar de fumar y una intervención breve desarrollada en el marco de la atención a la salud mental diaria, mejoraría el nivel de preparación. La disfunción neurocognitiva asociada con el TB podría limitar la disposición de los pacientes a dejar de fumar.

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability.

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

Effective Remodelling of Human Osteoarthritic Cartilage by sox9 Gene Transfer and Overexpression upon Delivery of rAAV Vectors in Polymeric Micelles.

Recombinant adeno-associated virus (rAAV) vectors are well suited carriers to provide durable treatments for human osteoarthritis (OA). Controlled release of rAAV from polymeric micelles was already shown to increase both the stability and bioactivity of the vectors while overcoming barriers, precluding effective gene transfer. In the present study, we examined the convenience of delivering rAAV vectors via poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) polymeric (PEO-PPO-PEO) micelles to transfer and overexpress the transcription factor SOX9 in monolayers of human OA chondrocytes and in experimentally created human osteochondral defects. Human osteoarthritic (OA) chondrocytes and human osteochondral defect models were produced using human OA cartilage obtained from patients subjected to total knee arthroplasty. Samples were genetically modified by adding a rAAV-FLAG-h sox9 vector in its free form or via polymeric micelles for 10 days relative to control conditions (unmodified cells). The effects of sox9 overexpression in human OA cartilage samples were monitored by biochemical, histological, and immunohistochemical analyses. Delivery of rAAV-FLAG-h sox9 via polymeric micelles enhanced the levels of sox9 expression compared with free vector administration, resulting in increased proteoglycan deposition and in a stimulated cell proliferation index in OA chondrocytes. Moreover, higher production of type II collagen and decreased hypertrophic events were noted in osteochondral defect cultures when compared with control conditions. Controlled therapeutic rAAV sox9 gene delivery using PEO-PPO-PEO micelles is a promising, efficient tool to promote the remodelling of human OA cartilage.

PEO-PPO-PEO Tri-Block Copolymers for Gene Delivery Applications in Human Regenerative Medicine-An Overview.

Lineal (poloxamers or Pluronic®) or X-shaped (poloxamines or Tetronic®) amphiphilic tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO-PEO) have been broadly explored for controlled drug delivery in different regenerative medicine approaches. The ability of these copolymers to self-assemble as micelles and to undergo sol-to-gel transitions upon heating has endowed the denomination of "smart" or "intelligent" systems. The use of PEO-PPO-PEO copolymers as gene delivery systems is a powerful emerging strategy to improve the performance of classical gene transfer vectors. This review summarizes the state of art of the application of PEO-PPO-PEO copolymers in both nonviral and viral gene transfer approaches and their potential as gene delivery systems in different regenerative medicine approaches.

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment.

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.

Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae.

Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.

Peripheral blood aspirates overexpressing IGF-I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes.

Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single-step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) in human peripheral blood aspirates via rAAV-mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type-II collagen, SOX9) activities in the samples relative to control (reporter rAAV-lacZ) treatment over extended periods of time (at least 21 days, the longest time-point evaluated). Interestingly, IGF-I gene transfer also triggered hypertrophic, osteo- and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF-I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries.

Hydrogels for precision meniscus tissue engineering: a comprehensive review.

The meniscus plays a pivotal role to preserve the knee joint homeostasis. Lesions to the meniscus are frequent, have a reduced ability to heal, and may induce tibiofemoral osteoarthritis. Current reconstructive therapeutic options mainly focus on the treatment of lesions in the peripheral vascularized region. In contrast, few approaches are capable of stimulating repair of damaged meniscal tissue in the central, avascular portion. Tissue engineering approaches are of high interest to repair or replace damaged meniscus tissue in this area. Hydrogel-based biomaterials are of special interest for meniscus repair as its inner part contains relatively high proportions of proteoglycans which are responsible for the viscoelastic compressive properties and hydration grade. Hydrogels exhibiting high water content and providing a specific three-dimensional (3D) microenvironment may be engineered to precisely resemble this topographical composition of the meniscal tissue. Different polymers of both natural and synthetic origins have been manipulated to produce hydrogels hosting relevant cell populations for meniscus regeneration and provide platforms for meniscus tissue replacement. So far, these compounds have been employed to design controlled delivery systems of bioactive molecules involved in meniscal reparative processes or to host genetically modified cells as a means to enhance meniscus repair. This review describes the most recent advances on the use of hydrogels as platforms for precision meniscus tissue engineering.

TGF-ß gene transfer and overexpression via rAAV vectors stimulates chondrogenic events in human bone marrow aspirates.

Genetic modification of marrow concentrates may provide convenient approaches to enhance the chondrogenic differentiation processes and improve the repair capacities in sites of cartilage defects following administration in the lesions. Here, we provided clinically adapted recombinant adeno-associated virus (rAAV) vectors to human bone marrow aspirates to promote the expression of the potent transforming growth factor beta (TGF-ß) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF-ß gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF-ß/mg total proteins after 21 days), allowing to durably enhance the levels of cell proliferation, matrix synthesis, and chondrogenic differentiation. Strikingly, in the conditions applied here, application of the candidate TGF-ß vector was also capable of reducing the hypertrophic and osteogenic differentiation processes in the aspirates, showing the potential benefits of using this particular vector to directly modify marrow concentrates to generate single-step, effective approaches that aim at improving articular cartilage repair in vivo.

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay.

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg(2+). We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K(+) and Mg(2+) and was inhibited by Na(+). GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.

In the beginning, Escherichia coli assembled the proto-ring: an initial phase of division.

Cell division in Escherichia coli begins by assembling three proteins, FtsZ, FtsA, and ZipA, to form a proto-ring at midcell. These proteins nucleate an assembly of at least 35 components, the divisome. The structuring of FtsZ to form a ring and the processes that effect constriction have been explained by alternative but not mutually exclusive mechanisms. We discuss how FtsA and ZipA provide anchoring of the cytoplasmic FtsZ to the membrane and how a temporal sequence of alternative protein interactions may operate in the maturation and stability of the proto-ring. How the force needed for constriction is generated and how the proto-ring proteins relate to peptidoglycan synthesis remain as the main challenges for future research.

Nonviral gene transfer into human meniscal cells. Part II: effect of three-dimensional environment and overexpression of human fibroblast growth factor 2.

PURPOSE: Our aim was to study the effect of three-dimensional (3D) environment and overexpression of human fibroblast growth factor 2 (FGF-2) on meniscal fibrochondrocytes in vitro. METHODS: Human meniscal fibrochondrocytes were transfected with expression plasmid vectors carrying the Photinus pyralis luciferase gene, the Escherichia coli ß-galactosidase gene or a human FGF-2 cDNA. Modified fibrochondrocytes were cultivated in 3D alginate hydrogel or cell pellets or in 2D monolayer culture. RESULTS: The levels of luciferase activity showed a peak at day two and returned to baseline levels by day 11, regardless of the type of cultivation. Both 3D environments supported the secretion of human FGF-2 protein upon FGF-2 transfection. Overexpression of human FGF-2 by genetically modified human meniscal fibrochondrocytes stimulated proliferation but not glycosaminoglycan synthesis only in 3D culture. Culture in alginate spheres resulted in a larger difference in cell numbers compared with pellet cultures. CONCLUSIONS: Three-dimensional alginate spheres are well suited for the culture of genetically modified human meniscal fibrochondrocytes. These data are of value for cell-based approaches to meniscal repair using genetically modified human meniscal fibrochondrocytes overexpressing human FGF-2.