The potential of a spheroid tumor model composed of cells in different proliferative and metabolic states for the development of new anticancer strategies has been amply demonstrated. However, there is little or no information in the literature on the problems of reproducibility of data originating from experiments using 3D models. Our analyses, carried out using a novel open source software capable of performing an automatic image analysis of 3D tumor colonies, showed that a number of morphology parameters affect the response of large spheroids to treatment. In particular, we found that both spheroid volume and shape may be a source of variability. We also compared some commercially available viability assays specifically designed for 3D models. In conclusion, our data indicate the need for a pre-selection of tumor spheroids of homogeneous volume and shape to reduce data variability to a minimum before use in a cytotoxicity test. In addition, we identified and validated a cytotoxicity test capable of providing meaningful data on the damage induced in large tumor spheroids of up to diameter in 650 μm by different kinds of treatments.

New neurons in the adult dentate gyrus are widely held to incorporate into hippocampal circuitry via a stereotypical sequence of morphological and physiological transitions, yet the molecular control over this process remains unclear. We studied the role of brain-derived neurotrophic factor (BDNF)/TrkB signaling in adult neurogenesis by deleting the full-length TrkB via Cre expression within adult progenitors in TrkB lox/lox mice. By 4 weeks after deletion, the growth of dendrites and spines is reduced in adultborn neurons demonstrating that TrkB is required to create the basic organization of synaptic connections. Later, when new neurons normally display facilitated synaptic plasticity and become preferentially recruited into functional networks, lack of TrkB results in impaired neurogenesis-dependent long-term potentiation and cell survival becomes compromised. Because of the specific lack of TrkB signaling in recently generated neurons a remarkably increased anxiety-like behavior was observed in mice carrying the mutation, emphasizing the contribution of adult neurogenesis in regulating mood-related behavior.BDNF ͉ LTP ͉ neurogenesis ͉ plasticity ͉ dendritogenesis T he life-long generation of new neurons is well documented in the subgranule zone of the dentate gyrus in the hippocampus (1-4). Dentate gyrus neurons result from local self-replicating radial glia-like stem cells (5). Once generated, the vast majority of neurons remain located on the hilar side of the granule layer and attempt to connect into the existing neuronal network, finally receiving afferent input from perforant path fibers (6, 7) and providing efferent output to CA3 cells (8, 9). While new neurons are held to incorporate into the preexisting circuitry via a stereotypical sequence of morphological transitions (10), the molecular mechanisms regulating the functional integration and/or survival of newborn neurons are not yet fully understood. There is growing evidence of the role of neuronal activity in this process. New neurons sense neuronal activity through ambient ␥-aminobutyric acid (GABA) before receiving, in sequence, GABAergic and glutamatergic inputs. Defects in the GABA responsiveness of newborn neurons, such as that obtained by inducing the conversion of GABA-mediated depolarization into hyperpolarization, lead to marked deficits in dendritic arborization and synapse formation (11), suggesting that network activity controls key morphological transitions required for the connectivity of adult-born neurons. At the initiation of connectivity glutamatergic inputs control newborn neuron survival (12). Indeed, access to afferent inputs may be the key for their life and death decisions. A central hypothesis arising from this regulation is that adult-born neurons could contribute to the formation of new circuits in tune with network needs, which, in turn, relates to the functional incorporation of adult-born neurons into hippocampal circuits. Hence, around connectivity time and later adult-born neurons become preferentially recruited into...

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Neurotrophins play an important role in modulating activitydependent neuronal plasticity. In particular, threshold levels of brain-derived neurotrophic factor (BDNF) are required to induce long-term potentiation (LTP) in acute hippocampal slices. Conversely, the administration of exogenous BDNF prevents the induction of long-term depression (LTD) in the visual cortex. A long-standing missing link in the analysis of this modulatory role of BDNF was the determination of the time-course of endogenous BDNF secretion in the same organotypic preparation in which LTP and LTD are elicited. Here, we fulfilled this requirement in slices of perirhinal cortex. Classical theta-burst stimulation patterns evoking LTP lasting >180 min elicited a large increase in BDNF secretion that persisted 5-12 min beyond the stimulation period. Weaker theta-burst stimulation patterns leading only to the initial phase of LTP (Ϸ35 min) were accompanied by a smaller increase in BDNF secretion lasting <1 min. Sequestration of BDNF by TrkB-IgG receptor bodies prevented LTP. Low-frequency stimulations leading to LTD were accompanied by reductions in BDNF secretion that never lasted beyond the duration of the stimulation. N eurotrophins, in particular brain-derived neurotrophic factor (BDNF), play an important modulatory role in activitydependent neuronal plasticity (1-5). The physiological relevance of this action is based on the observation that BDNF differentially modulates activity-dependent changes in synaptic strength, as reflected by long-term potentiation (LTP) and long-term depression (LTD), which are cellular paradigms for learning and memory (6). Hippocampal LTP is strongly impaired in both BDNF (7-9) and TrkB (10, 11) knock-out mice. Conversely, LTD in the visual cortex is facilitated by blocking anti-BDNF Abs or TrkB-IgG receptor bodies (12). It is of particular interest that in the hippocampus of BDNF knock-out mice (7) the extent of LTP reduction is the same in both homozygote and heterozygote animals, indicating that a minimal, critical concentration of BDNF has to be available to fulfill its modulatory functions. LTP could be rescued by adenoviral-mediated (re)expression of BDNF in the CA1 region (8) or administration of exogenous BDNF (9). Relatively detailed studies have been performed on the regulation of BDNF synthesis (13-17) and secretion (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The studies concerned with the regulation of BDNF secretion have been conducted in reductionistic systems, such as synaptosomes (18) and dissociated neuronal cultures in which BDNF secretion was evoked by high potassium depolarization or exogenous neurotransmitter administration (19-24). Recent studies using primary cultures of hippocampal neurons were designed specifically to investigate the relationship between electrical stimulation parameters and BDNF secretion. However, to reach this goal either the overexpression of BDNF (25, 26) or long stimulation periods in nontranduced cultures (27) were necessary to permit the determination of BDNF secret...

Abbreviations used in this paper: BDNF, brain-derived neurotrophic factor; GFAP, glial fi brillary acidic protein; Map2, microtubule-associated protein 2; MDC, monodansylcadaverine; QD, quantum dot; TBS, -burst stimulation; TeNT, tetanus neurotoxin; TIRF, total internal refl ection fl uorescence; TrkB, tropomyosinrelated kinase B receptor; TrkB-t, truncated TrkB; Vamp2, vesicle-associated membrane protein 2.The online version of this article contains supplemental material.

The use of protein cross-linking agents during bonding procedures has been recently proposed to improve bond durability. This study aimed to use zymography and in situ zymography techniques to evaluate the ability of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linker to inhibit matrix metalloproteinase (MMP) activity. The hypotheses tested were that: (1) bonding procedures increase dentin gelatinolytic activity and (2) EDC pre-treatment prevents this enzymatic activity. The zymographic assay was performed on protein extracts obtained from dentin powder treated with Optibond FL or Scotchbond 1XT with or without 0.3M EDC pre-treatment. For in situ zymography, adhesive/dentin interfaces were created with the same adhesives applied to acid-etched dentin slabs pre-treated or not with EDC conditioner. Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesive application, while the use of EDC as a primer inactivated dentin gelatinases. Results of in situ zymograpy showed that hybrid layers of tested adhesives exhibited intense collagenolytic activity, while almost no fluorescence signal was detected when specimens were pre-treated with EDC. The correlative analysis used in this study demonstrated that EDC could contribute to inactivate endogenous dentin MMPs within the hybrid layer created by etch-and-rinse adhesives.

Pre-lamin A undergoes subsequent steps of post-translational modification at its C-terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre-lamin A processing in nuclei, induced by expression of mutated pre-lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non-farnesylated) pre-lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl-K9-histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre-lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre-lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins.

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.