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Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the Drosophila sleep homeostat (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation). We report that dFB neurons can distinguish between different types of waking and can change their physiological response-properties accordingly. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons themselves can determine their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Here imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca2+ data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca2+ influx, while somatic neuropeptide release is triggered by the biochemical signal IP3. Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron dependent behaviors. Thus, compartment specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.Significance StatementIt is believed that electrical activity simultaneously stimulates widespread release sites in single neurons to elicit neuropeptide dependent behaviors. However, optically detecting neuropeptide release in the intact brain shows that clock neurons release neuropeptides from different sites at different times of the day. This is possible because one neuronal compartment, the soma, uses biochemical signaling instead of electrical activity to evoke release. Disrupting somatic release affects specific features of circadian locomotor activity and sleep. Thus, neuropeptide release is elicited by independent triggers from distinct parts of clock neurons to engage different regions of behavior regulating circuitry. This strategy for expanding the connectome may be used for other neuropeptide dependent behaviors, such as feeding and pain perception.

To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.-Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

Despite regulatory issues surrounding the use of animal-derived cell culture supplements, most clinical cardiac cell therapy trials using mesenchymal stromal cells (MSCs) still rely on fetal bovine serum (FBS) for cell expansion before transplantation. We sought to investigate the effect of human serum from heart failure patients (HFS) on cord blood MSCs (CB-MSCs) during short-term culture under regular conditions and during simulated acute and chronic stress. Cell survival, proliferation, metabolic activity, and apoptosis were quantified, and gene expression profiles of selected apoptosis and cell cycle regulators were determined. Compared to FBS, HFS and serum from healthy donors (CS) showed similar effects by substantially increasing cell survival during chronic and acute stress and by increasing cell yields 5 days after acute stress. Shortly after the termination of acute stress, both HFS and CS resulted in a marked decrease in apoptotic cells. Transcriptome analysis suggested a decrease in TNF-mediated induction of caspases and decreased activation of mitochondrial apoptosis. Our data confirm that human serum from both healthy donors and heart failure patients results in increased cell yields and increased resistance to cellular stress signals. Therefore, we consider autologous serum a valid alternative to FBS in cell-based therapies addressing severe heart disease.

Neuronal circuits can be re-modeled by Hebbian plasticity, synaptic scaling and, under some circumstances, activity-dependent respecification of cell-surface receptors. Although the impact of sleep on Hebbian plasticity and synaptic scaling are well studied, sleep’s role in receptor respecification remains unclear. We demonstrate that high sleep-pressure quickly reprograms the Drosophila wake-promoting large-ventrolateral clock-neurons to express the Pigment Dispersing Factor receptor. The addition of this signaling input into the circuit is associated with increased waking and early mating success. The respecification of Pigment Dispersing Factor receptor in both young and adult large ventrolateral neurons requires two dopamine receptors and activation of the transcriptional regulator nejire (CREB-binding protein). These data identify receptor-respecification as an important mechanism to sculpt circuit function to match sleep levels with demand.

Objective Enriched populations of proliferative induced cardiomyocyte precursors (iCMPs) generated from cardiac fibroblasts (CF) could represent a potential population for use in therapeutic application. We developed a protocol for generation of iCMPs from cardiac fibroblasts by genetic reprogramming followed by transcriptional selection and induced maturation. Methods Cardiac fibroblasts were reprogrammed into induced cardiomyocyte precursor cells (iCMPs) via transduction with cardiomyogenesis-related transcription factors Gata4, Mef2c, TBX5 and Myocd. Pure population of iCMPs were obtained by molecular beacons (MB) based selection of MYH6/7 expression. The transcriptome was profiled by RNA sequencing. For maturation iCMPs were treated with 5'-azacytidine and cultured in medium containing ascorbic acid and TGFβ1. The cells were monitored regularly for formation of sarcomere structures, cardiac marker expression and contractions. iCMPs were transplanted into MI mice and monitored for 6 weeks. At the end of 6 weeks, the mice were sacrificed and hearts were explanted and cryopreserved to examine the heart structure and scare size. Cardiac function was monitored by echocardiography. Results iCMPs expressed troponin T, α-actinin and myosin heavy chain (MHC) protein observed by immunocytology. These iCMPs could robustly proliferative and maintain a stable phenotype. Global transcriptome analysis revealed that the iCMP gene expression profile is unique from those of the parental CFs and adult CMs. With Gene Ontology (GO) analysis, we found that iCMPs show upregulation of genes associated with cardiac development, differentiation and morphogenesis while they showed downregulation of genes associated to cell-proliferation in comparison to their parental CFs. Evaluation of selected gene sets showed downregulation of non-myocyte genes, upregulation of transcription factors and upregulation of certain functional and structural genes like ion channel genes and contractile genes. Maturation studies showed that iCMPs gradually changed morphology after stimulation with 5-Aza, as seen in immunofluorescence staining. Cells expressing cTnT showed formation of sarcomeric striations. In vivo translational studies showed reduction of heart function in control groups treated with PBS or CF whereas the iCMPs group showed an improvement in function, reflected by left ventricular ejection fraction and fractional shortening as evaluated by echocardiography. Additionally we noticed an improvement in left ventricular wall thickness at diastole in mice transplanted with iCMPs. Conclusions Gene expression pattern suggests that these iCMPs represent an intermediate state of cardiogenic population that can be expanded to yield therapeutic cell doses. When transplanted to mouse hearts following myocardial infarction, they improved cardiac function.

OBJECTIVES: Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways. METHODS: Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex ® immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells ( poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK) 1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic). RESULTS: CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs. CONCLUSIONS: The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 μm (15–157 μm) by laser diffraction, and 57 μm (20–182 μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, −72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) inDrosophila. Null mutants ofent2are lethal during late larval/early pupal stages, indicating thatent2is essential for normal development. Hypomorphic mutant alleles ofent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock downent2expression in specific regions of the CNS and show thatent2is required in the α/β lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated inent2mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR1) into theent2mutant background. The results of the present study provide the first evidence of a role forent2function inDrosophilaand suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

The amniotic epithelium consists of cells exhibiting mature epitelial cell characteristics, but also varying degrees of stemness. We tested the hypothesis that induction of epithelial-to-mesenchymal transition (EMT) in amniotic epitelial cells (AECs) derived from human placenta enhances their capacity to support the ischemic myocardium. In response to incubation with transforming growth factor-β1 (TGF-β1) protein, AECs lost their cobblestone morphology and acquired a fibroblastoid shape, associated with downregulation of E-cadherin, upregulation of N-cadherin, Akt phosphorylation, and intracellular periostin translocation. EMT—AECs displayed greatly enhanced mobility and secreted gelatinase activity compared with naive AECs. The surface presentation of CD105 and CD73 decreased, and RNA microarray analysis mirrored the loss of epithelial characteristics and transcriptional profile. Unmodified AECs and EMT—AECs were then injected intramyocardially in fully immunocompetent mice after permanent LAD ligation, and heart function was followed by MRI as well as 2D speckle tracking echocardiography after 4 weeks. EMT—AEC-treated infarct hearts displayed better global systolic function and improved longitudinal strain rate in the area of interest. Although no signals of human cells were detectable by histology, infarct size was smaller in EMT—AEC-treated hearts, associated with fewer TUNEL-positive cells and upregulation of periostin, while blood vessel density was increased in both ACE- and EMT—AEC-treated hearts. We conclude that EMT enhances the cardioprotective effects of human AECs.

Background/Aims: Cell-based therapies may be useful for treating ischemic diseases, but the underlying mechanisms are incompletely understood. We investigated the impact of cord blood mesenchymal stromal cell (CBMSC)- or fibroblast (FB)-secreted factors on starved endothelial cells and determined the relevant intracellular signaling pathways. Methods: HUVECs were subjected to glucose/serum deprivation (GSD) in hypoxia or normoxia, in presence of CBMSC- or FB-conditioned medium (CM). Viability and proliferation were determined via WST-8 conversion and BrdU incorporation. Apoptosis was quantified by annexin V/ethidium homodimer-III staining, nuclear fragmentation and cell morphology. mRNA expression and protein phosphorylation were determined by real-time qPCR and western blot. Experiments were repeated in presence of small-molecule inhibitors. Results: The negative impact of GSD was most pronounced at 21% O2. Here, medium of CBMSCs and FBs increased viability and proliferation and reduced apoptosis of HUVECs. This was associated with increased STAT3 and ERK1/2 phosphorylation and BCL-2 expression. Under STAT3 inhibition, the beneficial effect of CBMSC-CM on viability and BCL-2 expression was abolished. Conclusion: Factors released by CBMSCs protect endothelial cells from the deleterious impact of GSD by activation of the STAT3 survival pathway. However, this phenomenon is not CBMSC-specific and can be reproduced using juvenile fibroblasts.

Background Clinical cardiac cell therapy using autologous somatic stem cells is restricted by age and disease-associated impairment of stem cell function. Juvenile cells possibly represent a more potent alternative, but the impact of patient-related variables on such cell products is unknown. We therefore evaluated the behavior of neonatal cord blood mesenchymal stem cells (CB-MSC) in the presence of serum from patients with advanced heart failure (HF). Methods Human serum was obtained from patients with severe HF (n = 21) and from healthy volunteers (n = 12). To confirm the systemic quality of HF in the sera, TNF-α and IL-6 were quantified. CB-MSC from healthy neonates were cultivated for up to 14 days in medium supplemented with 10% protein-normalized human HF or control serum or fetal calf serum (FCS). Results All HF sera contained increased cytokine concentrations (IL-6, TNF-α). When exposed to HF serum, CB-MSC maintained basic MSC properties as confirmed by immunophenotyping and differentiation assays, but clonogenic cells were reduced in number and gave rise to substantially smaller colonies in the CFU-F assay. Cell cycle analysis pointed towards G1 arrest. CB-MSC metabolic activity and proliferation were significantly impaired for up to 3 days as measured by MTS turnover, BrdU incorporation and DAPI + nuclei counting. On day 5, however, CB-MSC growth kinetics approached control serum levels, though protein expression of cell cycle inhibitors (p21, p27), and apoptosis marker Caspase 3 remained elevated. Signal transduction included the stress and cytokine-induced JNK and ERK1/2 MAP kinase pathways. Conclusions Heart failure temporarily inhibits clonality and proliferation of “healthy” juvenile MSC in vitro. Further studies should address the in vivo and clinical relevance of this finding.

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

SYNOPSIS. Environmental stresses such as high temperature or low levels of oxygen can lead to structural destabilization of cells, disruption of cellular processes, and, in extreme cases, death. Previous experience of sub-lethal stress can lead to protection during a subsequent stress that may otherwise have been lethal. Synapses are particularly vulnerable to extreme environmental conditions and failure of function at this level may be the primary cause of organismal death. Prior heat shock induces enhanced thermotolerance at neuromuscular junctions in the locust extensor tibiae muscle and in abdominal muscles of larval Drosophila. Synaptic thermoprotection is associated with an increase in short-term plasticity at these synapses. Prior anoxic coma in locusts induces synaptic thermotolerance suggesting that the same protective pathways are activated. It is well established that diverse forms of stress induce the upregulation of cellular chaperones (heat shock proteins; HSPs) that mediate acquired protection. The mechanisms underlying HSP-mediated synaptic protection are currently unknown but evidence is accumulating that stabilization of the cytoskeleton may play an important role.

Neuropeptides are found in both mammals and invertebrates and can modulate neural function through activation of G-protein-coupled receptors (GPCRS). The precise mechanisms by which many of these GPCRs modulate specific signaling cascades to regulate neural function are not well defined. We usedDrosophila melanogasteras a model to examine both the cellular and behavioral effects of DPKQDFMRFamide, the most abundant peptide encoded by the dFMRF gene. We show that DPKQDFMRFamide enhanced synaptic transmission through activation of two G-protein-coupled receptors,Fmrf Receptor(FR) andDromyosupressin Receptor-2 (DmsR-2). The peptide increased both the presynaptic Ca2+response and the quantal content of released transmitter. Peptide-induced modulation of synaptic function could be abrogated by depleting intracellular Ca2+stores or by interfering with Ca2+release from the endoplasmic reticulum through disruption of either the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor. The peptide also altered behavior. Exogenous DPKQDFMRFamide enhanced fictive locomotion; this required both theFRandDmsR-2. Likewise, both receptors were required for an escape response to intense light exposure. Thus, coincident detection of a peptide by two GPCRs modulates synaptic function through effects of Ca2+-induced Ca2+release, and we hypothesize that these mechanisms are involved in behavioral responses to environmental stress.

Maintenance of synaptic transmission requires regulation of intracellular Ca2+in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca2+indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na+/Ca2+exchanger or removal of extracellular Ca2+prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca2+clearance—inactivation of the endoplasmic reticulum Ca2+-ATPase with thapsigargin and inhibition of the plasma membrane Ca2+-ATPase with high pH—significantly accelerated the temperature-induced rise in resting Ca2+concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca2+-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca2+] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca2+regulation. In each of these cases, Ca2+elevations evoked by an electrical stimulation of the nerve (evoked Ca2+responses) failed when resting Ca2+remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca2+-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca2+ entry and enhanced nerve-terminal growth. The impaired Ca2+ entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca2+ channels, or that it regulates the PI4Kβ pathway as described in other organisms. To determine whether Frq interacts with PI4K. with consequent effects on Ca2+ channels, we first characterized a PI4Kβ-null mutant and found that PI4Kβ was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kβ-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kβ. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the α1-subunit of voltage-gated Ca2+ channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca2+ entry through a functional interaction with the voltage-gated Ca2+-channel subunit; this interaction regulates neurotransmission and nerveterminal growth.

We examined the thermosensitivity of calcium regulation in Drosophila larval neuromuscular junctions, testing effects of prior heat shock and Hsp70 expression. Motor neurons were loaded with either the ratiometric indicator Fura-dextran or the nonratiometric indicator Oregon Green bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid to monitor parameters of calcium regulation as temperature increased. Nerve terminals treated to a prior heat shock, and those of transgenic flies expressing higher than normal levels of Hsp70, were better able to maintain near-normal resting calcium concentrations, calcium influx, and calcium clearance at higher temperatures. Synaptic transmission was also protected by prior heat shock and by higher than normal Hsp70 expression. Thus the thermal limit of synaptic transmission may be directly linked to the stability of calcium regulation.

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged inDrosophilamotor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro- angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) ofcspnull-mutantDrosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by anin vivostructure/function analysis. Expression of mutant CSP lacking the J domain atcspnull-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+levels. We detected no effects ofcspmutations on individual presynaptic Ca2+signals triggered by action potentials, suggesting that presynaptic Ca2+entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+levels at rest and during stimulation.

SUMMARYIntracellular symbiosis requires that the host satisfy the symbiont's metabolic requirements, including the elimination of waste products. The hydrothermal vent tubeworm Riftia pachyptila and the hydrocarbon seep worm Lamellibrachia cf luymesi are symbiotic with chemolithoautotrophic bacteria that produce sulfate and protons as end-products. In this report, we examine the relationship between symbiont metabolism and host proton equivalent elimination in R. pachyptilaand L. cf luymesi, and the effects of sulfide exposure on proton-equivalent elimination by Urechis caupo, an echiuran worm that lacks intracellular symbionts (for brevity, we will hereafter refer to proton-equivalent elimination as `proton elimination'). Proton elimination by R. pachyptila and L. cf luymesi constitutes the worms' largest mass-specific metabolite flux, and R. pachyptilaproton elimination is, to our knowledge, the most rapid reported for any metazoan. Proton elimination rates by R. pachyptila and L.cf luymesi correlated primarily with the rate of sulfide oxidation. Prolonged exposure to low environmental oxygen concentrations completely inhibited the majority of proton elimination by R. pachyptila,demonstrating that proton elimination does not result primarily from anaerobic metabolism. Large and rapid increases in environmental inorganic carbon concentrations led to short-lived proton elimination by R. pachyptila, as a result of the equilibration between internal and external inorganic carbon pools. U. caupo consistently exhibited proton elimination rates 5-20 times lower than those of L. cf luymesi and R. pachyptila upon exposure to sulfide. Treatment with specific ATPase inhibitors completely inhibited a fraction of proton elimination and sulfide and inorganic carbon uptake by R. pachyptila, suggesting that proton elimination occurs in large part via K+/H+-ATPases and Na+/H+-ATPases. In the light of these results, we suggest that protons are the primary waste product of the symbioses of R. pachyptila and L. cf luymesi, and that proton elimination is driven by symbiont metabolism, and may be the largest energetic cost incurred by the worms.

The authors regret that an error appeared in the above article. When reading the manufacturer’s certificate of the Luminex cytokine/growth factor screening assay, a comma (thousand separator) was mistaken for a decimal separator. Hence, the analyte concentrations given in the article as pg/ml are in fact ng/ml. The paragraph of the subsection ‘Growth factors and cytokines in conditioned medium’ under RESULTS should read: In fibroblast-conditioned medium, the total protein content was 26 ± 10% lower than in CBMSC medium (CBMSC 27 ± 2 vs fibroblasts 20 ± 2 μg/ml, P < 0.05). The concentrations of selected growth factors and cytokines are shown in Fig. 2. The concentrations of VEGF (CBMSC 6.0 ± 0.9; fibroblast 4.9 ± 0.7 ng/ml), EGF (CBMSC 0.83 ± 0.09; fibroblast 0.65 ± 0.06 ng/ml), and Angiopoetin-2 (CBMSC 0.44 ± 0.04; fibroblast 0.49 ± 0.06 ng/ml) were similar, CBMSC medium contained more HGF (CBMSC 0.36 ± 0.05 vs fibroblast 0.04 ± 0.01 ng/ml, P < 0.001), and fibroblast medium more bFGF (CBMSC 0.08 ± 0.02 vs fibroblast 0.59 ± 0.14 ng/ml, P < 0.05) and IL-6 (CBMSC 0.15 ± 0.03 vs fibroblast 5.8 ± 1.0 ng/ml, P < 0.001). IL-10 and TNFα were not detectable in either medium. In addition, the Y-axis in Figure 2 should indicate ng/ml rather than pg/ml.

This paper presents a rationale for using videotape records in evaluation research. Although videotaping is commonly used in ethnographic and sociometric analysis, the evaluation literature does not show the application of these recording techniques to program evaluations. The authors outline a model for exploring the effectiveness of video in qualitative program evaluation.

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