In 2012, the Israeli Ministry of Health issued national guidelines for antibiotic stewardship. The guidelines require that all general hospitals, post-acute care hospitals and the ambulatory care sectors of health maintenance organizations report annual consumption of antibiotics. Data regarding antibiotic use in post-acute care hospitals are scarce.To describe antibiotic consumption in post-acute care and geriatric hospitals in Israel in 2014.Hospitals used their computerized pharmacy databases to report all systemic antibacterials  dispensed. Hospitals also reported patient days, by ward, for 2014. We calculated antibiotic consumption as Defined Daily Doses (DDDs) per 1000 patient-days (PD). To overcome differences in case mix between institutions, we limited our analysis to 4 common ward types: ventilation /respiratory rehabilitation, sub-acute care, rehabilitation, and complex nursing.Seven of Israel’s 15 post-acute care and geriatric hospitals reported at the ward level. Median use of systemic antibacterials in DDD/1000 PDs was 206 (range: 130-313) in complex nursing wards, 232 (range: 172-323) in ventilation wards, 244 (range: 154-379) in rehabilitation wards, and 348 (range: 272-600) in sub-acute wards.  The most commonly used antibiotic group was penicillins with beta-lactamase inhibitors in ventilation and complex nursing wards and cephalosporins in sub-acute and rehabilitation wards. Fluoroquinolones accounted for 14% -19% of all antibiotic use, depending on ward type. Individual hospitals were consistently either below the median or above the median for all ward types.Antibiotic consumption in the major ward types of post-acute care and geriatric hospitals in Israel is substantial, amounting to over one-fourth of the amount used in acute care hospitals’ medical wards. Our data underestimate total antibiotic use in this patient population because patients may be temporarily transferred to general hospitals for treatment of complex infections. The consistently high or low use in each hospital suggests that antibiotic use is influenced by institutional culture or formal policies that dictate a permissive or restrictive approach.None declared.


No particular regimen is considered standard therapy for widespread metastatic melanoma, although surgery is the primary choice for regional nodal metastases. Systemic interleukin-2 (IL-2) is an effective immunotherapy for melanoma, but standard doses are associated with severe toxicity. We report a patient who was treated with intralesional low-dose IL-2 and V-beam pulsed dye laser for the treatment of scalp melanoma metastases. This treatment resulted in rapid regression of metastatic tumors with limited adverse effects. No particular regimen is considered standard therapy for widespread metastatic melanoma, and effectiveness of single-agent interventions has been questioned . Here, w2), and was treated with wide local excision and sentinel lymph node biopsy. Two lymph nodes harvested were negative. Half a year later, multiple in-transit metastases appeared on his scalp without signs of distant metastases. The lesions were surgically excised. However, multiple in-transit metastases continued to develop in the region rapidly. Thereafter, surgical excision and topical imiquimod 5% were initiated resulting in complete resolution of one lesion, whereas two others persisted.The patient was a 61-year-old man. Twenty-two years ago, a primary melanoma was removed from his neck. Eleven years later, a second primary melanoma developed on his scalp, and was surgically excised. Seven years later, a third melanoma  developed on the scalp (Clark level III, Breslow thickness 0.9 mm, and mitotic rate 2/mmThe patient was referred to our institution A. The meThe management of recurrent melanoma remains challenging. This patient with prolonged but localized in-transit melanoma showed remarkably improved clinical outcome when intralesional IL-2 was added to the interventions. The synergistic effect of IL-2 with V-beam laser is supported by animal models that show enhanced antitumor immunity with judicious combination of immunologic stimulation and laser devitalization of tumor nodules [IL-2 induced vascular leakage is advantageous in localized treatment because local edema may initiate tumor necrosis that stimulates the immune response. Furthermore, intralesional injection of IL-2 achieves much higher local concentration and limits systemic adverse effects. Therefore a greater therapeutic effect could be expected. Indeed, studies indicated that intralesional IL-2 therapy was more effective than systemic, perilesional, and distant site IL-2 treatments . One repPreferential absorption of laser radiance by hemoglobin and the subsequent conversion of light energy into thermal energy may cause blood vessel coagulation , which mOur results indicate that this combined therapy is a promising strategy for the treatment of in-transit metastatic melanoma. Further study in a larger patient population is warranted.


Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.The zebrafish ( Usintyrosine hydroxylase locus in four of 275 potential F0 zebrafish founders. Subsequently, Shin et al. reported the efficient TALEN-mediated knock-in of donor DNA templates by optimizing the length of the homology arms and the configuration of the donor DNA construct [sox2 locus, and 29 of 363 F0 founders produced sfGFP-positive F1 embryos. Furthermore, tandem dimeric Tomato (tdTomato) was integrated into the gfap locus, and five of 44 F0 founders produced tdTomato-positive F1 embryos [et al. reported the CRISPR/Cas9-mediated knock-in of a donor vector [albino mutant , which carries a nonsense mutation in the slc45a2 gene essential for melanin production. Because the alb mutant embryos exhibit a pale phenotype with pigment defects, the phenotypic rescue by the HR-mediated knock-in of a donor vector containing the wild-type slc45a2 gene is easily determined by the appearance of the pigmented cells. They demonstrated that the frequency of phenotypic rescue was increased using circular donor DNA containing CRISPR target sites. In the rescue experiment, three of 28 F0 founders produced pigmented embryos with the wild-type phenotype [Genome editing technologies (TALEN and CRISPR/Cas9) were applied to perform HR-mediated integration of donor DNA templates because the HR of the donor vector at the target site is largely enhanced by the production of site-specific DSBs. The eGFP reporter donor containing approximately 1 kb of homology arms was integrated at the target site using HR in zebrafish ; howeveronstruct . They usr vector . To examhenotype . Althouget al. reported the efficient CRISPR/Cas9-mediated knock-in of donor vectors at targeted sites in zebrafish [Recently, site-specific insertion via homology-independent repair mechanisms, presumably mediated by NHEJ, was developed. When the targeted genomic locus and the donor vector containing the ZFN or TALEN target sequences were simultaneously cleaved by ZFN or TALEN, the NHEJ-mediated insertion of donor DNA was observed in cultured cells at the target site ,39. One ebrafish . In thisebrafish . When thet al. improved the NHEJ-mediated knock-in of donor vectors for establishing transgenic zebrafish [evx2 gene. Furthermore, the introduction of the hsp promoter usually enhances the basal expression of the integrated reporter/driver gene. Immunostaining using an anti-Evx2 antibody revealed that the eGFP expression generally overlapped with Evx2 protein expression. However, the expression of the Evx2 protein was not detected in some of the eGFP-positive cells, presumably owing to the enhanced leaky expression of the hsp-dependent promoter activity. Using this strategy, the authors consistently established stable knock-in lines in four endogenous loci  [More recently, Kimura ebrafish . They prvglut2a) . Importavglut2a) . The CRItyrosinase locus, the NHEJ-mediated knock-in of the donor vector in the forward direction was detected in 53% of the injected F0 embryos using PCR, while the addition of 10–40 bp homology arms in the donor vector increased its efficiency to 85%. Genomic sequence analysis revealed that the MMEJ-mediated knock-in with precise targeted integration was 60%–77% of analyzed knock-in events. Therefore, the introduction of short homology arms promotes precise targeted integration of exogenous genes. In order to eliminate the undesired integration of the backbone vector sequences, we designed an improved donor vector that configures the eGFP and polyA signal between two bait sequences in the case of the keratin type 1 c19e/krtt1c19e locus (It has been recently shown that DSBs can be repaired by MMEJ that uses the existing microhomology sequences (five to 25 bases) around the broken DNA ends . Both Dr9e locus . We obseGenetic manipulation in whole organisms plays an important role in basic, applied and medical sciences. Recent advances in genome editing technologies  have enabled researchers to make precise genomic modifications that were previously difficult or impossible in various organisms including humans. In this review, we summarized the targeted integration of exogenous genes using genome editing technologies mediated by DNA repair mechanisms, such as NHEJ, MMEJ and HR. Because integration directions become random and various indel mutations occur at the junction in the process of NHEJ-mediated knock-in, we recommend that MMEJ-mediated knock-in technology is suitable for the precise targeted integration of donor DNA templates. Furthermore, the construction of a donor vector for MMEJ-mediated knock-in is much easier than that of HR-mediated knock-in. Therefore, we predict that the MMEJ-mediated targeted integration could be widely applied in various organisms. Importantly, genome editing technologies can be used to precisely model human genetic disorders in animals including zebrafish, which could lead to novel findings with respect to the molecular mechanisms of human diseases. Furthermore, these technologies make it easier to correct serious genetic disorders in affected patients and in induced pluripotent stem (iPS) cells derived from patients. Further innovation of genome editing technologies will accelerate various applications in basic biology, biotechnology and medicine, including potential therapeutic strategies for human genetic disorders.


RS)-2,6-dichloro-9--9H-purine], a small anti-tumor compound, demonstrated selectivity on cancer cells and showed an inhibitory effect over kinases involved in carcinogenesis, proliferation and angiogenesis. The cytotoxic effects of Bozepinib were observed in both breast and colon cancer cells expressing different receptor patterns. Bozepinib inhibited HER-2 signaling pathway and JNK and ERKs kinases. In addition, Bozepinib has an inhibitory effect on AKT and VEGF together with anti-angiogenic and anti-migratory activities. Moreover, the modulation of pathways involved in tumorigenesis by Bozepinib was also evident in microarrays analysis. Interestingly, Bozepinib inhibited both mamo- and colono-spheres formation and eliminated ALDH+ CSCs subpopulations at a low micromolar range similar to Salinomycin. Bozepinib induced the down-regulation of c-MYC, β-CATENIN and SOX2 proteins and the up-regulation of the GLI-3 hedgehog-signaling repressor. Finally, Bozepinib shows in vivo anti-tumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting sub-acute toxicity. These findings support further studies on the therapeutic potential of Bozepinib in cancer patients.Identification of novel anticancer drugs presenting more than one molecular target and efficacy against cancer stem-like cells (CSCs) subpopulations represents a therapeutic need to combat the resistance and the high risk of relapse in patients. In the present work we show how Bozepinib [( Cancer represents the second cause of death by disease in the world, being colorectal and breast cancer two diseases with important incidence in most countries . Some ofThe mortality rate of breast and colorectal cancer is high because of disease recurrence, which remains the major therapeutic barrier in these types of cancer. Although chemotherapy or radiotherapy can kill most bulky tumor cells and provide temporary remission, relapse occurs in most cases, as explained in the recently proposed cancer stem cell hypothesis , 9. ThisRS)-4,1-benzoxazepin-purines against human breast and colon cancer cell lines -9H-purine, named as Bozepinib, presents very low inhibitory concentration 50 (IC50) values in both breast and colon cancer cells, being the most potent structure that we have reported -9H-purine (Bozepinib) was synthesized as previously described  when necessary. Significance was accepted at P < 0.05 in all cases.


The focal adhesion protein leupaxin (LPXN) is overexpressed in a subset of prostate cancers (PCa) and is involved in the progression of PCa. In the present study, we analyzed the LPXN-mediated adhesive and cytoskeletal changes during PCa progression. We identified an interaction between the actin-binding protein caldesmon (CaD) and LPXN and this interaction is increased during PCa cell migration. Furthermore, knockdown of LPXN did not affect CaD expression but reduced CaD phosphorylation. This is known to destabilize the affinity of CaD to F-actin, leading to dynamic cell structures that enable cell motility. Thus, downregulation of CaD increased migration and invasion of PCa cells. To identify the kinase responsible for the LPXN-mediated phosphorylation of CaD, we used data from an antibody array, which showed decreased expression of TGF-beta-activated kinase 1 (TAK1) after LPXN knockdown in PC-3 PCa cells. Subsequent analyses of the downstream kinases revealed the extracellular signal-regulated kinase (ERK) as an interaction partner of LPXN that facilitates CaD phosphorylation during LPXN-mediated PCa cell migration. In conclusion, we demonstrate that LPXN directly influences cytoskeletal dynamics via interaction with the actin-binding protein CaD and regulates CaD phosphorylation by recruiting ERK to highly dynamic structures within PCa cells. Prostate cancer is the most common cancer type and the third most common cause of cancer-related death among men in developed countries . Many prCancer cell adhesion has been known to be a critical feature of this metastatic spread, and understanding the underlying mechanisms of metastasis in PCa will result in the identification of biomarkers for the advanced stages of PCa. Reduction of focal adhesions and their high turnover is required for cell motility, cell migration and cell invasion. Focal adhesions are important cellular structures that build a structural link between the extracellular matrix (ECM) and the actin cytoskeleton. Their components take part in cell signaling downstream of integrin activation induced by ECM proteins such as fibronectin and collagen .One of those focal adhesion proteins is leupaxin (LPXN), which belongs to the paxillin protein family. Characterized by their subcellular localization at focal adhesion sites and their specific domain structure composed of LIM domains and LD motifs, paxillin family proteins are mainly known for protein-protein interactions . OriginaRecently, we found that LPXN was overexpressed in approximately 20% of prostate cancer patients and that LPXN expression correlated with the differentiation stage of these tumors . DownregCALD1). CaD is known to regulate the actin-myosin interaction and thereby actin cytoskeletal stabilization in a Ca2+/Calmodulin and phosphorylation-dependent manner [CALD1 gene encodes five different CALD1 transcripts, resulting in two major isoforms: a high-molecular-mass isoform (h-CaD) that is expressed in smooth muscle cells and a low-molecular-mass isoform (l-CaD) expressed in non-muscle cells. The regulation of CaD is important for proper cell function because decreased expression of l-CaD has been found in many cancer cell types [One protein that is essential for this process and during the regulation of smooth muscle and non-muscle contraction is the protein caldesmon 1 .To investigate the influence of LPXN expression on the adhesive characteristics of PCa cells, we performed a cell adhesion assay. After downregulation of LPXN expression in PC-3 and DU 145 cells using a specific siRNA, cells were plated on glass slides coated with fibronectin (FN), rat tail collagen (Col), bovine serum albumin (BSA) or gelatin (Gel). Adhered cells were fixed after 2 hours of incubation, and the cytoskeleton was visualized using FITC-conjugated phalloidin. Cell numbers and cell size were analyzed using confocal fluorescence microscopy. We observed that cells with LPXN knockdown showed reduced adhesion on all substrates in comparison to control cells Figure . The strAs summarized in Figure CALD1). Subsequently, we verified the specificity of this interaction by a direct yeast two-hybrid experiment with full-length constructs of LPXN and CaD  pulldown assays to identify the LPXN protein domains responsible for this interaction. Therefore, PC-3 cells were transfected with EGFP-CALD1. Several LPXN fusion proteins were used to pull down EGFP-CALD1 from isolated PC-3 protein lysates: GST-LPXN, containing full-length LPXN, GST-LPXN-LD, harboring only the four LD motifs of LPXN (aa 32-176) and GST-LPXN-LIM, harboring only the four LIM domains of LPXN  and CaD (green) at focal adhesion sites and the F-actin filaments Figure .in situ proximity ligation assay (PLA) on PC-3 cells using specific LPXN and CaD antibodies, respectively. Interaction of the two proteins is indicated by the red dots  has been shown to play an important role in the formation and regulation of the actin- cytoskeletal network. To assess the function and relevance of CaD in PCa cells, CaD expression was downregulated in PC-3 and DU 145 cells using two different CALD1-specific siRNAs (siCALD1-A and siCALD1-B). qRT-PCR and western blot analyses showed a strong downregulation of CaD after siRNA treatment in comparison to control transfected cells (siLuc) Figure . A transFrom the literature, CaD is also known to regulate the cell cycle by promoting M-phase entry . To addrBecause it is known that LPXN can translocate to the nucleus and activate transcription factors such as the androgen receptor  and the We further analyzed protein lysates that were isolated from LPXN overexpressing primary cells . These c2+-calmodulin [The affinity of CaD for actin is regulated by phosphorylation and by binding of Calmodulin , 17, 18.We next investigated the biological relevance of the LPXN-CaD interaction during migration of PCa cells. Scratches were applied to confluent PC-3 cell layers. Cells were fixed immediately after scratching and after 18 hours of migration, and proximity ligation assays (PLA) were performed using LPXN- and CaD-specific antibodies. As summarized in Figure To study LPXN interaction with phosphorylated pS534) CaD, a pS534-specific antibody was used in the PLA. Fluorescent microscopy also revealed increased interaction of phosphorylated CaD with LPXN in migrating cells  and p38 MAPK , 20.To investigate which kinase phosphorylates CaD in a LPXN-dependent manner, we first analyzed the signaling pathways influenced by LPXN using the antibody array KAM 850 (Kinexus) with protein lysates from PC-3 cells after LPXN knockdown together with protein from control transfected PC-3 cells. We found the TGF-beta activated kinase 1 (TAK-1), among others, to be downregulated in response to LPXN knockdown Table . TAK-1 hin situ PLA, we were able to detect interaction of LPXN and ERK in PC-3 and DU 145 cells -specific inhibitor PD98059 after LPXN knockdown. PD98059 blocks activation of ERK by preventing phosphorylation through its upstream kinase MEK1. Compared with the control transfected cells (siLuc), no reduction of CaD phosphorylation was observed after treatment with the P38 inhibitor. Only the knockdown of LPXN expression reduced CaD phosphorylation in P38 inhibitor-treated cells. Instead, CaD phosphorylation was markedly decreased as a consequence of ERK inhibition. Furthermore, LPXN knockdown decreased pERK levels but did not enhance the effect of PD98059 on CaD phosphorylation. Figure . This obCell adhesion is fundamentally important in development and tissue homeostasis. Deregulation of this complex process leads to detachment of tumor cells from the tissue of origin and is an important determinant of their metastatic capacity. In previous studies, we showed that LPXN directly influences the migration and invasion of PCa cells  and thatTaken together, these results suggest that LPXN might have important roles in the formation of focal adhesion sites. This is supported by the fact that LPXN was originally found to be expressed in highly motile cells such as macrophages, osteoclasts and leukocytes, whereas in other tissues, LPXN was not detectable , 5. In aTo leave their place of origin, tumor cells must lose adhesion as well as migrate and even penetrate and invade surrounding tissues. Recently, we demonstrated elevated expression levels of LPXN in approximately 20% of prostate cancer patients and showed that knockdown of LPXN by RNAi reduces migration and invasion of PC-3 and DU 145 PCa cell lines . HoweverHere, we identified the actin-binding protein caldesmon (CaD) as an interaction partner of LPXN. In addition, these proteins showed similar expression patterns and co-localization on the subcellular level was observed in PC-3 and DU 145 cells. CaD is known to play an essential role in actin cytoskeletal dynamics and cell contraction, but little is known regarding its functions in cancer cells. Most evidence today is based on studies that have been conducted on h-CaD, which is the high molecular mass isoform of l-CaD and is exclusively expressed in smooth muscle cells, where it takes part in muscle contraction. Because both isoforms are generated from the same gene, their domain sequences are identical, and their biochemical characteristics are expected to be quite similar. h-CaD has been reported to have tumor-supporting  as well In our experiments, we show increased migration and invasion of PC-3 and DU 145 cells after CaD knockdown. This data is supported by two recent independent studies that achieved the same results in MDA-MB-231 breast cancer cells  and AGS Taken together, CaD represents a suitable candidate protein that controls actin stabilization and thereby migration and invasion dynamics downstream of LPXN. In our model, LPXN represents a mediator and regulator of CaD phosphorylation through interaction with ERK, a mechanism that may explain the aggressive growth of LPXN-overexpressing PCa cells.Several lines of evidence indicate that the function of CaD is regulated by calmodulin (CaM) binding and phosphorylation , 38. A nInterestingly, the p38 MAPK has also been reported to phosphorylate CaD at these specific sites. However, we show that p38 MAPK activity was not affected by downregulation of LPXN, whereas the phosphorylation levels of ERK were reduced after LPXN knockdown in PC-3 and DU 145 cells. This is consistent with a study that showed no effect of the p38 MAPK-specific inhibitor, SB203580, on phosphorylation of S789 in h-CaD (corresponding to S534 in l-CaD) in rat vascular smooth muscle cells .According to these data and our findings, p38 MAPK might have distinct functions in CaD phosphorylation but does not play a role in the migration-dependent phosphorylation of CaD. Therefore, we excluded p38 MAPK from our further analyses and focused on ERK-mediated phosphorylation of CaD. In the present study, we hypothesized a possible mechanism for LPXN-mediated migration of PCa cells, in which LPXN acts as an adapter protein that mediates phosphorylation of l-CaD at S534 by the recruitment of ERK Figure . In the In summary, LPXN plays an important role in the adhesion of PCa cells. We found that LPXN acts as a mediator of ERK-dependent CaD phosphorylation and might thereby regulate actin-cytoskeletal dynamics. Pathological conditions caused by LPXN overexpression might thus promote migration and invasion of PCa cells.LNCaP, PC-3 and DU 145 cells  were grown in RPMI 1640 medium  containing 10% fetal calf serum and 1.2% penicillin/streptomycin. Transient transfection was carried out using the X-tremeGENE HP DNA Transfection Reagent  according to the manufacturer's instructions. For inhibition of p38 and MEK1/2, cells were serum-starved overnight and treated with 10 μM SB203580 or 20 μM PD98059, respectively. For stimulation of CaD phosphorylation, cells were stimulated with 10% FCS in the medium for 10 min, and protein was subsequently isolated.Before cells were plated, glass slides were coated with 20 μg/cm² bovine serum albumin, 1% gelatin, or rat-tail collagen for 1 hour or with fibronectin (FN)  according to the manufacturer's instructions. PC-3 and DU 145 cells were plated on coated and uncoated glass slides . Adhesive cells were washed once with DPBS (PAN) and fixed after 1, 2 or 24 hours, as indicated. After staining of the actin cytoskeleton using fluorescein isothiocyanate (FITC)-conjugated phalloidin , cells were counted and cell size was determined by fluorescent microscopy (Olympus BX60) with CellSense® software.EcoRI and XhoI restriction sites of the pCMV-Myc vector  to produce a Leupaxin-Myc fusion protein. Likewise, full-length cDNA of human l-Caldesmon (NM_004342) was cloned into the pEGFP-C1 vector using the XhoI and EcoRI restriction sites and into pCMV-Myc vector using the SfiI and XhoI restriction sites  to produce EGFP-Caldesmon and Caldesmon-Myc fusion proteins, respectively. Fusion constructs were expressed in PC-3, DU 145 and LNCaP cells to analyze subcellular localization. For GST pulldown, full-length cDNA of human Leupaxin as well as LD motifs (aa 32-176) and LIM (aa 173-417) domains were cloned into the EcoRI and XhoI sites of the bacterial expression vector pGEX-4T3  to produce GST-LPXN, GST-LPXN-LD and GST-LPXN-LIM fusion proteins.The full-length cDNA of human Leupaxin (NM_004811) was cloned in frame into the Yeast two-hybrid screening was performed using LPXN as bait pGBKT7-LPXN) and the prostate cDNA library cloned into pGADT7 . Yeast colonies were selected on minimal synthetic dropout (SD) plates lacking amino acids leucine, tryptophan, histidine and adenine (SD –LTHA). Growing yeast colonies were used to isolate plasmid DNA, which was sequenced using vector specific primers. The direct yeast two-hybrid experiments were carried out as described previously [-LPXN andFor co-immunoprecipitation, a pCMV-Myc-CaD or pCMV-Myc-LPXN construct, which contained either the full-length cDNA of CaD (NM_004342) or LPXN (NM_004811), respectively, was generated and transiently transfected into PC-3 cells. For immunoprecipitation, cell lysate of PC-3 cells was incubated with an anti-CaD or anti-LPXN antibody followed by a western blot analysis using a LPXN-specific or CaD-specific antibody, respectively.E. coli DH5α and then purified. Concurrently PC-3 cells were transfected with pEGFP-Caldesmon for 48 h to achieve overexpression of CaD. Isolated protein and the purified GST-fusion proteins were incubated together. The interaction of leupaxin and CaD permits binding of the (GST-LPXN)-(EGFP-CaD) complex to the glutathione-Sepharose. As a negative control, protein from transfected PC-3 cells was incubated with GST protein. After elution of the bound protein, the precipitates were immunoblotted using a CaD-specific antibody. The success of the purification was checked using an anti-GST antibody.GST-fusion proteins were expressed in 32P)-labeled cDNA probes for human β-actin and human CALD1  were hybridized to the membrane in Rapid-hyb buffer  at 65°C for 16 h. The filters were washed, and the hybridization signals were quantified with a Molecular Imager FX using Quantity One software .Total RNA was isolated from PC-3, DU 145 and LNCaP cells with the RNeasy Mini Kit  according to the manufacturer's instructions and used for subsequent northern blot analysis, as previously described . The  were performed using the DuoLink® II Fluorescence Kit  according to the manufacturer's instructions. Briefly, fixed cells were blocked with 3% BSA in PBS for 30 min at 37°C. All subsequent incubation steps were performed in a humidity chamber. Cells were incubated with specific primary antibodies raised in different species at suitable concentrations to detect proteins of interest. Secondary antibodies conjugated to specific plus and minus oligonucleotide probes, as indicated, were incubated with the reaction for 1 hour at 37°C. The linker oligonucleotide sequence and ligase were added and incubated for 30 min at 37°C to prepare for rolling-circle amplification of the annealed PLA probes. Rolling-circle amplification was carried out for 100 min at 37°C. Finally, the amplified sequences were detected using fluorescently labeled oligonucleotides. Protein proximity is indicated by distinct fluorescent spots analyzed by fluorescent microscopy using the FluoView1000 confocal laser-scanning microscope . Antibodies to detect Leupaxin-Caldesmon/pS789-Caldesmon and Leupaxin-ERK/pERK interactions were purchased as follows: polyclonal anti-Leupaxin N-terminal SAB4200010 ; monoclonal mouse anti-purified l-Caldesmon , polyclonal rabbit anti-p-Caldesmon (Ser789) ; rabbit polyclonal p44/42 MAPK (ERK1/2) and rabbit monoclonal phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) D13.14.E4 .Transfection of PC-3 and DU 145 cells was accomplished as previously described . AdditioIn vitro cell migration was analyzed as described previously [In vitro cell invasion of PC-3 and DU 145 cells was determined in BioCoat Matrigel Invasion Chambers  as described previously [eviously . In brieeviously .3 siRNA-transfected PC-3 or DU 145 cells were plated in 96-well plates, and proliferation was determined using the MTS assay (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) Promega) according to the manufacturer's instructions.For these assays, 3 × 10


Bulk Regional Viral Injection (BReVI). Our strategy involves a large volume adeno-associated virus (AAV) injection in the targeted brain region of neonatal Cre driver mice. Using the mouse olfactory bulb (OB) as a model system, we tested the ability of BReVI to broadly and selectively label tufted cells, one of the two principal neuron populations of the OB, in CCK-IRES-Cre mice. BReVI resulted in labeling of neurons throughout the injected OB, with no spatial bias toward the injection site and no evidence of damage. The specificity of BReVI labeling was strikingly similar to that seen previously using immunohistochemical staining for cholecystokinin (CCK), an established tufted cell marker. Hence, the CCK-IRES-Cre line in combination with BReVI can provide an important tool for targeting and manipulation of OB tufted cells. We also found robust Cre-dependent reporter expression within three days of BReVI, which enabled us to assess developmental changes in the number and laminar distribution of OB tufted cells during the first three postnatal weeks. Furthermore, we demonstrate that BReVI permits structural and functional imaging in vivo, and can be combined with transgenic strategies to facilitate multi-color labeling of neuronal circuit components. BReVI is broadly applicable to different Cre driver lines and can be used to regionally manipulate genetically defined populations of neurons in any accessible brain region.The ability to label and manipulate specific cell types is central to understanding the structure and function of neuronal circuits. Here, we have developed a simple, affordable strategy for labeling of genetically defined populations of neurons throughout a targeted brain region:  The recent development of a large repertoire of Cre driver lines  has greaBulk Regional Viral Injection (BReVI) to achieve population labeling throughout the targeted brain region of Cre driver mice, beginning in early postnatal life. We tested BReVI primarily in the olfactory bulb (OB), the initial site of odor processing in the mammalian brain. Here, the principal neurons of the OB, the mitral and tufted cells, process odor information received from olfactory sensory neurons in the nose before transmitting it to olfactory cortex. While often considered to be functionally equivalent, mitral and tufted cells in fact transmit temporally distinct signals to spatially segregated areas of olfactory cortex (in vivo. The neuropeptide cholecystokinin (CCK) is expressed in tufted but not mitral cells . We also demonstrate that BReVI can be used for both structural and functional imaging +/-;ROSA-CAG-LSL-tdTomato+/-  or OMP-nd #8601 ]. Mice wAAV was obtained from Penn Vector Core. Viruses used were AAV1.CAG.Flex.tdTomato.WPRE.bGH , AAV1.CAG.Flex.GCaMP6f.WPRE , and AAV1.CAG.Flex.eGFP.WPRE.bGH . P0–P3 mouse pups were cryo-anesthetized  in an icMice were deeply anesthetized with 200 mg/kg ketamine and 20 mg/kg xylazine (Vedco) and transcardially perfused with ice-cold PBS followed by 4% PFA. Brains were dissected out and post-fixed overnight in 4% PFA at 4°C, cryopreserved in 30% sucrose in PBS for 24 h at 4°C, embedded in 10% gelatin, fixed/cryopreserved in 15% sucrose/2% PFA in PBS overnight, and flash frozen in 2-methyl butane on dry ice. Coronal sections were cut using a cryostat  at 25 or 40 μm. Sections were mounted directly in Vectashield containing DAPI (Vector Labs) or ProLong Diamond containing DAPI (Life Technologies), or stored at -80°C.Free-floating sections were blocked in 2% normal donkey serum/0.1% Triton X-100 for 1 h, then incubated with rabbit anti-Iba1 primary antibody  and then anti-rabbit-AF647 secondary antibody  in 2% normal donkey serum/0.05% Tween 20 for 1h each at RT before mounting in Vectashield or ProLong Diamond.+/-;Thy1-YFP-G+/-;TH-Cre+/- tissue were acquired using a Leica TCS SP5 equipped with HCX PLAN FLUOTAR 10x/0.3NA air and HCX PL APO 40x/1.25NA oil immersion objectives and spectral detectors. GFP and YFP fluorescence were collected sequentially and unmixed using LASAF software (Leica).Images of CCK-IRES-Cre tissue were acquired with a Nikon A1R confocal microscope equipped with Plan Fluor 10x/0.3NA air and Apo 60x/1.4NA oil immersion objectives. Images of TH-Cre and OMP-GFPFigure 4, tdTomato-labeled neurons in CCK-IRES-Cre mice were counted in 40x confocal z-stacks. Iba1-stained microglia and labeled granule cells in TH-Cre mice were counted in 10x confocal images. For the comparison of labeled neuron density with CCK-IRES-Cre;ROSA-CAG-LSL-tdTomato mice, tdTomato-expressing neurons were counted in 10x confocal images of 25 μm sections from P14 CCK-IRES-Cre mice injected with AAV1-FLEX-tdTomato at P0. Counts for P14 CCK-IRES-Cre;ROSA-CAG-LSL-tdTomato mice were made from images in the Allen Developing Mouse Brain Atlas (Laminar position was assigned according to the following criteria: cells in the glomerular layer (GL) were juxtaglomerular, i.e., encircled a glomerulus; the superficial EPL (sEPL) was the superficial half of the EPL, containing dense tdTomato-labeled lateral dendrites; the deep EPL (dEPL) was the deep half of the EPL extending to the border of the MCL; the MCL was defined as the dense band of somata identified by DAPI staining; the internal plexiform layer (IPL) was immediately deep to the MCL with sparse nuclei and tdTomato-labeled axons in older mice; and the granule cell layer (GCL) commenced where nuclei became more densely packed, deep to the IPL. For the data in in Atlas . Linear 2) and ∼1 mm diameter cranial windows were implanted over the right OB as described (2) were imaged using a Leica TCS SP5 microscope using a HCX APO 20x/1.0NA water-immersion objective lens and a Chameleon Vision II laser (Coherent) mode-locked at 910 nm. tdTom emission was collected at >600 nm and GCaMP6f emission at 500–550 nm.Mice were anesthetized using isoflurane  was dissolved in mineral oil (1% volume/volume) and delivered to the anesthetic air stream using a PV-820 Pneumatic PicoPump (World Precision Instruments). The timing of odorant delivery and image acquisition were controlled using LASAF software (Leica). Each imaging trial lasted 20 s, consisting of 3 s of baseline, 3 s of odor presentation, and 14 s after stimulus termination. Images  were collected at three frames/s and there was a 90 s interval between trials.t-tests , one-way ANOVA (soma diameter) or two-way ANOVA (cell counts in CCK-IRES-Cre mice injected with AAV1-Flex-tdTomato). All statistical tests were performed using Prism 6 (GraphPad).Data were compared using paired We assessed the ability of BReVI, which comprises a single large-volume AAV injection in the neonatal brain, to label a genetically defined population of neurons throughout the targeted brain region. We tested BReVI in the mouse OB, which has a radial, laminated structure that makes it ideally suited to assess the spatial extent of AAV-mediated labeling in three dimensions. We focused on labeling of tufted cells, one of the two principal neuron populations of the OB, which were genetically targeted using the CCK-IRES-Cre knock-in line  and AAV Figure 1A). Labeled axons formed a dense band in the IPL, and were more sparsely distributed in the GCL , while labeled lateral dendrites were in the superficial half of the EPL, characteristic of tufted cells (in situ hybridization (ISH) in the rodent OB . To estimate the proportion of CCK-expressing neurons that were transduced by AAV, we compared the density of labeled neurons in mice injected at P0 with AAV1-FLEX-tdTomato  with that in age-matched CCK-IRES-Cre;ROSA-CAG-LSL-tdTomato mice . Overall, approximately 90% of the number of neurons labeled in the Cre reporter line cross were labeled in mice injected with AAV at P0, indicating that the majority of Cre-expressing neurons were transduced with AAV.We performed freehand injections of 1 μl of AAV1-Flex-tdTomato near the dorsal surface of the right OB in P0 CCK-IRES-Cre mice. Three weeks post-injection, we observed tdTomato-expressing neurons, predominantly concentrated in the deep GL, EPL, and MCL in OB sections . The laminar distribution of labeled neurons was very similar to that seen following P0 injection. Interestingly, we noted that in P0-injected mice, tufted cells throughout the OB contralateral to the injection site were also brightly labeled . In marked contrast, in P3-injected mice, there was little or no labeling of tufted cells in the contralateral (uninjected) OB . Some labeling of centrifugal fibers in the GCL of the contralateral OB was seen following P3 injection, which presumably arises from retrograde infection of bilaterally projecting CCK-expressing neurons in the anterior olfactory nucleus  and control contralateral OBs . Microglia had similar ramified morphologies in both OBs , suggesting that AAV injection did not result in microglial activation. Therefore, we concluded that BReVI provides a reliable method for labeling of genetically defined populations of neurons throughout the mouse OB.We performed BReVI in a total of 25 neonatal CCK-IRES-Cre mice, with a 100% survival rate and no need to foster pups post-injection. We also examined serial cryostat sections through the OBs of these mice, and found no evidence of either spatial bias in tufted cell labeling within the OB, or of mechanical tissue damage to the OB resulting from the injection. Furthermore, the density of Iba1-labeled microglia was similar in the injected , although their numbers and laminar positions differed from those of tdTomato-labeled neurons in older mice . Therefore, we quantified the density of tdTomato-labeled neurons in different layers of the OB during the first three weeks of postnatal life . In both the GL and sEPL, the density of tdTomato-labeled neurons, which correspond to external/superficial and middle tufted cells, increased dramatically between P7 and P14 . In the dEPL, the location of internal tufted cells, tdTomato-labeled cell density increased gradually from P3 to P14, and then stabilized . This more gradual developmental change meant that differences between age groups mostly did not reach statistical significance . Interestingly, there was also a high density of tdTomato-labeled neurons in the MCL in 2–3 week-old mice . Many of these MCL-residing neurons had large somata , which in combination with the lack of labeled secondary dendrites in the dEPL suggests that these may be type II mitral cells . Finally, scattered tdTomato-labeled neurons in the IPL and GCL were present at all ages . These cells had small somata  and hence are likely to be granule cells or deep short-axon cells that transiently expressed CCK during their development. We did not observe any differences in the density or laminar distribution of tdTomato-labeled neurons in 3-week-old mice injected at P0 vs. P3 . Overall, we concluded that BReVI-mediated labeling in CCK-IRES-Cre mice enables developmental analysis of OB tufted cells.We found that brightly labeled tdTomato-expressing neurons were present throughout the OB as early as three days post-injection . At higher magnification, the structure of apical dendritic tufts in the GL  and lateral dendrites in the EPL  can clearly be visualized. Hence, BReVI is a useful technique to enable structural analysis of genetically defined neurons in vivo.Imaging of fine neuronal structures deep in the living brain requires strong expression of fluorescent proteins . To assein vivo. We injected P3 CCK-IRES-Cre mice with AAV1-Flex-GCaMP6f. Three weeks later, GCaMP6f fluorescence was detectable in a subset of tufted cells . Many tufted cells in fixed tissue did not exhibit GCaMP6f fluorescence, consistent with the low resting fluorescence of GCaMP6 variants . In preliminary odor stimulation trials, we identified a glomerulus that exhibited robust responses to odor stimulation with 1% EB, and was surrounded by other glomeruli that did not respond to this odor . We then quantified responses of both external tufted cells, and tufted cell dendrites, to EB stimulation. The external tufted cell located at the periphery of the EB-responsive glomerulus exhibited a significant response to EB stimulation, whereas other more distant external tufted cells showed no change in fluorescence during odor stimulation . All regions of interest (ROIs) within the EB-responsive glomerulus showed robust, large magnitude fluorescence increases in response to EB stimulation, whereas ROIs in neighboring glomeruli showed no response . Notably, ΔF/F was much greater for tufted cell dendrites than for the responsive external tufted cell . Overall, these data demonstrate that BReVI-mediated labeling with GCaMP6f enables functional imaging of neuronal responses in vivo.We next assessed whether BReVI drives expression of a functional reporter sufficiently strongly to detect sensory responses variants . We thenFigure 7A). Again, labeling was evident throughout the extent of the OB, and showed no spatial bias toward the dorsally located injection site . Consistent with fluorescent reporter expression in TH-Cre;flox-reporter mice, some small neurons in the MCL, whose dendrites terminated in the EPL, were also brightly labeled , their density was much lower than in TH-Cre;ROSA-CAG-LSL-tdTomato mice .To confirm that BReVI can also be used to label other neuronal types in the OB, we performed P0 injections of AAV1-Flex-GFP in TH-Cre mice to label dopaminergic neurons, one of the regenerating populations of OB interneurons . We obsein vivo 2-photon time-lapse imaging at 1 h intervals for 3 h. This enabled individual dendritic branches to be resolved and tracked over time, with no discernable photo-bleaching during the 3 h imaging session . To test whether BReVI can be combined with transgenic and knock-in lines to facilitate labeling of multiple components of the glomerular circuit, we injected P2 OMP-GFP+/-;Thy1-YFP-G+/-;TH-Cre+/- mice with AAV1-Flex-tdTomato. Eight weeks post-injection, we observed strong expression of tdTomato in dopaminergic neurons in the GL, in addition to GFP-expressing olfactory sensory neuron axons and YFP-expressing mitral cells arising from knock-in/transgene-mediated expression . Hence, we labeled three important components of the OB circuit with spectrally distinct fluorescent proteins, while circumventing the extra generation of breeding (and associated time and expense) required for introduction of a fourth transgene.We next injected TH-Cre mice with AAV1-Flex-GFP at P2 and implanted cranial windows 6 weeks later. We performed Figure 8A), anterior cingulate, prelimbic, infralimbic, and motor cortex . A small number of somata in the same regions of the left hemisphere were also labeled. The tdTomato expression pattern matched that seen previously using ISH , consistent with previous reports of CCK immunoreactivity in the corpus callosum and striatum . It is possible that granule cells are poorly transduced by AAV1-Flex-GFP, or that cis-acting sequences prevent GFP expression from the viral construct. Alternatively, the transient transcription from the TH promoter that presumably drives reporter expression in TH-Cre;ROSA-LSL-tdTomato mice may be largely complete by birth, such that postnatal BReVI labels only a small number of GCL neurons in TH-Cre mice.We have developed BReVI, a simple, cost-effective strategy for reporter gene expression in large numbers of neurons throughout a particular brain region. BReVI requires minimal equipment, is quick to perform (∼30 min per litter), and all injected pups survived the procedure with no evidence of injection site damage. In the OB, which comprises radial layers that enabled us to assess the spread of viral infection in three dimensions, neurons were labeled throughout the entire injected OB. Indeed, reporter gene expression appeared to be uniform in Cre-expressing neurons for an ∼2 mm radius around the injection site, in both the OB and in neocortex. Injections could be performed up to P3, and we observed strong reporter expression as early as 3 days post-injection, providing the opportunity to label and manipulate specific neuronal types both for developmental studies and for slice electrophysiology experiments, which typically use juvenile mice. Furthermore, expression was strong enough to permit elopment . One sucelopment . We founFigure 8) demonstrate that BReVI has broad applicability and can be used to drive reporter expression in any accessible brain region. Similarly, stereotaxic BReVI could be used to target deeper brain structures for population labeling. However, it should be noted that BReVI is complementary to existing strategies for AAV-mediated expression and is not ideal for all purposes. In circumstances where it is essential to confine reporter expression to a discrete locus, for example when assessing the function or connectivity of a spatially restricted group of neurons, then small volume stereotaxic AAV injections in adult mice would be a better experimental strategy.Although we predominantly tested BReVI in the OB, the results of our injections in frontal cortex (We used BReVI to evaluate the specificity of tufted cell labeling in the OBs of CCK-IRES-Cre mice. The pattern of Cre-dependent reporter expression was strikingly similar to that seen by anti-CCK immunohistochemistry , with soGiven the tdTomato-labeled neurons in the MCL, does the CCK-IRES-Cre line specifically label OB tufted cells? First, it is clear that only a small subset of principal neurons in the MCL are labeled in CCK-IRES-Cre mice, therefore we believe that this line can be used to distinguish between principal neuron sub-types. Second, the distinction between mitral and internal tufted cells can be ambiguous; indeed, internal tufted cells have also been termed ‘displaced mitral cells’ . ClassifWe found that the number of tdTomato-labeled tufted cells in the GL, EPL, and MCL increased dramatically between P3 and P14, and then stabilized. There was a particularly sharp increase in tufted cell density between P7 and P14; overall, the number of tdTomato-labeled neurons increased 3.8-fold over the course of the second postnatal week. This finding is consistent with a previous report that external tufted cell numbers increase during postnatal development , ruling CC designed experiments, acquired, analyzed, and interpreted data and wrote the manuscript. BG acquired data. LB contributed to experimental design. All authors critically revised the work for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current report assessed the effects of low-level proton irradiation in inbred adult male Fischer 344 and Lewis rats performing an analog of the human Psychomotor Vigilance Test (PVT), commonly utilized as an object risk assessment tool to quantify fatigue and sustained attention in laboratory, clinical, and operational settings. These strains were used to determine if genetic differences in dopaminergic function would impact radiation-induced deficits in sustained attention. Exposure to head-only proton irradiation (25 or 100 cGy) disrupted rPVT performance in a strain-specific manner, with 25 cGy-exposed Fischer 344 rats displaying the most severe deficits in sustained attention ; Lewis rats did not display behavioral deficits following radiation. Fischer 344 rats displayed greater tyrosine hydroxylase and dopamine transporter levels in the frontal cortex compared to the Lewis rats, even though radiation exposure increased both of these proteins in the Lewis rats only. Tyrosine hydroxylase was decreased in the parietal cortex of both rat strains following radiation exposure, regardless of proton dose. Strain-specific cytokine changes were also found in the frontal cortex, with the Lewis rats displaying increased levels of putative neurotrophic cytokines . These data support the hypothesis that basal dopaminergic function impacts the severity of radiation-induced deficits in sustained attention. Very little is known about the short- and long-term biological consequences associated with exposure to high energy and charge (HZE) and proton radiation. In addition to changing an astronaut’s risk of cancer, it is acknowledged that such radiation may have cumulative deleterious effects in multiple tissues, including the central nervous system (CNS). Ground-based studies demonstrate that radiation can induce behavioral changes in rodents, including impaired performance in motor tasks and deficits in spatial learning and memory –3. Thoug56Fe exposure in rodents  and a significant within-subjects interaction of Week X Strain X Radiation Dose ; significant between-subjects main-effect of Radiation Dose  was also found. No other significant effects or interactions were apparent . Tukey-corrected post-hocs showed a significant decrease in percent correct scores for the 25 cGy-exposed F344 rats at Weeks 13, 14, 17–24, 28–31, and 34 post-irradiation when compared to F344 and LEW sham controls, and Week 33 for the F344 sham controls . The 25 While not a statistically significant decrease, the 100 cGy-exposed F344 rats did show a subtle decrease over time in percent correct scores, with a weekly mean of 81% correct immediately following radiation exposure to a weekly mean of 67% correct at 34 weeks post-exposure. Importantly, this group of rats was consistently below the 75% correct criterion starting at Week 28 post-irradiation, suggesting that these rats displayed a less-severe performance deficit compared to the 25 cGy-exposed rats. In comparison, both exposed groups of LEW rats maintained percent correct scores at or above 75% correct throughout the post-exposure period.For false alarm rates, a repeated-measures ANOVA with a Greenhouse-Geisser correction determined a significant within-subject main-effect of Week  and a significant within-subjects Week X Strain X Radiation Dose interaction ; a significant between-subjects main-effect of Radiation Dose  was also found. No other main-effects or interactions were significant . Tukey-corrected post-hocs showed a significant increase in false alarm rates in the 25 cGy-exposed F344 rats at Weeks 13, 14, 17–24, 28–31, 33, and 34 compared to F344 and LEW sham controls . The 25 For median reaction time, a repeated-measures ANOVA with a Greenhouse-Geisser correction revealed only a significant within-subjects main-effect of Week ; the effect of Strain and Strain X Radiation Dose interaction were not significant . Levels of this protein were significantly decreased in 25 cGy-exposed rats, regardless of strain, when compared to sham-irradiated controls and 100 cGy exposed rats .Five different proteins were examined in the frontal cortices of all F344 and LEW rats: tyrosine hydroxylase (TOH), the rate-limiting enzyme for dopamine synthesis; the dopamine transporter (DAT); Akt and phospho-Akt  = 11.673, p < 0.05], but not Strain (p = 0.913) or the Strain X Radiation Dose interaction (p = 0.162). TOH was significantly decreased in the parietal cortex of all irradiated rats, regardless of proton dose, when compared to sham-irradiated controls  = 18.693, p < 0.05] and Strain X Radiation Dose interaction , but no main-effect of Radiation Dose (p = 0.641). On average, pAktser473 decreased with increasing radiation dose in the LEW rats and increased with increasing radiation dose in the F344 rats. These changes were significantly different in the rats exposed to 100 cGy, with F344 rats displaying significantly more of this protein compared to sham-irradiated controls and both LEW proton-exposed groups (p ≤ 0.029), but not F344 rats exposed to 25 cGy (p = 0.563). LEW rats exposed to 100 cGy protons displayed a significant decrease in this protein compared to both 25 and 100 cGy-exposed F344 rats , but were not different from sham-irradiated controls of either strain or LEW rats exposed to 25 cGy .Three different proteins were examined in the parietal cortices of all F344 and LEW rats: TOH, Akt, and pAktTwenty-nine different cytokines were assessed with the array in the frontal cortex of F344 and LEW rats and are listed in The results indicate that F344 rats exposed to head-only protons at 25 cGy show significant decreases in accuracy and significant increases in impulsive responding, while LEW rats do not. Importantly, 100 cGy-exposed F344 rats did display a decrease in rPVT performance over the post-exposure period, even though this difference was not significantly different from F344 sham controls. For example, the 100 cGy-exposed F344 rats displayed a more subtle decrease in accuracy compared to the 25 cGy-exposed F344 rats, and the 100 cGy-exposed F344 rats were consistently below the 75% correct criterion from starting at Week 28. While this change was not significantly different from the F344 sham controls, performance consistently below the 75% correct criterion could be considered a performance deficit. In comparison, both of the irradiated LEW groups maintained accuracy values at or above 75% correct for the duration of the post-exposure period.increase in TOH levels in the frontal cortex. This increase in frontal cortex TOH in LEW rats was also accompanied by an increase in the DAT protein. No changes in frontal cortex DAT levels were apparent in the irradiated F344 rats. Thus, radiation exposure induced brain protein changes in a strain-specific manner, suggesting that the LEW rats were able to adapt to or mitigate the radiation-induced damage, which resulted in a lack of behavioral deficits following head-only proton exposure.These behavioral deficits (or lack thereof) were accompanied by strain-specific changes in various proteins in the frontal and parietal cortices, two brain areas important for sustained attention and performance of the PVT in humans. Specifically, radiation exposure decreased TOH in the parietal cortex in all irradiated groups, but only the LEW rats displayed a concomitant In a previous report from our laboratory, proton-induced rPVT deficits including decreased accuracy and increased impulsive responding, were associated with increased whole-brain DAT protein levels in 100 cGy-exposed, but not 25 cGy-exposed outbred Long-Evans rats. Further, these behavioral and neurochemical changes were evident in approximately 40% of the exposed cohort, which suggests the presence of individual differences in sensitivity to the deleterious effects of proton radiation on neurobehavioral performance. On the surface, the data in the current report appear to contradict these previous results, given that the LEW rats displayed increased DAT levels, but did not display radiation-induced deficits in rPVT performance throughout the 34-week post-IR testing period; however, the differences in basal dopaminergic tone between these inbred strains could underlie these results and have important implications for interpretation of our previous report in light of pre-irradiation differences in the dopaminergic neurotransmitter system.in vivo DAT activity in various brain regions [greater frontal cortex DAT levels than the LEW sham controls prior to adjusting all data to a change from each strain’s sham control values , a dose and energy similar to those used in the current study, in addition to deficits in the conditioned passive avoidance task . Thus, DAs expected, the pattern of cytokine changes in the frontal cortex was strain-specific. For example, the standard proinflammatory cytokines such as IL-1b, IL-6, and TNF-α were not consistently elevated in either strain; IL-6 was significantly decreased in the 25 cGy-exposed F344 rats, but unchanged in the irradiated LEW rats. Interestingly, several cytokines that are considered neurotrophic and/or important for cognitive function were differentially altered in the F344 and LEW rats and could provide promising potential therapeutic leads in future studies. In particular, CNTF was two-fold higher in 25 cGy-exposed LEW rats compared to the LEW sham controls. This cytokine could be important for the maintenance of control-like rPVT performance in this group, given that administration of CNTF reportedly improves cognitive function and/or motor performance in preclinical models of Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotropic lateral sclerosis Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


ECG-gated cardiac MRI is the gold standard for volumetric evaluation of patients, and clinically, ejection fraction is used as a surrogate for function. We hypothesized that the use of arrhythmia rejection in the presence of ectopic beats compromises the accuracy of hemodynamic measurements. Real-time MRI, coupled with ECG telemetry and semi-automated LV endocardial segmentation, can be used to identify multiple beat morphologies and derive global hemodynamic measurements for each beat.Short-axis golden angle radial bSSFP projections (8000 projections/slice) were reconstructed using Gadgetron  with 34 projections per frame, and slice volume was measured via segmentation of LV endocardial contour with ITK-SNAP. ECG was synchronously recorded with the acquisition, and each image frame was assigned a time point on the ECG (~22 seconds/slice). QRS detection was used to identify cardiac cycles, and similar beats (across slices) were grouped via RR-duration of the beat of interest as well as the previous (loading) beat. The use of the loading beat duration allowed for accurate grouping of premature ventricular contractions (PVCs). For each beat morphology, global volume was obtained by summation of slice volume curves allowing for hemodynamic evaluation via EDV, ESV, SV, and EF.10 patients were imaged: 5 in sinus rhythm, 3 with PVCs with 19%, 24%, and 33% prevalence, 1 in regular bigeminy, and 1 in irregular bigeminy. All beats detected beats were assigned a morphology, excluding the first beat of each acquisition since the loading conditions of these beats were unknown. A single beat morphology was observed in patients in sinus rhythm while patients with rhythm disturbances demonstrated multiple beat morphologies with large variations in measured volumes and EF. Figure ECG-gated cardiac MRI is unable to capture the hemodynamics associated with arrhythmic events. As a result, values such as EF are acquisition - dependent (desired RR-duration determines arrhythmia rejection). By combining real-time volume measurements with ECG recordings, we categorized beat morphologies and provided a more comprehensive evaluation of ventricular function during arrhythmia.We would like to acknowledge the National Institutes of Health for support through grants: F31-HL120580, R00-HL108157, R01-EB014346, R01-HL103723, R01-HL63954, T32-HL007954, T32-EB009384.


Mutations activating the receptor tyrosine kinase c-Kit occur commonly in melanomas arising on mucosal membranes and acral skin. Clinical studies have demonstrated that selective inhibition of c-Kit is effective in treating patients with c-Kit mutant gastrointestinal stromal tumors, but c-Kit inhibitor activity has been disappointing in c-Kit mutant melanoma patients. Activated c-Kit utilises phosphatidylinositol 3-kinase (PI3K) signalling as the dominant effector of cell proliferation and survival with the mitogen-activated protein kinase (MAPK) cascade serving as an ancillary survival pathway. We confirmed that these pathways are re-activated in melanoma cells with acquired resistance to c-Kit inhibitors and that these resistant sublines remain sensitive to the concurrent inhibition of MAPK and PI3K signalling. These findings suggest that durable responses in c-Kit mutant melanoma may require combination therapies that selectively inhibit critical downstream proliferative and survival pathways. We also discuss the interaction between targeted therapies and anti-tumor immune responses and the need to consider immunotherapies in new combinatorial treatment approaches. The constitutive activation of the c-Kit receptor tyrosine kinase via somatic mutations is uncommon in melanoma (3% are c-Kit mutant), but occurs in approximately 20% of melanomas arising from acral skin  or mucosal surfaces and less frequently in cutaneous melanomas found in chronically sun-damaged skin . c-Kit mThe importance of mutant c-Kit as a therapeutic target has been demonstrated in gastrointestinal stromal tumors (GIST), which frequently harbour activating c-Kit mutations . The treINK4a variants, may diminish c-Kit inhibitor responses [Q61K mutation was associated with c-Kit inhibitor resistance in a c-Kit mutant melanoma [The diminished clinical efficacy of imatinib-based therapy in c-Kit-mutant melanoma patients may reflect the high genetic mutation load found in melanomas and the distribution of activating c-Kit mutations. The average somatic mutation rate of 30 mutations per Mb in three c-Kit mutant melanomas is high compared to other tumors and pre-existing alterations, including activating H-RAS mutations or loss-of-function p16esponses . For insmelanoma . Furthermelanoma , and varmelanoma , 7. Sevemelanoma , dasatinmelanoma  and nilomelanoma  are actiTargeting signalling effectors downstream of driver oncogenes may be an effective, alternative therapeutic strategy. For instance, the pharmacological inhibition of MEK (the downstream target of BRAF) improves overall survival in patients with BRAF-mutant metastatic melanoma, when compared with chemotherapy . The actL576P mutant M230 melanoma cell line after prolonged exposure to these c-Kit inhibitors. The genetic effectors of resistance included additional secondary c-Kit mutations (A829P or T670I) and c-Kit independent mechanisms [A829p mutant rendered melanoma cells resistant to imatinib and sunitinib, but these cells remained sensitive to nilotinib and dasatinib. In contrast, sublines with the second-site c-KitT670I mutation showed resistance to imatinib, nilotinib and dasatinib, but responded to sunitinib. As expected sublines with c-Kit independent mechanisms were resistant to all tested c-Kit inhibitors. Importantly, regardless of the resistance mechanism, all c-Kit inhibitor resistant M230 sublines re-activated MAPK and PI3K signalling and remained sensitive to the concurrent inhibition of these two pathways.The fundamental roles of MAPK and PI3K signalling were also demonstrated in c-Kit mutant melanoma cell models with acquired resistance to c-Kit inhibitors imatinib and nilotinib . Multiplchanisms . Given mchanisms , 17. Secchanisms , and conOur work highlights the central role of the PI3K and MAPK cascades in c-Kit mutant melanoma, and the clinical potential of concurrently inhibiting these pathways. This treatment strategy is also being explored in patients with BRAF-mutant melanoma and may prove effective in circumventing acquired drug resistance. Recent reports have confirmed that BRAF-inhibitor treated melanoma patients rapidly develop multiple, independent mechanisms of resistance that can exist within a single progressing tumor , 20. We Recent reports have also demonstrated that the therapeutic efficacy of targeted therapies, including c-Kit and BRAF inhibitors may depend on anti-tumor T-cell responses. For instance, BRAF-mutant melanoma patients treated with BRAF inhibitors exhibit increased tumor infiltration by CD8+ lymphocytes early during therapy . SimilarMolecular targeted therapies for the treatment of c-Kit mutant melanoma have been disappointing. Clinical responses are likely diminished by pre-existing genetic alterations and early resistance is heterogeneous and frequently involves the re-activation of downstream c-Kit signalling pathways. The current management of c-Kit mutation-positive melanoma patients involves enrolment in clinical trials using a c-Kit inhibitor or immunotherapy, and there is minimal data for efficacy of immune-modulators in c-Kit mutant melanomas . To achi


In this recent study, we established the concept that the antigenicity/immunogenicity of ALDH+/ALDHhigh or ALDEFLUOR-/ALDHlow cells were isolated from tumor cells. ALDHlow lysate-pulsed DCs (ALDHlow -DC) or ALDHhigh lysate-pulsed DCs  were used as vaccines. To detect T and B cells in PBMCs, PBMCs were stained with PerCP mouse anti-human CD3 and FITC mouse anti-human CD19. Before sensitization in vitro, T or B cells were activated respectively with immobilized anti-human CD3 and anti-human CD28 in complete medium (CM) containing hrIL-2 or lipopolysaccharide plus anti-human CD45. Statistical analyses were performed to compare various interactions of the DC vaccine-primed/sensitized T, B cells with autologous ALDHhigh CSC vs. ALDHlow HNSCC target cells.Patients with HNSCC enrolled in the University of Michigan Special Project of Research Excellence (SPORE) were recruited to collect tumor and peripheral blood samples. T or B cells were purified from the PBMCs using anti-CD3-coupled or anti-CD19-coupled microbeads respectively with a MACS separator. Primary tumor samples were digested and collected. ALDEFLUORhigh cells isolated from cultured HNSCC cells (CSC-DC) could sensitize autologous T, B lymphocytes in vitro, which was evident by cytokine production, CTL activity, and antibody secretion of these primed T, B cells in response to ALDHhigh CSCs. In contrast, DCs pulsed with lysate of ALDHlow cells from the same HNSCC patient (ALDHlow-DC) resulted in limited sensitization/priming of autologous T, B lymphocytes to produce IFNg, lyse CSCs, and secrete IgM and IgG in response to ALDHhigh CSCs.DCs generated from the PBMC and pulsed with the lysate of ALDHhigh CSCs vs. ALDHlow cells isolated from the tumor specimen of patients with HNSCC, which indicates the existence of unique CSC antigens in the ALDHhigh population. In addition, this study demonstrates that it is feasible to generate DCs from the PBMCs and isolate ALDHhigh CSCs from tumor cells of the patients with HNSCC to prepare CSC-DC vaccines for clinical application.These results demonstrated significant differences in the antigenicity/immunogenicity between ALDH


Plasmodium falciparum sporozoites (CPS-immunization) induces sterile protection from malaria. Antibody responses have long been known to contribute to naturally acquired immunity against malaria, but their association with sterile protection after whole sporozoite immunization is not well established. We therefore studied the induction and kinetics of malaria parasite antigen-specific antibodies and memory B-cells (MBCs) during CPS-immunization and their correlation with protection from challenge infection.Immunization of healthy volunteers during receipt of chemoprophylaxis with P. falciparum by performing standardized MBC enzyme-linked immunospot and enzyme-linked immunosorbent assays on peripheral blood mononuclear cells and plasma samples from 38 Dutch volunteers enrolled in 2 randomized controlled clinical trials.We assessed humoral reactivity to 9 antigens representing different stages of the life cycle of MBCs and antibodies recognizing pre-erythrocytic and cross-stage antigens were gradually acquired during CPS-immunization. The magnitude of these humoral responses did not correlate with protection but directly reflected parasite exposure in CPS-immunization and challenge.Humoral responses to the malarial antigens circumsporozoite protein, liver-stage antigen-1, apical membrane antigen-1, and merozoite surface protein-1 do not to predict protection from challenge infection but can be used as sensitive marker of recent parasite exposure.NCT01236612 and NCT01218893. Plasmodium falciparum [Plasmodium parasites have a complex multistage life cycle, initiated by anopheline mosquitoes depositing sporozoites into the skin of the vertebrate host, which then migrate to the liver, where they establish a clinically silent infection of hepatocytes. After maturation, merozoites egress from hepatocytes into the bloodstream, where they invade and cyclically replicate within erythrocytes. During blood-stage infection, clinical pathology becomes apparent and can be severe. A safe, affordable, and effective vaccine to supplement other intervention strategies would dramatically benefit public health [Malaria remains a major global health burden leading to widespread morbidity and mortality, which is mainly caused by lciparum . Apicompc health , but a vc health , due to c health .Malaria subunit vaccine development has thus far yielded disappointing results, with RTS,S the only vaccine candidate tested in phase 3 clinical trials. This circumsporozoite protein (CSP)–based vaccine showed an encouraging 50% sterile protection in malaria-naive adult volunteers , 7. In cAntibodies play a critical role in preventing infection by a large range of pathogens . ImmediaBoth clinical trials from which samples for this study were obtained received approval by the Central Committee for Research Involving Human Subjects of the Netherlands  and were registered at ClinicalTrials.gov . The study team complied with the Declaration of Helsinki and good clinical practice, including monitoring of data. Volunteers enrolled in both studies provided written informed consent.P. falciparum–infected mosquitoes on 3 occasions, which was previously shown to provide sterile protection from future mosquito challenge [intravenous injection of parasitized erythrocytes. Study B [P. falciparum–infected mosquitoes. All immunized and control subjects in this study were subjected to mosquito challenge. This study setup allowed us to longitudinally assess humoral responses in healthy, previously malaria-naive, adult volunteers before and after exposure to different P. falciparum life cycle stages in the context of CPS-immunization, in the presence of prophylactic drug levels, and after challenge infection. After challenge, sterile protection was defined as the absence of parasitized erythrocytes on thick blood smears until 21 days after challenge. Volunteers for whom parasites were detected on a thick blood smear were immediately treated with antimalarial drugs, and all remaining volunteers received presumptive drug treatment 21 days after challenge. Importantly, the amount of exposure to blood-stage parasites during both immunization and challenge was characterized for each volunteer retrospectively by quantitative polymerase chain reaction (qPCR) analysis. For further details about study groups, outcomes and sample collection see supplementary information.To determine the generation of malaria-specific MBC and antibody responses in individuals who received CPS-immunization and those with primary infection, we used peripheral blood mononuclear cells (PBMCs) and plasma samples from 2 single-center randomized controlled clinical trials Figure . In studhallenge , 12. The Study B  was a CP7 cells/mL in fetal calf serum (Gibco)/10% dimethyl sulfoxide (Merck), using Mr Frosty freezing containers , and stored in vapor-phase nitrogen. Plasma samples were stored in aliquots at −20°C and rethawed no more than twice.PBMCs were isolated by density centrifugation, cryopreserved at 10Staphylococcus aureus Protein A, Toll-like receptor 9 ligand ODN 2006, and recombinant human interleukin 10 to promote development of MBCs into antibody-secreting cells (ASCs) [Supplementary Materials.The generation of malaria-specific MBCs was assessed by an MBC enzyme-linked immunospot assay . Cryopres (ASCs) . ASCs wes (ASCs) . For a dU test (2-tailed and nonparametric). Differences between values from >2 times points were analyzed by the Friedman test with the Dunn multiple comparison post hoc test . Differences between values for ≥2 different groups over time were analyzed by a repeated-measures, mixed-model, 2-way analysis of variance with the Bonferroni post hoc test. Correlations were analyzed by Spearman correlation (nonparametric). Differences with a P value of <.05 were considered statistically significant.Data were analyzed using GraphPad Prism v6. Differences between values at 2 time points were analyzed by the Wilcoxon matched-pairs signed rank test . Unmatched data between 2 groups were analyzed by the Mann–Whitney P. falciparum antigens representing either the pre-erythrocytic or the erythrocytic part of the Plasmodium life cycle. CSP and liver-stage antigen 1 (LSA-1) are exclusively expressed by sporozoites and liver-stage parasites, whereas most proteins, including exported protein 1 (EXP-1), thrombospondin-related anonymous protein (TRAP), merozoite surface protein 1 (MSP-1) and MSP-2, apical membrane antigen 1 (AMA-1), and glutamate-rich protein (GLURP), are most highly abundant in blood-stage parasites but are also expressed in late liver-stages (cross-stage antigens). Erythrocyte binding protein 175 (EBA-175), on the other hand, is only expressed in blood-stage parasites. Between 19 and 21 weeks after the last CPS-immunization . Antibodies for the cross-stage antigens EXP-1, MSP-1, and MSP-2 were induced, whereas no significant induction of AMA-1, GLURP, and TRAP antibodies was observed . All immunized volunteers developed CSP-specific antibodies (above group background), and antibodies were detectable in 74% of volunteers for LSA-1. Over half (53%) of the volunteers had MSP-1–specific antibodies above background after CPS-immunization, while 42% and 37% developed antibodies against EXP-1 and MSP-2, respectively. Immunization also gave rise to CSP-specific MBCs in 95% of volunteers, and 42% of volunteers also developed MSP-1–specific MBCs, while no MBCs specific for any other selected antigen were observed (data not shown).Malaria-specific antibody and MBC responses induced by the highest-dose regimen of CPS-immunization . Antibody responses peaked after the third immunization but then contracted over the following 15 weeks, until C − 1 . Importantly, a different pattern was observed for the acquisition of MBCs : CSP- and MSP-1-specific MBCs were also acquired in a stepwise fashion during CPS-immunization. However, for the majority of volunteers, they did not contract after the third immunization in the absence of parasite exposure but instead remained stable or even increased during the >4-month period until C − 1. These patterns of CSP- and MSP-1–specific antibody and MBC acquisition were seen irrespective of immunization dose .We next studied the kinetics of acquisition of antibody and MBC responses to the strongest recognized antigens, CSP and MSP-1, over the course of CPS-immunization Figure A. Antibo1 Figure A. Imports Figure B: CSP- aAn important question is whether the magnitude of CPS-induced humoral responses to the investigated antigens reflects the degree of parasite exposure and whether they are predictive of sterile protection from challenge infection. We therefore compared immune responses in volunteers who received different immunization doses Figure  and did A). CPS-immunization with different numbers of infectious mosquito bites also led to a different amount of blood-stage exposure measured by qPCR after the first immunization . This is consistent with the dose-response relation for completed pre-erythrocytic and early erythrocytic development, which is in turn reflected in the induction of MSP-1 antibodies . The level of blood-stage parasitemia was reduced after the second and completely absent after the third CPS-immunization in volunteers subsequently sterilely protected from mosquito challenge .In volunteers protected from mosquito challenge, there was a trend toward higher antibody responses to CSP with increasing amounts of immunizing infectious mosquito bites, consistent with the different doses of pre-erythrocytic parasites experienced Figure A. CPS-imn Figure B. This is Figure A. The lee Figure B.C). This was probably a result of the greater parasite exposure during the second and third immunization, reflected by the significantly higher level of blood-stage parasitemia, compared with protected volunteers .In the group receiving the lowest immunization dose (bites from 5 mosquitoes on 3 occasions), half of the volunteers were unprotected from mosquito challenge. Unprotected volunteers had significantly higher antibody responses against both CSP and MSP-1 after the third immunization, compared with protected volunteers receiving the same number of immunizing mosquito bites Figure C. This ws Figure D.E).Across all immunized volunteers in this dose de-escalation trial Figure , magnitu4These data clearly demonstrate that the magnitude of the humoral response against CSP, LSA-1, AMA-1, and MSP-1 after CPS-immunization does not predict protection from challenge infection but accurately reflects parasite exposure (both the number of infectious mosquito bites and the amount of blood-stage parasitemia) during CPS-immunization.Supplementary Table 1). A total of 22 immunized volunteers across both clinical trials were protected from mosquito challenge , but responses specific for the analyzed cross-and blood-stage antigens were not (Supplementary Table 1). One volunteer, however, who was classified as protected from mosquito challenge on the basis of the absence of parasites on a thick blood smear, was retrospectively found to have developed qPCR-detectable parasitemia (457 parasites/mL) at day 21 after challenge, just prior to receipt of presumptive drug treatment [5A).Humoral immune responses acquired during CPS-immunization were boosted dependent on parasite exposure by different challenge regimens and protection status .Seven CPS-immunized volunteers were unprotected and thus experienced blood-stage parasitemia (as well as pre-erythrocytic parasites) after mosquito challenge Figure . In line5C and Supplementary Table 1).Furthermore, 9 CPS-immunized volunteers were subjected to and not protected from injection of parasitized erythrocytes Figure  and, thu5Supplementary Table 2), albeit at lower levels than after mosquito challenge of primed CPS-immunized volunteers. In the majority of volunteers, a single infection was not sufficient to induce a detectable MBC response.Interestingly, a single mosquito challenge of malaria-naive controls from studies A and B also induced CSP- and MSP-1–reactive antibodies in 80% and 90% of volunteers, respectively  and cross-stage (MSP-1) antigens, which is consistent with exposure mainly to pre-erythrocytic parasite antigens during CPS-immunization. Exposure to erythrocytic-stage parasites is limited by chloroquine in this immunization regimen; hence, no humoral responses against AMA-1, GLURP, and EBA175, which are mainly expressed during the erythrocytic stages of the parasite, were detectable. This recognition profile is in line with data from a previous CPS-immunization study, in which antibody reactivity toward CSP but not toward schizont lysate or GLURP was observed in most volunteers . Furtherlunteers .P. falciparum–specific antibodies and MBCs after CPS-immunization was associated with cumulative parasite exposure, defined as the number of immunizations, the immunization dose, and the level of blood-stage parasitemia experienced. Curiously, correlation of CPS-induced humoral responses with cumulative blood-stage exposure during immunization was not only observed for MSP-1, which is expressed in erythrocytic parasites, but also for CSP, which is often considered a sporozoite-specific antigen. However, it was shown that CSP-expression is not confined to the sporozoite stage but continues until the late stages of liver infection for P. falciparum [P. berghei and P. yoelii [The abundance of lciparum , as well. yoelii , 26. SinP. falciparum erythrocyte membrane protein 1 than women in their first pregnancy, also indicating a successful generation and maintenance of humoral immune memory [Antibody and MBC levels expanded gradually after the first, second, and third CPS-immunization, a kinetic seen independently of the level of parasite exposure during immunization. Antibody titers declined in the absence of parasite exposure between immunization and challenge, likely because of the physiological contraction of short-lived plasma cell responses, whereas MBC levels were largely stably maintained or even increased further. That MBCs are much more long-lived than quickly waning serological responses was recently also reported in African children no longer exposed to malaria . Furthere memory . An intre memory , 30, is Challenge infection strongly boosted antibody responses acquired during immunization. This suggests an efficient differentiation of MBCs into ASCs upon antigen re-encounter, even in volunteers experiencing patent blood-stage parasitemia during challenge infection. Therefore, low-grade blood-stage exposure during challenge infection in humans does not lead to an ablation of previously generated MBCs or impaired survival of resulting antibody-producing cells, unlike previous findings for high-level erythrocytic parasitemias in rodent malaria models , 32.P. falciparum antigens was dependent on exposure to different parasite life cycle stages during challenge. Mosquito challenge boosted pre-erythrocytic and cross-stage responses, while exposure to blood-stage parasites boosted responses to cross-stage and erythrocytic antigens. Of note, the subunit vaccine candidate TRAP [P. falciparum protein, was also increased by blood-stage exposure during challenge, strengthening reports of TRAP or TRAP-like protein expression in erythrocytic parasites [P. falciparum–infected erythrocytes for 1 day only led to a strong boosting of the MSP-1 antibody response. By contrast, MSP-1 antibody levels in volunteers with qPCR-confirmed absence of blood-stage parasitemia after challenge were not boosted. In turn, a lack of boosting of MSP-1–specific antibody responses after challenge infection may be used as an indicator of sterile pre-erythrocytic protection. Taken together, antibody and MBC responses observed in our study depended directly on the amount and life cycle stage of parasite exposure during both CPS-immunization and challenge.Boosting of humoral responses to the different ate TRAP , often carasites , 35. We arasites . This smP. falciparum transmission dynamics in a naturally exposed population [P. falciparum sporozoite challenge [The immunodominant antigens in our study, CSP and MSP1, induced antibodies in the majority of volunteers already after a single CPS-immunization or mosquito challenge and can thus be used as markers of recent parasite exposure. Indeed, CSP antibodies can be used to assess pulation . Howeverpulation , 38 werehallenge , RTS,S ohallenge , 7. The hallenge –43. Our hallenge . Analysihallenge . In RAS-hallenge .+ T-cells with a cytotoxic phenotype (CD107a+) [Finally, while the magnitude of individual or combined humoral responses does not predict protection, the actual functional capacity of the resulting antibodies could do so. We have recently shown that antibodies induced by CPS-immunization can reduce the traversal of sporozoites through hepatocytes in vitro, as well as during liver-stage infection and development in vivo in human liver-chimeric mice . HoweverCD107a+) . An inteP. falciparum. Despite their efficient acquisition, the magnitude of acquired humoral immune responses to the set of immunodominant malarial antigens analyzed in this study did not predict sterile protection from challenge infection. Instead, it proved to be a sensitive indicator of the degree and nature of parasite-antigen exposure during both immunization and challenge. The magnitude of acquired antigen-specific humoral responses alone can therefore not be used as a surrogate marker of protective efficacy. Other indicators and assessments of responses to novel antigens are hence necessary to predict protection.In conclusion, CPS-immunization induced antibody and MBC responses mainly against pre-erythrocytic and cross-stage antigens of The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.Supplementary materials are available at


Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. Here we show that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. We propose that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. Our findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions.The Lethal giant larvae (Lgl) protein was discovered in  •Aurora kinases directly phosphorylate Lgl during mitosis•Aurora phosphorylation of Lgl drives relocalization to the cytoplasm at mitosis•Relocalization of Lgl promotes mitotic spindle orientation in epithelia•Removal of Lgl from the plasma membrane may promote Pins-Dlg interactions Bell et al. describe a new role for the Lgl tumor suppressor in mitotic spindle orientation. They show that Aurora kinases phosphorylate Lgl to drive a dramatic relocalization of the protein during mitosis that is necessary for normal orientation of mitotic spindles in epithelial cells. Drosophila  consensus motif for Auroras, suggesting that the middle site may be an exclusive site for aPKC A. Consislgl mutant clones with wild-type Lgl-GFP, with Aurora-insensitive LglASA-GFP, or with a membrane-tethered myristylated Lgl-GFP (myrLgl-GFP) that cannot be removed from the plasma membrane by either aPKC or Auroras. We find that Lgl-GFP rescues cell polarity in lgl mutant wing discs, as expected , an apical protein that can bind to Pins/LGN and orient the spindle  Figure K. After pins mutant phenotype is weaker in imaginal discs than follicle cells, which suggests the existence of another Dlg-binding protein that may act in parallel with Pins in this tissue.In the case of symmetrically dividing epithelial cells, a similar mechanism operates to regulate Lgl localization, which is then essential for spindle orientation in the plane of the epithelium L. aPKC pIn conclusion, the Lgl tumor suppressor is regulated by similar mechanisms in both symmetric and asymmetric divisions. The aPKC kinase controls polarization of Lgl, whereas the Aurora A and B kinases control mitotic relocalization of Lgl to the cytoplasm in both epithelia and neuroblasts. Mitotic relocalization of Lgl is essential to promote mitotic spindle orientation in epithelia, but not in neuroblasts. Future work on Lgl and its role as a tumor suppressor should consider not only its function at the cell cortex but also its role in regulating the mitotic spindle. Finally, Aurora kinases are known to be overexpressed in many human cancers, and the Lgl protein may be an important target for Auroras in promoting tumor formation and progression.FRT42B aPKCpsu141, FRT42B aPKCpsu417, FRT42B aPKCpsu265 [FRT82B aurA14641 [Crb-GFP [UAS-Baz-mCherry [UAS-Lgl-GFP, UAS-Lgl3A-GFP [UAS-Dlg-GFP [UAS-Par6-GFP [Scrib-GFP [UAS-AurA-GFP [FRT40A lgl4, aurA87Ac-3, lgl334, FRT42B aPKCK06403, Df(3R)Exel6163, dlg1, dlgM52 (Bloomington Drosophila Stock Centre); and UAS-AurB-RNAi (KK library). UAS-myrLgl-GFP, UAS-LglASA, and UAS-LglASA-GFP constructs were generated for this study. Additional methods are described in the The following strains were used: KCpsu265 ; FRT82B [Crb-GFP ; UAS-Baz-mCherry ; UAS-Lglgl3A-GFP ; UAS-Dlg-Dlg-GFP ; UAS-ParPar6-GFP ; Scrib-Gcrib-GFP ; UAS-AurAurA-GFP ; FRT40A


However, cellular therapies can pose significant risks, including graft-vs.-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The “ideal” suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of “all” and “only” the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase and inducible-caspase-9.Adoptive T-cell therapy can involve donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte expanded  Donor T cells within the HSC product or infused post-transplant as DLI have been associated with potentially fatal graft-vs.-host-disease (GVHD) Copelan, , whereasUnlike small molecules or biologics, cell therapies have a very long, or even an indefinite half-life, therefore since toxicity can be progressive a safety switch is needed in order to eliminate the infused cells in case of adverse events. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The “ideal” suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of “all” and “only” the cells responsible for the unwanted toxicity.This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the more extensively validated suicide genes in the clinical setting: herpes-simplex-thymidine-kinase (HSV-TK) and inducible-caspase-9 (iCasp9).Suicide gene technologies can be broadly classified based upon their mechanism of action in metabolic , dimerization inducing, and therapeutic monoclonal antibody mediated. The ideal agent for suicide gene activation should be biologically inert, have an adequate bio-availability and bio-distribution profiles, and be characterized by intrinsic acceptable or absent toxicity.GDEPT  eliminate cells by inducing apoptosis , or human CD20 codon optimized suicide gene. In this study, activation of HSV-TK, iCasp9, and CD20 ultimately resulted in equally effective destruction of transduced T cells. However, while iCasp9 and CD20 effected immediate cell-death induction, HSV-TK-expressing T cells required 3 days of exposure to ganciclovir to reach full effect, and mTMPK-transduced cells showed lower T-cell killing at all time-points  effect: the iCasp9 and the HSV-TK suicide genes. Clinical results are summarized in Table Bonini et al. demonstrated the efficacy of HSV-TK suicide gene modified T cells in controlling GVHD in 8 patients who received DLI after HSCT for disease relapse or EBV post-transplant lymphoproliferative disease (PTLD). Importantly, a GVL effect attributed to the DLI was demonstrated in five patients  molecule, serving as selectable marker to ensure ≥90% purity , or matched related settings, .Given the successful abrogation of GVHD In both the HSV-TK and iCasp9 studies, infusion of suicide gene modified cells aided non-gene modified T cell immune reconstitution (Ciceri et al., TCR (Robbins et al., Innovative technologies offer compelling opportunities for the optimization of gene therapy based approaches. Successful clinical validation of suicide gene strategies to control GVHD after allo-HSCT is now in advanced phase clinical studies, and introduction of suicide genes in conjunction with CAR modification of T cells for cancer immunotherapy is now undergoing phase 1 clinical testing.in vitro and in vivo models (Huber et al., Suicide genes have also been employed as cytotoxic strategy, Finally, non-integrating vectors (Banasik and McCray, All the authors contributed to conception, acquisition, and analysis of data, participated in the manuscript draft preparation, revision and approved and revised the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Because IFN-I has a key role in both the innate and adaptive immune responses, it is believed that heightened levels of this cytokine and the many genes it regulates may underlie the immune hyperreactivity and autoimmunity of SLE. The mechanism of IFN-I hyperproduction in SLE is under intense study. An important lead has emerged from our laboratory implicating the RIG-I antiviral pathway as a possible cause. In particular, the mitochondrial adaptor protein MAVS is a key intermediary in the RIG-I/MDA5 pathway, where viral RNA triggers a conformational change in RIG-I, leading to MAVS activation with subsequent IFN production. It has been reported that MAVS may form large prion-like aggregates, which might stimulate IFN-I production in a potent and prolonged fashion. We wondered whether such aggregates might be detectable Peripheral blood mononuclear cells (PBMCs) were isolated from patients fulfilling ACR criteria for SLE, from healthy controls, and from patients with rheumatoid arthritis (RA). Mitochondrial lysates were prepared and MAVS aggregation was identified with a semi-denaturing agarose gel and confirmed by confocal immunofluorescent microscopy.P < 0.01 by chi-square. 64.7% aggregation-positive patients had active SLE disease , while only 10% of the aggregation-negative patients had active disease.Twenty-two of 61 SLE patients showed clear MAVS aggregation, with essentially all of their MAVS protein in a high molecular weight aggregated form. None of the RA patients and only three of 33 healthy controls had abnormal MAVS. Clinical data analysis revealed that 82.4% MAVS-aggregate-positive SLE patients (mean age 46) had anti-SSA antibodies, compared with 40% MAVS-aggregate-negative patients (mean age 44), Our findings are consistent with the notion that activation of the RIG-I pathway through inappropriate or persistent MAVS aggregation may lead to increased IFN-I production, immune stimulation, and systemic autoimmunity in SLE.