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ملاحظات حول الإخراج : 130 درسا في القيادة من مقعد المخرج
\"ملاحظات حول الإخراج : 130 درسا في القيادة من مقعد المخرج\" هو كتاب تعليمي يجمع بين خبرات وتجارب اثنين من المخرجين المسرحيين المرموقين، فرنك هاوزر ورسل ريتشل. يقدم الكتاب مجموعة من الدروس والنصائح العملية التي يمكن أن تكون مفيدة لكل من يعمل في مجال الإخراج، سواء في المسرح أو السينما أو حتى في المجالات الإبداعية الأخرى التي تتطلب القيادة والإدارة، يتناول الكتاب في البداية مفهوم القيادة من منظور المخرج، موضحا كيف أن الإخراج لا يتعلق فقط بإدارة التفاصيل الفنية، بل أيضا بإلهام الفريق، وتحفيز الممثلين، والتعامل مع التحديات التي قد تنشأ أثناء الإنتاج، يحتوي الكتاب على 130 درسا قصيرا، يقدم كل منها نصيحة أو ملاحظة عملية حول جوانب مختلفة من عملية الإخراج. تتنوع هذه الدروس بين كيفية التعامل مع الممثلين، وإدارة الوقت، واتخاذ القرارات السريعة، إلى كيفية خلق بيئة عمل إيجابية وتحفيز الفريق لتحقيق أفضل أداء.
Book
Feeding-induced changes in allatostatin-A and short neuropeptide F in the antennal lobes affect odor-mediated host seeking in the yellow fever mosquito, Aedes aegypti
Aedes aegypti is a model species in which the endogenous regulation of odor-mediated host seeking behavior has received some attention. Sugar feeding and host seeking in female A. aegypti are transiently inhibited following a blood meal. This inhibition is partially mediated by short neuropeptide F (sNPF). The paired antennal lobes (ALs), as the first processing centers for olfactory information, has been shown to play a significant role in the neuropeptidergic regulation of odor-mediated behaviors in insects. The expression of sNPF, along with other peptides in the ALs of A. aegypti, indicate parallel neuromodulatory systems that may affect olfactory processing. To identify neuropeptides involved in regulating the odor-mediated host seeking behavior in A. aegypti, we use a semi-quantitative neuropeptidomic analysis of single ALs to analyze changes in the levels of five individual neuropeptides in response to different feeding regimes. Our results show that the level of sNPF-2, allatostatin-A-5 (AstA-5) and neuropeptide-like precursor-1-5 (NPLP-1-5), but not of tachykinin-related-peptides and SIFamide (SIFa), in the AL of female mosquitoes, changes 24 h and 48 h post-blood meal, and are dependent on prior access to sugar. To assess the role of these neuropeptides in modulating host seeking behavior, when systemically injected individually, sNPF-2 and AstA-5 significantly reduced host seeking behavior. However, only the injection of the binary mixture of the two neuropeptides lead to a host seeking inhibition similar to that observed in blood fed females. We conclude that modulation of the odor mediated host seeking behavior of A. aegypti is likely regulated by a dual neuropeptidergic pathway acting in concert in the ALs.
Journal Article
Improvement in fingerprint detection using Tb(III)-dipicolinic acid complex doped nanobeads and time resolved imaging
•Tb dipicolinic acid complex doped silica nanobeads as a fingerprint powder.•Functionalized the surface to increase lipophilicity for selective binding.•Optimised doping efficiency of complex into nanobeads.•Utilised time resolved imaging to successfully detect latent fingerprints.
This paper deals with the synthesis and application of lanthanide complex doped nanobeads used as a luminescent fingerprint powder. Due to their special optical properties, namely a long emission lifetime, sharp emission profiles and large Stokes shifts, luminescent lanthanide complexes are useful for discriminating against signals from background emissions. This is a big advantage because latent fingerprints placed on multicoloured fluorescent surfaces are difficult to develop with conventional powders. The complex of 2,6-dipicolinic acid (DPA) and terbium ([Tb(DPA)3]3−) is used for this purpose. Using the Stöber process, this complex is incorporated into a silica matrix forming nanosized beads (230–630nm). It is shown that the [Tb(DPA)3]3− is successfully incorporated into the beads and that these beads exhibit the wanted optical properties of the complex. A phenyl functionalisation is applied to increase the lipophilicity of the beads and finally the beads are used to develop latent fingerprints. A device for time resolved imaging was built to improve the contrast between developed fingerprint and different background signals, whilst still detecting the long lasting luminescence of the complex. The developed fingerprint powder is therefore promising to develop fingerprints on multicoloured fluorescent surfaces.
Journal Article
An evolutionary genomics view on neuropeptide genes in Hydrozoa and Endocnidozoa (Myxozoa)
Background
The animal phylum Cnidaria consists of six classes or subphyla: Hydrozoa, Scyphozoa, Cubozoa, Staurozoa, Anthozoa, and Endocnidozoa. Cnidarians have an early evolutionary origin, diverging before the emergence of the Bilateria. Extant members from this phylum, therefore, are important resources for understanding the evolution of the nervous system. Cnidarian nervous systems are strongly peptidergic. Using genomics, we have recently shown that three neuropeptide families (the X
1
PRX
2
amides, GRFamides, and GLWamides) are wide-spread in four (Scyphozoa, Cubozoa, Staurozoa, Anthozoa) out of six cnidarian classes or subphyla, suggesting that these three neuropeptide families emerged in the common cnidarian ancestor. In the current paper, we analyze the remaining cnidarian class, Hydrozoa, and the subphylum Endocnidozoa, to make firm conclusions about the evolution of neuropeptide genes in Cnidaria.
Results
We analyzed sixteen hydrozoan species with a sequenced genome or transcriptome, using a recently developed software program for discovering neuropeptide genes. These species belonged to various hydrozoan subclasses and orders, among them the laboratory models
Hydra
,
Hydractinia
, and
Clytia
. We found that each species contained three to five neuropeptide families. A common feature for all hydrozoans was that they contained genes coding for (i) X
1
PRX
2
amide peptides, (ii) GRFamide peptides, and (iii) GLWamide peptides. These results support our previous conclusions that these three neuropeptide families evolved early in evolution. In addition to these three neuropeptide families, hydrozoans expressed up to two other neuropeptide gene families, which, however, were only occurring in certain animal groups. Endocnidozoa (Myxozoa) are microscopically small endoparasites, which are strongly reduced. For long, it was unknown to which phylum these parasites belonged, but recently they have been associated with cnidarians. We analyzed nine endocnidozoan species and found that two of them (
Polypodium hydriforme
and
Buddenbrockia plumatellae
) expressed neuropeptide genes. These genes coded for neuropeptides belonging to the GRFamide and GLWamide families with structures closely resembling them from hydrozoans.
Conclusions
We found X
1
PRX
2
amide, GRFamide, and GLWamide peptides in all species belonging to the Hydrozoa, confirming that these peptides originated in the common cnidarian ancestor. In addition, we discovered GRFamide and GLWamide peptide genes in some members of the Endocnidozoa, thereby linking these parasites to Hydrozoa.
Journal Article
PACAP-38 and PACAP(6–38) Degranulate Rat Meningeal Mast Cells via the Orphan MrgB3-Receptor
Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than PACAP-27 in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed PACAP(6-38) to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6-38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor.
Journal Article
CCHamide-2 Is an Orexigenic Brain-Gut Peptide in Drosophila
The neuroendocrine peptides CCHamide-1 and -2, encoded by the genes ccha1 and -2, are produced by endocrine cells in the midgut and by neurons in the brain of Drosophila melanogaster. Here, we used the CRISPR/Cas9 technique to disrupt the ccha1 and -2 genes and identify mutant phenotypes with a focus on ccha-2 mutants. We found that both larval and adult ccha2 mutants showed a significantly reduced food intake as measured in adult flies by the Capillary Feeding (CAFE) assay (up to 72% reduced food intake compared to wild-type). Locomotion tests in adult flies showed that ccha2 mutants had a significantly reduced locomotor activity especially around 8 a.m. and 8 p.m., where adult Drosophila normally feeds (up to 70% reduced locomotor activity compared to wild-type). Reduced larval feeding is normally coupled to a delayed larval development, a process that is mediated by insulin. Accordingly, we found that the ccha2 mutants had a remarkably delayed development, showing pupariation 70 hours after the pupariation time point of the wild-type. In contrast, the ccha-1 mutants were not developmentally delayed. We also found that the ccha2 mutants had up to 80% reduced mRNA concentrations coding for the Drosophila insulin-like-peptides-2 and -3, while these concentrations were unchanged for the ccha1 mutants. From these experiments we conclude that CCHamide-2 is an orexigenic peptide and an important factor for controlling developmental timing in Drosophila.
Journal Article
De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission
Background
The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa
Tripedalia cystophora.
Results
Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from
T. cystophora.
We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel
T. cystophora
neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans
Alatina alata, Carybdea xaymacana, Chironex fleckeri,
and
Chiropsalmus quadrumanus.
Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in
T. cystophora
. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides.
Conclusions
By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from
T. cystophora
that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.
Journal Article
Isolation and Functional Characterization of Calcitonin-Like Diuretic Hormone Receptors in Rhodnius prolixus
Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixusCT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogasterCT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC50 = 200-300 nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15 nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.
Journal Article
Comparison of Different Analytical Methods for the On-Site Analysis of Traces at Clandestine Drug Laboratories
While fast and reliable analytical results are crucial for first responders to make adequate decisions, these can be difficult to establish, especially at large-scale clandestine laboratories. To overcome this issue, multiple techniques at different levels of complexity are available. In addition to the level of complexity their information value differs as well. Within this publication, a comparison between three techniques that can be applied for on-site analysis is performed. These techniques range from ones with a simple yes or no response to sophisticated ones that allows to receive complex information about a sample. The three evaluated techniques are immunoassay drug tests representing easy to handle and fast to explain systems, ion mobility spectrometry as state-of-the-art equipment that needs training and experience prior to use and ambient pressure laser desorption with the need for a highly skilled operator as possible future technique that is currently under development. In addition to the measurement of validation parameters, real case samples are investigated to obtain practically relevant information about the capabilities and limitations of these techniques for on-site operations. Results demonstrate that in general all techniques deliver valid results, but the bandwidth of information widely varies between the investigated techniques.
Journal Article
Expression Patterns of the Drosophila Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain
The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, in situ hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly Drosophila melanogaster. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.
Journal Article